Chapter 3: Mechanisms of Toxicity

Depending primarily on the degree and route of exposure, chemicals may adversely affect the function and/or structure of living organisms. The qualitative and quantitative characterization of these harmful or toxic effects is essential for an evaluation of the potential hazard posed by a particular chemical. It is also valuable to understand the mechanisms responsible for the manifestation of toxicity— that is, how a toxicant enters an organism, how it interacts with target molecules, and how the organism deals with the insult.

An understanding of the mechanisms of toxicity is of both practical and theoretical importance. Such information provides a rational basis for interpreting descriptive toxicity data, estimating the probability that a chemical will cause harmful effects, establishing procedures to prevent or antagonize the toxic effects, designing drugs and industrial chemicals that are less hazardous, and developing pesticides that are more selectively toxic for their target organisms. Elucidation of the mechanisms of chemical toxicity has led to a better understanding of fundamental physiologic and biochemical processes ranging from neurotransmission (eg, curare-type arrow poisons) through deoxyribonucleic acid (DNA) repair (eg, alkylating agents) to transcription, translation, and signal transduction pathways (eg, chemicals acting through transcription factors [TFs], such as the aryl hydrocarbon receptor [AhR]). Pathologic conditions such as cancer and Parkinson disease are better understood because of studies on the mechanism of toxicity of chemical carcinogens and 1,2,3,6-tetrahydro-1-methyl-4-phenylpyridine (MPTP), respectively. Continued research on mechanisms of toxicity will undoubtedly continue to provide such insights.

This chapter reviews the cellular mechanisms that contribute to the manifestation of toxicities. Although such mechanisms are also dealt with elsewhere in this volume, they are discussed in detail in this chapter in an integrated and comprehensive manner. We provide an overview of the mechanisms of chemical toxicity by relating a series of events that begins with exposure, involves a multitude of interactions between the invading toxicant and the organism, and culminates in a toxic effect. This chapter focuses on mechanisms that have been identified definitively or tentatively in humans or animals.

As a result of the huge number of potential toxicants and the multitude of biological structures and processes that can be impaired, there are a tremendous number of possible toxic effects. Correspondingly, there are various pathways that may lead to toxicity (Fig. 3-1). A common course is when a toxicant delivered to its target reacts with it, and the resultant cellular dysfunction manifests itself in toxicity. An example of this route to toxicity is that taken by the puffer fish poison, tetrodotoxin. After ingestion, this poison reaches the voltage-gated Na⁺ channels of neurons (step 1). Interaction of tetrodotoxin with this target (step 2a) results in blockade of Na⁺ channels, inhibition of the activity of motor neurons (step 3), and ultimately skeletal muscle paralysis. No repair mechanisms can prevent the onset of such toxicity.

Sometimes a xenobiotic does not react with a specific target molecule but rather adversely influences the biological (micro) environment, causing molecular, organellar, cellular, or organ dysfunction leading to deleterious effects. For example, 2,4-dinitrophenol, after entering the mitochondrial matrix space (step 1), collapses the outwardly directed proton gradient across the inner membrane by its mere presence there (step 2b), causing mitochondrial dysfunction (step 3), which is manifest in toxic effects such as hyperthermia and seizures. Chemicals that precipitate in renal tubules and block urine formation represent another example for such a course (step 2b).

The most complex path to toxicity involves more steps (Fig. 3-1). First, the toxicant is delivered to its target or targets (step 1), after which the ultimate toxicant interacts with endogenous target molecules (step 2a), triggering perturbations in cell function and/or structure (step 3), which initiate repair mechanisms at the molecular, cellular, and/or tissue levels as well as adaptive mechanisms to diminish delivery, boost repair capacity, and/or compensate for dysfunction (step 4). When the perturbations induced by the toxicant exceed repair and adaptive capacity or when repair and adaptation becomes malfunctional, toxicity occurs. Tissue necrosis, cancer, and fibrosis are examples of chemically induced toxicities whose development follow this 4-step course.

Theoretically, the intensity of a toxic effect depends primarily on the concentration and persistence of the ultimate toxicant at its site of action. The ultimate toxicant is the chemical species that reacts with the endogenous target molecule (eg, receptor, enzyme, DNA, microfilamental protein, lipid) or critically alters the biological (micro) environment, initiating structural and/or functional alterations that result in toxicity. Often the ultimate toxicant is the original chemical to which the organism is exposed (parent compound). In other cases, the ultimate toxicant is a metabolite of the parent compound or a reactive oxygen or nitrogen species (ROS or RNS) generated during the biotransformation of the toxicant. Occasionally, the ultimate toxicant is an unchanged or altered endogenous molecule (Table 3-1).

The concentration of the ultimate toxicant at the target molecule depends on the relative effectiveness of the processes that increase or decrease its concentration at the target site (Fig. 3-2). The accumulation of the ultimate toxicant at its target is facilitated by its absorption, distribution to the site of action, reabsorption, and toxication (metabolic activation). Conversely, presystemic elimination, distribution away from the site of action, excretion, and detoxication oppose these processes and work against the accumulation of the ultimate toxicant at the target molecule.

Absorption versus Presystemic Elimination

Absorption

Absorption is the transfer of a chemical from the site of exposure, usually an external or internal body surface (eg, skin, mucosa of the alimentary and respiratory tracts), into the systemic circulation. Whereas transporters may contribute to the gastrointestinal (GI) absorption of some chemicals (eg, salicylate and valproate by monocarboxylate transporters, some β-lactam antibiotics and ACE inhibitor drugs by peptide transporters (PEPT), Fe²⁺, Cd²⁺, as well as some other divalent metal ions by the divalent metal ion transporter, and arsenate by phosphate transporters), the vast majority of toxicants traverse epithelial barriers and reach the blood capillaries by diffusing through cells. The rate of absorption is related to the concentration of the chemical at the absorbing surface, which depends on the rate of exposure and the dissolution of the chemical. It is also related to the area of the exposed site, the characteristics of the epithelial layer through which absorption takes place (eg, the thickness of the stratum corneum in the skin), the intensity of the subepithelial microcirculation, and the physicochemical properties of the toxicant. Lipid solubility is usually the most important property influencing absorption. In general, lipid-soluble chemicals are absorbed more readily than are water-soluble substances.

Presystemic Elimination

During transfer from the site of exposure to the systemic circulation, toxicants may be eliminated. This is not unusual for chemicals absorbed from the GI tract because they must first pass through the GI mucosal cells, liver, and lung before being distributed to the rest of the body by the systemic circulation. The GI mucosa and the liver may eliminate a significant fraction of a toxicant during its passage through these tissues, decreasing its systemic availability. For example, ethanol is oxidized by alcohol dehydrogenase in the gastric mucosa (Lim et al., 1993), cyclosporine is returned from the enterocyte into the intestinal lumen by multidrug resistance protein (MDR1, also known as P-glycoprotein, an ATP-dependent xenobiotic transporter) and is also hydroxylated by cytochrome P450 (CYP3A4) in these cells (Lin et al., 1999), morphine is glucuronidated in intestinal mucosa and liver, and manganese is taken up from the portal blood into liver and excreted into bile. Such processes may prevent a considerable quantity of chemicals from reaching the systemic blood. Thus, presystemic or first-pass elimination reduces the toxic effects of chemicals that reach their target sites by way of the systemic circulation. In contrast, the processes involved in presystemic elimination may contribute to injury of the digestive mucosa, liver, and lungs by chemicals such as ethanol, iron salts, α-amanitin, and paraquat because these processes promote their delivery to those sites.

Distribution To and Away from the Target

Toxicants exit the blood during the distribution phase, enter the extracellular space, and may penetrate into cells. Chemicals dissolved in plasma water may diffuse through the capillary endothelium via aqueous intercellular spaces and transcellular pores called fenestrae and/or across the cell membrane. Lipid-soluble compounds move readily into cells by diffusion. In contrast, highly ionized and hydrophilic xenobiotics (eg, tubocurarine and aminoglycosides) are largely restricted to the extracellular space unless specialized membrane carrier systems are available to transport them.

During distribution, toxicants reach their site or sites of action, usually a macromolecule on either the surface or the interior of a particular type of cell. Chemicals also may be distributed to the site or sites of toxication, usually an intracellular enzyme, where the ultimate toxicant is formed. Some mechanisms facilitate, whereas others delay, the distribution of toxicants to their targets.

Mechanisms Facilitating Distribution to a Target

Distribution of toxicants to specific target sites may be enhanced by (1) the porosity of the capillary endothelium, (2) specialized membrane transport, (3) accumulation in cell organelles, and (4) reversible intracellular binding.

Porosity of the Capillary Endothelium

Endothelial cells in the hepatic sinusoids and in the renal peritubular capillaries have larger fenestrae (50–150 nm in diameter) that permit passage of even protein-bound xenobiotics. This favors the accumulation of chemicals in the liver and kidneys.

Specialized Transport Across the Plasma Membrane

Specialized ion channels and membrane transporters can contribute to the delivery of toxicants to intracellular targets. For example, aquaglyceroporin channels may mediate influx of arsenite, which is present at physiologic pH as uncharged As(OH)₃, voltage-gated Ca²⁺ channels permit the entry of cations such as lead or barium ions into excitable cells, and Na⁺,K⁺-ATPase promotes intracellular accumulation of thallous ion. By mimicking Na⁺, Li⁺ may enter excitable cells through voltage-gated Na⁺ channels and, from the tubular fluid, into the principal cells of the renal collecting duct via the epithelial Na⁺ channels. Paraquat enters into pneumocytes via hitherto unspecified transporters and an MPTP metabolite (MPP⁺) is taken up into extrapyramidal dopaminergic neurons by the dopamine transporter. Organic cation transporters (OCT) mediate the uptake of small-molecular-weight organic cations (eg, metformin, cimetidine, and cisplatin) from blood into liver cells (by OCT1) and into renal proximal tubular cells (by OCT2). The hepatocellular uptake of α-amanitin is mediated by the Na-dependent bile acid transporter (NTCP). Specific organic anion-transporting polypeptide (OATP) transporters mediate the hepatic uptake and hepatotoxicity of microcystin, a cyclic heptapeptide toxin produced by blue-green algae. In mice, Oatp1b2 (the rodent ortholog of OATP1B1/1B3) is essential, as Oatp1b2-null mice are resistant to microcystin-induced liver injury (Lu et al., 2008). Organic anion transporters such as human OAT1 and OAT3 mediate renal tubular uptake of ochratoxin and mercuric ion (the latter as the dicysteine conjugate Cys-Hg-Cys), whereas both OAT1 and amino acid transporters can carry methylmercury as its cysteine conjugate CH₃-Hg-Cys, and an MPTP metabolite (MPP⁺) enters into extrapyramidal dopaminergic neurons by means of the dopamine transporter. Endocytosis of some toxicant–protein complexes, such as Cd-metallothionein (MT) or hydrocarbons bound to the male rat–specific α_2u-globulin, by renal proximal tubular cells can also occur. Particles, such as asbestos and manufactured nanomaterial, may also enter cells by endocytosis, depending on their size and shape. Lipoprotein receptor–mediated endocytosis contributes to entry of lipoprotein-bound toxicants into cells equipped with such transporters. Membrane recycling can internalize cationic aminoglycosides associated with anionic phospholipids in the brush border membrane of renal tubular cells (Laurent et al., 1990). This process may also contribute to cellular uptake of heavy metal ions. Such uptake mechanisms facilitate the entry of toxicants into specific cells, rendering those cells targets. Thus, carrier-mediated uptake of paraquat by pneumocytes and internalization of aminoglycosides by renal proximal tubular cells expose those cells to toxic concentrations of those chemicals.

Accumulation in Cell Organelles

Amphipathic xenobiotics with a protonable amine group and lipophilic character accumulate in lysosomes as well as mitochondria and cause adverse effects there. Lysosomal accumulation occurs by pH trapping, that is, diffusion of the amine (eg, amiodarone, amitriptyline, fluoxetine) in unprotonated form into the acidic interior of the organelle, where the amine is protonated, preventing its efflux. The entrapped amine inhibits lysosomal phospholipases, impairing degradation of lysosomal phospholipids and causing phospholipidosis. Mitochondrial accumulation takes place electrophoretically. The amine is protonated in the intermembrane space (to where the mitochondria eject protons). The cation thus formed will then be sucked into the matrix space by the strong negative potential there (−220 mV), where it may impair β-oxidation and oxidative phosphorylation. By such mechanisms, the valued antiarrhytmic drug amiodarone is entrapped in the hepatic lysosomes and mitochondria, causing phospholipidosis (Kodavanti and Mehendale, 1990) and microvesicular steatosis with other liver lesions (Fromenty and Pessayre, 1997), respectively. The cationic metabolite of MPTP (MPP⁺) also electrophoretically accumulates in the mitochondria of dopaminergic neurons, causing mitochondrial dysfunction and cell death, whereas highly lipophilic local anesthetics (eg, tetracaine, bupivacaine), when overdosed or inadvertently injected into a blood vessel, accumulate in cardiac mitochondria, compromising mitochondrial energy production and causing cardiac failure. Human equilibrative nucleoside transporter 1 (ENT1) in the mitochondrial inner membrane appears responsible for targeting fialuridine (an already withdrawn thymidine nucleoside analogue antiviral drug) into human mitochondria, where it inhibits mitochondrial DNA synthesis, thereby inducing hepatotoxicity. The fact that ENT1 is not localized in rodent mitochondria may account for the dramatic difference in mitochondrial toxicity of fialuridine between humans and rodents (Lee et al., 2006).

Reversible Intracellular Binding

Binding to the pigment melanin, an intracellular polyanionic aromatic polymer, is a mechanism by which chemicals, such as organic and inorganic cations and polycyclic aromatic hydrocarbons, can accumulate in melanin-containing cells in retina, substantia nigra, and skin (Larsson, 1993). The release of melanin-bound toxicants is thought to contribute to the retinal toxicity associated with chlorpromazine and chloroquine, injury to substantia nigra neurons by MPTP and manganese, and the induction of melanoma by polycyclic aromatics. Keratins are the major structural proteins in the epidermis and its appendages (nail and hair), constituting up to 85% of fully differentiated keratinocytes (skin cells). As keratins are abundant in cysteine residues, they can sequester thiol-reactive metal ions and metalloid compounds, whose nail and hair contents are indicative of exposure. Release of keratin-bound arsenic in keratinocytes may adversely affect these cells, leading to dermal lesions common in arsenicism.

Mechanisms Opposing Distribution to a Target

Distribution of toxicants to specific sites may be hindered by several processes. The processes include (1) binding to plasma proteins, (2) specialized barriers, (3) distribution to storage sites such as adipose tissue, (4) association with intracellular binding proteins, and (5) export from cells.

Binding to Plasma Proteins

As long as xenobiotics such as DDT (an insecticide) and TCDD (often called dioxin, an environmental pollutant) are bound to high-molecular-weight proteins or lipoproteins in plasma, they cannot leave the capillaries by diffusion. Even if they exit the bloodstream through fenestrae, they have difficulty permeating cell membranes. Dissociation from proteins is required for most xenobiotics to leave the blood and enter cells. Therefore, strong binding to plasma proteins delays and prolongs the effects and elimination of toxicants.

Specialized Barriers

Brain capillaries have very low aqueous porosity because their endothelial cells lack fenestrae and are joined by extremely tight junctions. This blood–brain barrier prevents the access of hydrophilic chemicals to the brain except for those that can be actively transported. In the choroid plexus, where the capillaries are fenestrated, the choroidal epithelial cells are sealed together by tight junctions, forming the blood–cerebrospinal fluid barrier. Water-soluble toxicants also have restricted access to reproductive cells, which are separated from capillaries by other cells. The oocyte in the ovary is surrounded by multiple layers of granulosa cells, and the spermatogenic cells are supported by Sertoli cells that are tightly joined in the seminiferous tubules to form the blood–testis barrier (see Chap. 20). Transfer of hydrophilic toxicants across the placenta is also restricted. However, none of these barriers are effective against lipophilic substances.

Distribution to Storage Sites

Some chemicals accumulate in tissues (ie, storage sites) where they do not exert significant effects. For example, highly lipophilic substances such as chlorinated hydrocarbon insecticides concentrate in adipocytes, whereas lead is deposited in bone by substituting for Ca²⁺ in hydroxyapatite. Such storage decreases the availability of these toxicants for their target sites and acts as a temporary protective mechanism. However, insecticides may return to the circulation and be distributed to their target site, the nervous tissue, when there is a rapid lipid loss as a result of fasting. This contributes to the lethality of pesticide-exposed birds during migration or during the winter months, when food is restricted. The possibility that lead is mobilized from the bone during pregnancy is of concern.

Association with Intracellular Binding Proteins

Binding to nontarget intracellular sites also reduces the concentration of toxicants at the target site, at least temporarily. MT, a cysteine-rich cytoplasmic protein, serves such a function in acute cadmium intoxication (Klaassen et al., 1999).

Export from Cells

Intracellular toxicants may be transported back into the extracellular space. This occurs in brain capillary endothelial cells. In their luminal membrane, these cells contain ATP-dependent membrane transporters (ATP-binding cassette [ABC] transporters) such as the MDR1, or P-glycoprotein, which extrudes chemicals and contributes to the blood–brain barrier (Schinkel, 1999). Compared with normal mice, mice with disrupted mdr1a gene exhibit 100-fold higher brain levels of and sensitivity to ivermectin, a neurotoxic pesticide and human antihelmintic drug that is one of many P-glycoprotein substrates (Schinkel, 1999). The ooctye is also equipped with P-glycoprotein that provides protection against chemicals that are substrates for this efflux pump (Elbling et al., 1993). Hematopoietic stem cells (and perhaps other stem cells) are also protected by ABC transporters, such as MDR1, MRP1, and breast cancer resistance protein (BCRP), of which the latter confers these cells resistance to mitoxantrone. ABC transporters that export drugs were first identified in tumor cells that often overexpress them, thereby making these cells resistant to antitumor drugs these transporters pump out (see Chap. 5).

Excretion versus Reabsorption

Excretion

Excretion is the removal of xenobiotics from the blood and their return to the external environment. It is a physical mechanism, whereas biotransformation is a chemical mechanism for eliminating the toxicant.

For nonvolatile chemicals, the major excretory structures in the body are the renal glomeruli, which hydrostatically filter small molecules (<60 kDa) through their pores, and the proximal renal tubular cells and hepatocytes, which actively transport chemicals from the blood into the renal tubules and bile canaliculi, respectively. These cells are readily exposed to blood-borne chemicals through the large endothelial fenestrae; they have transporters of the solute carrier (SLC) family (eg, OAT, OCT, and OATP type) that mediate the basolateral uptake of particular chemicals and transporters of the ABC carrier family (eg, MRP and MDR type) that mediate the luminal export of certain chemicals (see Chap. 5). Renal transporters have a preferential affinity for smaller (<300 Da), and hepatic transporters for larger (>400 Da) amphiphilic molecules. A less common “excretory” mechanism consists of diffusion and partition into the excreta on the basis of their lipid content (see below) or acidity. For example, morphine is transferred into milk and amphetamine is transferred into gastric juice by nonionic diffusion. This is facilitated by pH trapping of those organic bases in those fluids, which are acidic relative to plasma (see Chap. 5).

The route and speed of excretion depend largely on the physicochemical properties of the toxicant. The major excretory organs—kidney and liver—can efficiently remove only highly hydrophilic, usually ionized chemicals such as organic acids and bases. The reasons for this are as follows: (1) in the renal glomeruli, only compounds dissolved in plasma water can be filtered; (2) transporters in hepatocytes and renal proximal tubular cells are specialized for secretion of highly hydrophilic organic acids and bases; (3) only hydrophilic chemicals are freely soluble in the aqueous urine and bile; and (4) lipid-soluble compounds are readily reabsorbed by transcellular diffusion.

There are no efficient elimination mechanisms for nonvolatile, highly lipophilic chemicals such as polyhalogenated biphenyls and chlorinated hydrocarbon insecticides. If they are resistant to biotransformation, such chemicals are eliminated very slowly and tend to accumulate in the body on repeated exposure. Three rather inefficient processes are available for the elimination of such chemicals: (1) excretion by the mammary gland after the chemical is dissolved in milk lipids; (2) excretion in bile in association with biliary micelles and/or phospholipid vesicles; and (3) intestinal excretion, an incompletely understood transport from blood into the intestinal lumen. Volatile, nonreactive toxicants such as gases and volatile liquids diffuse from pulmonary capillaries into the alveoli and are exhaled.

Reabsorption

Toxicants delivered into the renal tubules may diffuse back across the tubular cells into the peritubular capillaries. This process is facilitated by tubular fluid reabsorption, which increases the intratubular concentration as well as the residence time of the chemical by slowing urine flow. Reabsorption by diffusion is dependent on the lipid solubility of the chemical. For organic acids and bases, diffusion is inversely related to the extent of ionization, because the nonionized molecule is more lipid-soluble. The ionization of weak organic acids, such as salicylic acid and phenobarbital, and bases, such as amphetamine, procainamide, and quinidine, is strongly pH-dependent in the physiologic range. Therefore, their reabsorption is influenced significantly by the pH of the tubular fluid. Acidification of urine favors the excretion of weak organic bases, whereas alkalinization favors the elimination of weak organic acids. Some organic compounds may be reabsorbed from the renal tubules by transporters. For example, PEPT can move peptidomimetic drugs, such as some β-lactam antibiotics and angiotensin-converting enzyme inhibitors, across the brush border membrane. Carriers for the physiologic oxyanions mediate the reabsorption of some toxic metal oxyanions in the kidney. Thus, chromate and molybdate are reabsorbed by the sulfate transporter, whereas arsenate is reabsorbed by the phosphate transporter.

Toxicants delivered to the GI tract by biliary, gastric, and intestinal excretion and secretion by salivary glands and the exocrine pancreas may be reabsorbed by diffusion across the intestinal mucosa. Because compounds secreted into bile are usually organic acids, their reabsorption is possible only if they are sufficiently lipophilic or are converted to more lipid-soluble forms in the intestinal lumen. For example, glucuronides of toxicants such as diethylstilbestrol (DES) and glucuronides of the hydroxylated metabolites of polycyclic aromatic hydrocarbons, chlordecone, and halogenated biphenyls are hydrolyzed by the β-glucuronidase of intestinal microorganisms, and the released aglycones are reabsorbed (Gregus and Klaassen, 1986). Glutathione conjugates of hexachlorobutadiene and trichloroethylene are hydrolyzed by intestinal and pancreatic peptidases, yielding the cysteine conjugates, which are reabsorbed and serve as precursors of some nephrotoxic metabolites (Anders, 2004).

Toxication versus Detoxication

Toxication

A number of xenobiotics (eg, strong acids and bases, nicotine, aminoglycosides, ethylene oxide, methylisocyanate, heavy metal ions, HCN, CO) are directly toxic, whereas the toxicity of others is due primarily to metabolites. Biotransformation to harmful products is called toxication or metabolic activation. With some xenobiotics, toxication confers physicochemical properties that adversely alter the microenvironment of biological processes or structures. For example, oxalic acid formed from ethylene glycol may cause acidosis and hypocalcaemia as well as obstruction of renal tubules by precipitation as calcium oxalate. Occasionally, chemicals acquire structural features and reactivity by biotransformation that allows for a more efficient interaction with specific receptors or enzymes. For example, the organophosphate insecticide parathion is biotransformed to paraoxon, an active cholinestrase inhibitor; the rodenticide fluoroacetate is converted in the citric acid cycle to fluorocitrate, a false substrate that inhibits aconitase; as a result of its CYP2E1-catalyzed oxidation, the general anesthetic methoxyflurane releases fluoride ion that inhibits several enzymes (including enolase in the glycolytic pathway) and that contributes to renal injury after prolonged anesthesia; some cephalosporin antibiotics (eg, cephoperazone) may cause hemorrhage because they undergo biotransformation with release of 1-methyltetrazole-5-thiol that inhibits vitamin K epoxide reductase and thus impairs activation of clotting factors; and fialuridine, an antiviral drug withdrawn from development because it produced lethal hepatotoxicity in patients involved in the clinical trial, is phosphorylated to the triphosphate, which inhibits DNA polymerase-γ and thus impairs synthesis of mitochondrial DNA (Lewis et al., 1996). Most often, however, toxication renders xenobiotics and occasionally other molecules in the body, such as oxygen and nitric oxide (^•NO), indiscriminately reactive toward endogenous molecules with susceptible functional groups. This increased reactivity may be due to conversion into (1) electrophiles, (2) free radicals, (3) nucleophiles, or (4) redox-active reactants.

Formation of Electrophiles

Electrophiles are molecules containing an electron-deficient atom with a partial or full positive charge that allows it to react by sharing electron pairs with electron-rich atoms in nucleophiles. The formation of electrophiles is involved in the toxication of numerous chemicals (Table 3-2) (see Chap. 6). Such reactants are often produced by insertion of an oxygen atom, which withdraws electrons from the atom it is attached to, making that electrophilic. This is the case when aldehydes, ketones, epoxides, arene oxides, sulfoxides, nitroso compounds, phosphonates, and acyl halides are formed (Table 3-2). In other instances, conjugated double bonds are formed, which become polarized by the electron-withdrawing effect of an oxygen, making one of the double-bonded carbons electron-deficient (ie, electrophilic). This occurs when α,β-unsaturated aldehydes and ketones as well as quinones, quinoneimines, and quinone methides are produced (Table 3-2). Formation of many of these electrophilic metabolites is catalyzed by cytochrome P450s (Leung et al., 2012).

Cationic electrophiles may be produced as a result of heterolytic bond cleavage. For example, methyl-substituted aromatics, such as 7,12-dimethylbenzanthracene, and aromatic amines (amides), such as 2-acetylaminofluorene, are hydroxylated to form benzylic alcohols and N-hydroxy arylamines (amides), respectively (Miller and Surh, 1994). These metabolites are then esterified, typically by sulfotransferases. Heterolytic cleavage of the C–O or N–O bonds of these esters results in a hydrosulfate anion and the concomitant formation of a benzylic carbonium ion or arylnitrenium ion, respectively (see Chap. 6). The antiestrogen tamoxifen undergoes similar activation by hydroxylation and sulfation to form a carbocationic metabolite (Kim et al., 2004). After being C-hydroxylated by CYP2E1, the hepatocarcinogen dimethylnitrosamine undergoes spontaneous decomposition, finally a heterolytic cleavage of the C–N bond to form methyl carbonium cation (⁺CH₃) (see Chap. 6). In a similar process, the tobacco-specific nicotine-derived nitrosamine ketone (NNK), the most potent carcinogen in tobacco, may generate either methyl carbonium cation or piridyloxobutyl carbonium cation, depending on the site of C-hydroxylation that initiates its decomposition. Formally, spontaneous heterolytic cleavage of the C–Cl bond results in formation of reactive episulfonium ion from the vesicant chemical warfare agent sulfur mustard (2,2′-bis-chloroethylsulfide). An episulfonium ion metabolite is also the ultimate toxicant produced from the fumigant 1,2-dibromoethane following its conjugation with glutathione to form S-(2-bromoethyl)glutathione conjugate, a so-called half-sulfur mustard (Anders, 2004). The oxidation of mercury (Hg⁰) to Hg²⁺ by catalase and reduction of Display Formula$CrO42−$ into DNA-reactive Cr³⁺ by ascorbate (Quievryn et al., 2002) are examples for formation of electrophilic toxicants from inorganic chemicals. Reduction of arsenate (containing As^V) to arsenite (containing As^III) by glutathione is greatly accelerated via incorporation of arsenate into arsenate esters or anhydrides. These arsenylated metabolites (eg, ribose-1-arsenate, glucose- 1-arsenate, ADP-arsenate) can be formed by phosphorolytic enzymes (eg, purine nucleoside phosphorylase and glycogen phosphorylase) and by ATP synthase, which accept arsenate instead of phosphate as their substrate. This is how such enzymes promote reduction of arsenate into the more toxic thiol-reactive arsenite (Gregus et al., 2009; Németi et al., 2010).

Formation of Free Radicals

A free radical is a molecule or molecular fragment that contains one or more unpaired electrons in its outer orbital. Radicals are formed by (1) accepting an electron, (2) losing an electron, or (3) homolytic fission of a covalent bond.

1. Xenobiotics such as paraquat, doxorubicin, and nitrofurantoin can accept an electron from reductases to give rise to radicals (Fig. 3-3). These radicals typically transfer the extra electron to molecular oxygen, forming a superoxide anion radical (Display Formula$O2•¯$) and regenerating the parent xenobiotic, which is ready to gain a new electron (Kappus, 1986). Through this “redox cycling,” one electron acceptor xenobiotic molecule can generate many Display Formula$O2•¯$ molecules. There are also endogenous sources of Display Formula$O2•¯$. This radical is generated in large quantities by NADPH oxidase (Nox) in activated macrophages and granulocytes during “respiratory burst,” and in smaller quantities by many other cells, such as endothelial cells and vascular smooth muscle cells, which also express Nox in the plasma membrane (Bokoch and Knaus, 2003). Growth factor receptor stimulation in these nonphagocytotic cells is coupled to Nox activation. Display Formula$O2•¯$ is also produced by the mitochondrial electron transport chain, especially by complexes I and III. The significance of Display Formula$O2•¯$ stems to a large extent from the fact that Display Formula$O2•¯$ is a starting compound in two toxication pathways (Fig. 3-4); one leads to formation of hydrogen peroxide (HOOH) and then hydroxyl radical (HO•), whereas the other produces peroxynitrite (ONOO⁻) and ultimately nitrogen dioxide (^•NO₂), and carbonate anion radical Display Formula$(CO2•¯)$.

2. Nucleophilic xenobiotics such as phenols, hydroquinones, aminophenols, aromatic amines (eg, benzidine), hydrazines, phenothiazines (eg, chlorpromazine; see Fig. 3-6), and thiols are prone to lose an electron and form free radicals in a reaction catalyzed by peroxidases (Aust et al., 1993). Some of these chemicals, such as catechols and hydroquinones, may undergo two sequential one-electron oxidations, producing first semiquinone radicals and then quinones. Quinones are not only reactive electrophiles (Table 3-2) but also electron acceptors with the capacity to initiate redox cycling or oxidation of thiols and NAD(P)H. Polycyclic aromatic hydrocarbons with sufficiently low ionization potential, such as benzo[a]pyrene and 7,12-dimethylbenzanthracene, can be converted via one-electron oxidation by peroxidases or cytochrome P450 to radical cations, which may be the ultimate toxicants for these carcinogens (Cavalieri and Rogan, 1992). Like peroxidases, oxyhemoglobin (Hb-FeII-O₂) can catalyze the oxidation of aminophenols to semiquinone radicals and quinoneimines. This is another example of toxication, because these products, in turn, oxidize ferrohemoglobin (Hb-FeII) to methemoglobin (Hb-FeIII), which cannot carry oxygen.

3. Free radicals are also formed by homolytic bond fission, which can be induced by electron transfer to the molecule (reductive fission). This mechanism is involved in the conversion of CCl₄ to the trichloromethyl free radical (Cl₃C^•) by an electron transfer from cytochrome P450 or the mitochondrial electron transport chain (reductive dehalogenation) (Recknagel et al., 1989). The Cl₃C^• reacts with O₂ to form the even more reactive trichloromethylperoxy radical (Cl₃COO^•) (Hippeli and Elstner, 1999).

The hydroxyl radical (HO^•), a free radical of paramount toxicological significance, is also generated by homolytic fission. Such a process yields large amounts of HO^• from water on ionizing radiation. Reductive homolytic fission of HOOH to the harmful HO^• and the harmless HO⁻ (hydroxide anion) is called the Fenton reaction (Fig. 3-4). This is catalyzed by transition metal ions, typically Fe(II), Cu(I), Cr(V), Ni(II), or Mn(II), and is a major toxication mechanism for HOOH and its precursor Display Formula$O2•¯$, as well as for transition metals. Moreover, the toxicity of chemicals, such as nitrilotriacetic acid, bleomycin, and orellanin (Hippeli and Elstner, 1999), which chelate transition metal ions is also based on Fenton chemistry, because chelation increases the catalytic efficiency of some transition metal ions. Complexation with some endogenous oligopeptides also increases the catalytic efficiency of weakly Fenton-active metal ions, such as Ni(II). The pulmonary toxicity of inhaled mineral particles such as asbestos and silica is caused, at least in part, by the formation of HO^• triggered by Fe ions on the particle surface (Vallyathan et al., 1998; Castranova, 2004). HOOH is a direct or indirect by-product of several enzymatic reactions, including monoamine oxidase, acyl-coenzyme A oxidase, xanthine oxidase (Harrison, 2002), aldehyde oxidase, and CYP2E1 (Caro and Cederbaum, 2004). It is produced in large quantities by spontaneous or superoxide dismutase (SOD)–catalyzed dismutation of Display Formula$O2•¯$.

Homolytic cleavage is also involved in free radical generation from ONOO⁻ (Squadrito and Pryor, 1998) (Fig. 3-4). The facile reaction of ONOO⁻ with the ubiquitous CO₂ yields nitrosoperoxycarbonate Display Formula$(ONOOCO2−)$, which can spontaneously homolyze into 2 radicals, the oxidant and nitrating agent nitrogen dioxide (^•NO₂) and the oxidant carbonate anion radical Display Formula$(CO2•¯)$. Thus, formation of ONOO⁻ and the latter radicals represents a toxication mechanism for Display Formula$O2•¯$ and ^•NO. As ^•NO is the product of nitric oxide synthase (NOS), this mechanism is especially relevant in and around cells that express NOS constitutively (ie, neurons and endothelial cells) as well as in and around cells that express the inducible form of NOS in response to cytokines. Inside the cell, a predominant site for formation of ONOO⁻ as well as its products Display Formula$CO3•¯$ and ^•NO₂ is the mitochondria, wherein the lipophilic ^•NO can readily diffuse and where the electron transport chain and the citric acid cycle produce Display Formula$O2•¯$ and CO₂, respectively (Denicola and Radi, 2005).

Formation of Nucleophiles

The formation of nucleophiles is a relatively uncommon mechanism for activating toxicants. Examples include the formation of cyanide from amygdalin, which is catalyzed by bacterial β-glucosidase in the intestine; from acrylonitrile after epoxidation and subsequent glutathione conjugation; and from sodium nitroprusside by thiol-induced decomposition. Carbon monoxide is a toxic metabolite of dihalomethanes that undergo oxidative dehalogenation. Hydrogen selenide, a strong nucleophile and reductant, is formed from selenite by reaction with glutathione or other thiols.

Formation of Redox-Active Reactants

There are specific mechanisms for the creation of redox-active reactants other than those already mentioned. Examples include the formation of the methemoglobin-producing nitrite from nitrate by bacterial reduction in the intestine or from esters of nitrous or nitric acids in reaction with glutathione. Dapsone hydroxylamine and 5-hydroxyprimaquine, hydroxylated metabolites of the respective drugs, produce methemoglobin by cooxidation (Fletcher et al., 1988). Reductants such as ascorbic acid and reductases such as NADPH-dependent flavoenzymes reduce Cr(VI) to Cr(V) (Shi and Dalal, 1990). Xenobiotic radicals formed in redox cycling (eg, those depicted in Fig. 3-3) as well as Display Formula$O2•¯$ can reduce Fe(III) bound to ferritin and consequently release it as Fe(II). Cr(V) and Fe(II) thus formed catalyze HO^• formation (Fig. 3-4).

In summary, the most reactive metabolites are electron-deficient molecules and molecular fragments such as electrophiles and neutral or cationic free radicals. Although some nucleophiles are reactive (eg, HCN, CO), many (eg, hydroquinones) are activated by conversion to electrophiles. Similarly, free radicals with an extra electron (eg, those shown in Fig. 3-3) cause damage by giving rise to the neutral HO^• radical after the formation and subsequent homolytic cleavage of HOOH.

Detoxication

Biotransformation that eliminates an ultimate toxicant or prevents its formation is called detoxication. In some cases, detoxication may compete with toxication for a chemical. Detoxication can take several pathways, depending on the chemical nature of the toxic substance.

Detoxication of Toxicants with No Functional Groups

In general, chemicals without functional groups, such as benzene and toluene, are detoxicated in 2 phases. Initially, a functional group such as hydroxyl or carboxyl is introduced into the molecule, most often by cytochrome P450 enzymes. Subsequently, an endogenous acid, such as glucuronic acid, sulfuric acid, or an amino acid, is added to the functional group by a transferase. With some exceptions, the final products are inactive, highly hydrophilic organic acids that are readily excreted. Carbonyl reduction, catalyzed by at least 5 enzymes (eg, 11-β-hydroxysteroid dehydrogenase-1, carbonyl reductase, and 3 members of aldo-keto reductase superfamily), initiates detoxication of the potent carcinogen NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The formed nicotine-derived nitrosamine alcohol is then readily glucuronidated at its hydroxyl moiety and is excreted into urine (Maser, 2004).

Detoxication of Nucleophiles

Nucleophiles are generally detoxicated by conjugation at the nucleophilic functional group. Hydroxylated compounds are conjugated by sulfation, glucuronidation, or rarely by methylation, whereas thiols are methylated or glucuronidated and amines and hydrazines are acetylated. These reactions prevent peroxidase-catalyzed conversion of the nucleophiles to free radicals and biotransformation of phenols, aminophenols, catechols, and hydroquinones to electrophilic quinones and quinoneimines. An alternative mechanism for the elimination of thiols, amines, and hydrazines is oxidation by flavin-containing monooxygenases (Krueger and Williams, 2005). Some alcohols, such as ethanol, are detoxicated by oxidation to carboxylic acids by alcohol and aldehyde dehydrogenases. A specific detoxication mechanism is the biotransformation of cyanide to thiocyanate by rhodanese or mercaptopyruvate sulfurtransferase.

Detoxication of Electrophiles

A general mechanism for the detoxication of electrophilic toxicants is conjugation with the thiol nucleophile glutathione (Ketterer, 1988). This reaction may occur spontaneously or can be facilitated by glutathione S-transferases. Metal ions—such as Ag⁺, Cd²⁺, Hg²⁺, and CH₃Hg⁺ ions—readily react with and are detoxicated by glutathione. Specific mechanisms for the detoxication of electrophilic chemicals include epoxide hydrolase-catalyzed biotransformation of epoxides and arene oxides to diols and dihydrodiols, respectively, and carboxylesterase-catalyzed hydrolysis of organophosphate ester pesticides. Others are two-electron reduction of quinones to hydroquinones by NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone oxidoreductase (NQO2), reduction of α,β-unsaturated aldehydes (eg, the lipid peroxidation product 4-oxonon-2-enal) to alcohols or to their saturated derivative by carbonyl reductase (Maser, 2006), or oxidation of α,β-unsaturated aldehydes to acids by aldehyde dehydrogenases. Complex formation of thiol-reactive metal ions by MT and the redox-active ferrous iron by ferritin are special types of detoxications. Covalent binding of electrophiles to proteins can also be regarded as detoxication provided that the protein has no critical function and does not become a neoantigen or otherwise harmful. Carboxylesterases, for example, inactivate organophosphates not only by hydrolysis but also by covalent binding.

Detoxication of Free Radicals

Because Display Formula$O2•¯$ can be converted into much more reactive compounds (Fig. 3-4), its elimination is an important detoxication mechanism. This is carried out by SOD, high-capacity enzymes located in the cytosol (Cu, Zn-SOD) and the mitochondria (Mn-SOD), which convert Display Formula$O2•¯$ to HOOH (Fig. 3-5). Subsequently, HOOH is reduced to water by catalase in the peroxisomes (also in the mitochondria in cardiac muscle), by the selenocysteine-containing glutathione peroxidases in the cytosol and mitochondria, and by peroxiredoxins in the cytosol, mitochondria, and endoplasmic reticulum (EPR) (Fig. 3-5) (Rhee et al., 2005).

No enzyme eliminates HO^•. Whereas some relatively stable radicals, such as peroxyl radicals (see Fig. 3-9), can readily abstract a hydrogen atom from glutathione, α-tocopherol (vitamin E), or ascorbic acid (vitamin C), thus becoming nonradicals, these antioxidants are generally ineffective in detoxifying HO^• (Sies, 1993). This is due to its extreme reactivity and thus short half-life (10⁻⁹ seconds), which provides little time for the HO^• to reach and react with antioxidants. Therefore, the only effective protection against HO^• is to prevent its formation by elimination of its precursor, HOOH, via conversion to water (Fig. 3-5).

ONOO⁻ (which is not a free radical oxidant) is significantly more stable than HO^• (half-life of about 1 second). Nevertheless, the small biological antioxidant molecules (glutathione, uric acid, ascorbic acid, α-tocopherol) are relatively inefficient in intercepting it, because ONOO⁻ rapidly reacts with CO₂ (Squadrito and Pryor, 1998) to form reactive free radicals (Fig. 3-4). More efficient are the selenocysteine-containing glutathione peroxidase and peroxiredoxins, which can reduce ONOO⁻ to nitrite (ONO⁻) the same way they reduce HOOH to water (Fig. 3-5). Selenoprotein P, which contains 10 selenocysteine residues and coats the surface of endothelial cells, also reduces ONOO⁻ and may serve as a protectant against this oxidant in blood. In addition, ONOO⁻ reacts with oxyhemoglobin, heme-containing peroxidases, and albumin, all of which could be important sinks for ONOO⁻. Furthermore, elimination of the 2 ONOO⁻ precursors—that is, ^•NO by reaction with oxyhemoglobin (to yield methemoglobin and nitrate) and Display Formula$O2•¯$ by SODs (see above)—is a significant mechanism in preventing ONOO⁻ buildup (Squadrito and Pryor, 1998).

Peroxidase-generated free radicals are eliminated by electron transfer from glutathione. This results in the oxidation of glutathione, which is reversed by NADPH-dependent glutathione reductase (Fig. 3-6). Thus, glutathione plays an important role in the detoxication of both electrophiles and free radicals.

Detoxication of Protein Toxins

Presumably, extracellular and intracellular proteases are involved in the inactivation of toxic polypeptides. Several toxins found in venoms, such as α- and β-bungarotoxin, erabutoxin, and phospholipase, contain intramolecular disulfide bonds that are required for their activity. These proteins are inactivated by thioredoxin, an endogenous dithiol protein that reduces the essential disulfide bond (Lozano et al., 1994).

When Detoxication Fails

Detoxication may be insufficient for several reasons:

1. Toxicants may overwhelm detoxication processes, leading to saturation of the detoxication enzymes, consumption of the cosubstrates, or depletion of cellular antioxidants such as glutathione, ascorbic acid, and α-tocopherol. This results in the accumulation of the ultimate toxicant.

2. Occasionally, a reactive toxicant inactivates a detoxicating enzyme. For example, ONOO⁻ incapacitates Mn-SOD, which normally would counteract ONOO⁻ formation (Murphy, 1999) (see Fig. 3-4).

3. Some conjugation reactions can be reversed. For example, 2-naphthylamine, a bladder carcinogen, is N-hydroxylated and glucuronidated in liver, with the glucuronide excreted into urine. While in the bladder, the glucuronide is hydrolyzed, and the released arylhydroxylamine is converted by protonation and dehydration to the reactive electrophilic arylnitrenium ion (Bock and Lilienblum, 1994). Isocyanates and isothiocyanates form labile glutathione conjugates from which they can be released. Thus, methylisocyanate readily forms a glutathione conjugate in the lung after inhalation. From there, the conjugate is distributed to other tissues, where the reactive electrophilic parent compound may be regenerated (Baillie and Kassahun, 1994). Such conjugates are considered transport forms of toxicants.

4. Sometimes detoxication generates potentially harmful by-products, such as the glutathione thiyl radical and glutathione disulfide, which are produced during the detoxication of free radicals (Fig. 3-6). Glutathione disulfide can form mixed disulfides with protein thiols, whereas the thiyl radical (GS^•), after reacting with thiolate (GS⁻), forms glutathione disulfide radical anion Display Formula$(GSSG2•¯)$, which can reduce O₂ to Display Formula$O2•¯$. Conjugation with glutathione may lead to HCN generation in the course of acrylonitrile biotransformation, the first step of which is epoxidation of acrylonitrile (H₂C=CH-CN). Whereas glutathione conjugation detoxifies this epoxide if it takes place at the carbon adjacent to the nitrile group (-CN), it causes release of this group as HCN if glutathione conjugates with the epoxide at the carbon not linked to the nitrile moiety.

Toxicity is typically mediated by a reaction of the ultimate toxicant with a target molecule (step 2a in Fig. 3-1). Subsequently, a series of secondary biochemical events occur, leading to dysfunction or injury that is manifest at various levels of biological organization, such as at the target molecule itself, cell organelles, cells, tissues and organs, and even the whole organism. Because interaction of the ultimate toxicant with the target molecule triggers the toxic effect, consideration is given to (1) the attributes of target molecules, (2) the types of reactions between ultimate toxicants and target molecules, and (3) the effects of toxicants on the target molecules (Fig. 3-7). Finally, consideration is given to toxicities that are initiated not by reaction of the ultimate toxicant with target molecules, but rather by alteration of the biological (micro)environment (step 2b in Fig. 3-1) in which critical endogenous molecules, cell organelles, cells, and organs function.

Attributes of Target Molecules

Practically all endogenous compounds are potential targets for toxicants. The identification and characteristics of the target molecules involved in toxicity constitute a major research priority, but a comprehensive inventory of potential target molecules is impossible. Nevertheless, the most prevalent and toxicologically relevant targets are macromolecules such as nucleic acids (especially DNA) and proteins. Among the small molecules, membrane lipids are frequently involved, whereas cofactors such as coenzyme A and pyridoxal rarely are involved.

To be a target, an endogenous molecule must possess the appropriate reactivity and/or steric configuration to allow the ultimate toxicant to enter into covalent or noncovalent reactions. For these reactions to occur, the target molecule must be accessible to a sufficiently high concentration of the ultimate toxicant. Thus, endogenous molecules that are exposed to reactive chemicals or are adjacent to sites where reactive metabolites are formed are frequently targets. Technical advances in the field of proteonomics make it increasingly possible to identify potential protein targets of reactive chemicals as chemical–protein adducts. A compendium of proteins adducted by reactive toxicant metabolites has been established at the University of Kansas (http://tpdb.medchem.ku.edu/tpdb.html). The first target for reactive metabolites is often the enzyme that catalyzes their production or the adjacent intracellular structures. For example, thyroperoxidase, the enzyme involved in thyroid hormone synthesis, converts some nucleophilic xenobiotics (such as methimazole, amitrole, and resorcinol) into reactive free radicals that inactivate thyroperoxidase (Engler et al., 1982). This is the basis for the antithyroid as well as the thyroid tumor–inducing effect of these chemicals. Carbon tetrachloride, which is activated by cytochrome P450, destroys this enzyme as well as the neighboring microsomal membranes (Osawa et al., 1995). Several mitochondrial enzymes are convenient targets for nephrotoxic cysteine conjugates such as dichlorovinyl cysteine, because these conjugates are converted to electrophiles by mitochondrial enzymes with cysteine conjugate β-lyase activity (eg, aspartate aminotransferase and branched chain amino acid aminotransferase) that can readily channel their reactive products to neighboring enzymes such as α-ketoglutarate dehydrogenase (Anders, 2004). Reactive metabolites that are unable to find appropriate endogenous reaction partners in close proximity to their site of formation may diffuse until they encounter such reactants. For example, hard electrophiles such as the arylnitrenium ion metabolite of N-methyl-4-aminoazobenzene react readily with hard nucleophilic atoms in nucleic acids, and thus target DNA in the nucleus, even though the electrophiles are produced in the cytoplasm. Vinyl chloride epoxide, formed in the hepatocytes from vinyl chloride, reaches its DNA targets in the neighboring endothelial cells (which are more sensitive to this genotoxin than the liver cells), initiating hepatic hemangiosarcoma.

Not all targets for chemicals contribute to the harmful effects. Thus, while carbon monoxide causes toxicity by binding to ferrohemoglobin, it also associates with the iron in cytochrome P450 with little or no consequence. Covalent binding of toxicants to various intracellular proteins, including enzymes and structural proteins, has been demonstrated, yet it is often uncertain which protein(s) is/are involved in binding that is toxicologically relevant (Cohen et al., 1997; Pumford and Halmes, 1997; Rombach and Hanzlik, 1999). Arylation of some hepatic mitochondrial proteins by acetaminophen (4′-hydroxyacetanilide) is thought to be causally related to the liver injury induced by this drug because the nonhepatotoxic regioisomer of acetaminophen (3′-hydroxyacetanilide) does not readily bind covalently to these proteins (Cohen et al., 1997; Jaeschke and Bajt, 2006). In contrast, arylation of a number of hepatic cytoplasmic proteins by acetaminophen is likely to be inconsequential because the nonhepatotoxic regioisomer of this drug also arylates those proteins (Nelson and Pearson, 1990). Covalent binding to proteins without adverse consequences may even represent a form of detoxication by sparing toxicologically relevant targets. This principle is best exemplified by covalent binding of organophosphate insecticides to plasma cholinesterase, which is a significant protective mechanism, as it counteracts phosphorylation of acetylcholinesterase, the target molecule. Thus, to conclusively identify a target molecule as being responsible for toxicity, it should be demonstrated that the ultimate toxicant (1) reacts with the target and adversely affects its function, (2) reaches an effective concentration at the target site, and (3) alters the target in a way that is mechanistically related to the observed toxicity.

Types of Reactions

The ultimate toxicant may bind to the target molecules noncovalently or covalently and may alter it by hydrogen abstraction, electron transfer, or enzymatically.

Noncovalent Binding

This type of binding can be due to apolar interactions or the formation of hydrogen and ionic bonds and is typically involved in the interaction of toxicants with targets such as membrane receptors, intracellular receptors, ion channels, and some enzymes. For example, such interactions are responsible for the binding of strychnine to the glycine receptor on motor neurons in the spinal cord, TCDD to the AhR, saxitoxin to sodium channels, phorbol esters to protein kinase C (PKC), and warfarin to vitamin K 2,3-epoxide reductase. Such forces also are responsible for the intercalation of chemicals such as acridine yellow and doxorubicin into the double helix of DNA. These chemicals are toxic because the steric arrangement of their atoms allows them to combine with complementary sites on the endogenous molecule more or less as a key fits into a lock. Noncovalent binding usually is reversible because of the comparatively low bonding energy.

Covalent Binding

Being practically irreversible, covalent binding is of great toxicological importance because it permanently alters endogenous molecules (Boelsterli, 1993). Covalent adduct formation is common with electrophilic toxicants such as nonionic and cationic electrophiles and radical cations. These toxicants react with nucleophilic atoms that are abundant in biological macromolecules, such as proteins and nucleic acids. Electrophilic atoms exhibit some selectivity toward nucleophilic atoms, depending on their charge-to-radius ratio. In general, soft electrophiles prefer to react with soft nucleophiles (low charge-to-radius ratio in both), whereas hard electrophiles react more readily with hard nucleophiles (high charge-to-radius ratio in both). Examples are presented in Table 3-3. Metal ions such as silver and mercury are also classified as soft electrophiles. These prefer to react covalently with soft nucleophiles (especially thiol groups). Conversely, hard electrophiles such as lithium, calcium, and barium react preferentially as cations with hard nucleophiles (eg, carboxylate and phosphate anions). Metals falling between these 2 extremes, such as chromium, zinc, and lead, exhibit universal reactivity with nucleophiles. The reactivity of an electrophile determines which endogenous nucleophiles can react with it and become a target.

A covalent reaction of special biological significance can take place between HOOH (a soft electrophile) and protein thiol groups, preferentially those with low pK_a value, that is, with a propensity of being in the strongly nucleophilic thiolate anion form (Prot-S⁻) at physiologic pH. This reaction produces protein sulfenic acid (Prot-S-OH):

Prot-SH + HOOH → Prot-S-OH + HOH.

The S atom in a sulfenic acid is electrophilic as it is made electron-deficient by the electron-withdrawing effect of O. Therefore, a protein–sulfenic acid can react with another thiol group of the same or a different protein or of glutathione (GSH), resulting in, respectively, formation of an intramolecular disulfide, intermolecular disulfide, or a mixed disulfide of the protein and glutathione (ie, glutathionylation of the protein):

Prot₁-S-OH + Prot₂-SH → Prot₁-S-S-Prot₂ + HOH;

Prot-S-OH + GSH → Prot-S-SG + HOH.

Not only HOOH but also hydroperoxides (eg, lipid hydroperoxides, LOOH; see Fig. 3-9) may enter into such reactions, producing protein–sulfenic acids, protein–disulfides, and glutathionylated proteins, which can be considered as posttranslational modifications with a role in redox signaling (Forman et al., 2004).

Neutral free radicals such as HO^•,•NO₂, and Cl₃C^• can also bind covalently to biomolecules. The addition of Cl₃C^• to double-bonded carbons in lipids or to lipid radicals yields lipids containing chloromethylated fatty acids. The addition of hydroxyl radicals to DNA bases results in the formation of numerous products, including 8-hydroxypurines, 5-hydroxymethylpyrimidines, and thymine and cytosine glycols (Breen and Murphy, 1995).

Nucleophilic toxicants are in principle reactive toward electrophilic endogenous compounds. Such reactions occur infrequently because electrophiles are rare among biomolecules. Examples include the covalent reactions of amines and hydrazides with the aldehyde pyridoxal, a cosubstrate for several enzymes, including glutamate decarboxylase. Carbon monoxide, cyanide, hydrogen sulfide, and azide form coordinate covalent bonds with iron in various heme proteins. Other nucleophiles react with hemoglobin in an electron transfer reaction (see below).

Hydrogen Abstraction

Neutral free radicals, such as those generated in reactions depicted in Fig. 3-4, can readily abstract H atoms from endogenous compounds, converting those compounds into radicals. Abstraction of hydrogen from thiols (R-SH) creates thiyl radicals (R-S^•), which on radical recombination with HO^• form sulfenic acids (R-S-OH) that are precursors of disulfides (R-S-S-R) (see above). Radicals can remove hydrogen from CH₂ groups of free amino acids or from amino acid residues in proteins and convert them to carbonyls. These carbonyls react covalently with amines, forming cross-links with DNA or other proteins. Hydrogen abstraction from deoxyribose in DNA yields the C-4′ radical, the first step to DNA cleavage (Breen and Murphy, 1995). Abstraction of hydrogen from fatty acids produces lipid radicals and initiates lipid peroxidation. As depicted in Fig. 3-8, nitration of tyrosine residues in proteins purportedly involves H abstraction followed by radical recombination, that is, covalent binding between the resultant tyrosyl radical and ^•NO₂ (Squadrito and Pryor, 1998).

Electron Transfer

Chemicals can oxidize Fe(II) in hemoglobin to Fe(III), producing methemoglobinemia. Nitrite can oxidize hemoglobin, whereas N-hydroxyl arylamines (such as dapsone hydroxylamine), phenolic compounds (such as 5-hydroxy primaquine), and hydrazines (such as phenylhydrazine) are cooxidized with oxyhemoglobin, forming methemoglobin and HOOH (Coleman and Jacobus, 1993).

Enzymatic Reactions

A few toxins act enzymatically on specific target proteins. For example, the plant toxins ricin and abrin are N-glycosidases; they hydrolyze a specific glycosidic bond in ribosomal RNA, blocking protein synthesis. Botulinum toxin acts as a Zn-protease; it hydrolyzes the fusion proteins that assist in exocytosis of the neurotransmitter acetylcholine in cholinergic neurons, most importantly motor neurons, causing paralysis. The lethal factor component of anthrax toxin is also a Zn-protease, which inactivates mitogen-activated protein kinase kinase (MAPKK), inducing cell death. Other bacterial toxins catalyze the transfer of ADP-ribose from NAD⁺ to specific proteins. Through such a mechanism, diphtheria toxin blocks the function of elongation factor 2 in protein synthesis and cholera toxin activates a G protein. Snake venoms contain hydrolytic enzymes that destroy biomolecules.

In summary, most ultimate toxicants act on endogenous molecules on the basis of their chemical reactivity. Those with more than one type of reactivity may react by different mechanisms with various target molecules. For example, quinones may act as electron acceptors and initiate thiol oxidation or free radical reactions that lead to lipid peroxidation, but they may also act as soft electrophiles and bind covalently to protein thiols. The lead ion acts as a soft electrophile when it forms coordinate covalent bonds with critical thiol groups in δ-aminolevulinic acid dehydratase, its major target enzyme in heme synthesis (Goering, 1993). However, it behaves like a hard electrophile or an ion when it binds to PKC or blocks calcium channels, substituting for the natural ligand Ca²⁺ at those target sites.

Effects of Toxicants on Target Molecules

Reaction of the ultimate toxicant with endogenous molecules may cause dysfunction or destruction; in the case of proteins, it may render them foreign (ie, an antigen) to the immune system.

Dysfunction of Target Molecules

Some toxicants activate protein target molecules, mimicking endogenous ligands. For example, morphine activates opioid receptors, clofibrate is an agonist on the peroxisome proliferator–activated receptor, and phorbol esters and lead ions stimulate PKC.

More commonly, chemicals inhibit the function of target molecules. Several xenobiotics—such as atropine, curare, and strychnine—block neurotransmitter receptors by attaching to the ligand-binding sites, whereas others interfere with the function of ion channels. Tetrodotoxin and saxitoxin, for example, inhibit opening of the voltage-activated sodium channels in the neuronal membrane, whereas DDT and the pyrethroid insecticides inhibit their closure. Some toxicants block ion transporters, others inhibit mitochondrial electron transport complexes, and many inhibit enzymes. Chemicals that bind to tubulin (eg, vinblastine, colchicine, paclitaxel, trivalent arsenic) or actin (eg, cytochalasin B, phalloidin) impair the assembly (polymerization) and/or disassembly (depolymerization) of these cytoskeletal proteins.

Protein function is impaired when conformation or structure is altered by interaction with the toxicant. Many proteins possess critical moieties, especially thiol groups, which are essential for catalytic activity or assembly to macromolecular complexes. Proteins that are sensitive to covalent and/or oxidative modification of their thiol groups include the enzymes glyceraldehyde 3-phosphate dehydrogenase (see Table 3-6) and pyruvate dehydrogenase (see Fig. 3-16), the Ca²⁺ pumps (see Fig. 3-17, Table 3-7), the DNA repair enzyme O⁶-methylguanine-DNA-methyltransferase, the DNA-methylating enzyme C⁵-cytosine-DNA-methyltransferase, ubiquitin-activating and -conjugating enzymes (E1 and E2), peroxiredoxins, protein tyrosine phosphatases (PTP, eg, cdc25), the lipid phosphatase PTEN (phosphatase and tensin homologue; see Fig. 3-12) (Rhee et al., 2005), caspases (see Fig. 3-19), the TF AP-1, and the electrophile sensor protein Keap1 (see Fig. 3-27), just to name a few. These and many other proteins are inactivated by thiol-reactive chemicals, causing impaired maintenance of the cell’s energy and metabolic homeostasis.

Binding of thiol-reactive chemicals to specific proteins may also initiate a signal. For example, thiol-reactive and oxidant chemicals, such as acrolein, methyl isocyanate, the lacrimator chloroacetophenone, and corrosive gases (eg, phosgene, chloropicrin, and chlorine), react with cysteine thiol groups of the TRPA1 receptor (a cation channel) in the membrane of sensory neurons. Excitation of these neurons located in the cornea and the mucous membranes of the eye and the respiratory tract elicits irritation, pain, lacrimation, bronchial secretion, sneezing, coughing, and bronchospasm (Bessac and Jordt, 2010). If moderate, these responses are alerting and protective, but are incapacitating and detrimental when exaggerated at high exposure. Keap1 may be regarded as an intracellular sensor of similar chemicals, as covalent and/or oxidative modification of thiol groups in Keap1 triggers the adaptive electrophile stress response, which is cytoprotective (see Fig. 3-27; Klaassen and Reisman, 2010). Conversion of the catalytic thiol group (-SH) in PTP and the lipid phosphatase PTEN to sulfenic acid group (-S-OH) on reaction with HOOH or lipid hydroperoxides (see reaction scheme above) inactivates these enzymes. PTPs keep protein kinase–mediated signal transduction pathways under control and PTEN controls the phosphatidilinositol-3-kinase (PI3K)–Akt pathway, both activated by growth factors (see Fig. 3-12). Therefore, inactivation of PTPs and PTEN permits these signalings to surge, thereby initiating, for example, a proliferative response. However, this concept may be an oversimplification because similar inactivation of cdc25 (another PTP) may compromise mitosis, as cdc25 normally dephosphorylates and activates cyclins 1 and 2, which are positive regulators of cell division (see Fig. 3-25). Formation of protein sulfenic acids by the oxidative stress product HOOH is thus considered as an initiating event of redox signaling (Forman et al., 2004). Redox signaling is controlled by the production of HOOH (eg, by Nox activated on growth factor receptor stimulation) and its enzymatic elimination. At low level, HOOH is removed effectively by peroxiredoxins (Fig. 3-5); however, these enzymes are incapacitated by overoxidation of their catalytic thiol groups at high HOOH levels (see Fig. 3-5). This modification can be reversed by sulfiredoxins (Lowther and Haynes, 2011). Furthermore, protein sulfenic acid formation is readily reversed by reaction of the sulfenic acid with a thiol (eg, glutathione), resulting in a protein disulfide (eg, protein-S-SG; see the scheme above), which in turn is reduced by thioredoxin or glutaredoxin as shown in Fig. 3-22, thereby regenerating PTPs and PTEN in their active thiol form. Therefore, peroxiredoxins, sulfiredoxins, thioredoxin, thioredoxin reductase, glutaredoxin, glutathione, and NADPH are all important negative regulators of oxidative signaling (Neumann et al., 2009). Protein tyrosine nitration (see Fig. 3-8) may also alter protein function or may interfere with signaling pathways that involve tyrosine kinases and phosphatases.

Toxicants may interfere with the template function of DNA. The covalent binding of chemicals to DNA causes nucleotide mispairing during replication. For example, covalent binding of aflatoxin 8,9-oxide to N-7 of guanine results in pairing of the adduct-bearing guanine with adenine rather than cytosine, leading to the formation of an incorrect codon and the insertion of an incorrect amino acid into the protein. Such events are involved in the aflatoxin-induced mutation of the Ras proto-oncogene and the p53 tumor suppressor gene (Eaton and Gallagher, 1994). 8-Hydroxyguanine (8-OH-Gua) and 8-hydroxyadenine are mutagenic bases produced by HO^• that can cause mispairing with themselves as well as with neighboring pyrimidines, producing multiple amino acid substitutions (Breen and Murphy, 1995). Cr(III) produced from chromate (Cr(VI)) via reduction by ascorbate, alone or complexed to ascorbate, forms a coordinate covalent bond with phosphate in DNA, thereby inducing a mutation (Quievryn et al., 2002). Chemicals such as doxorubicin, which intercalate between stacked bases in the double-helical DNA, push adjacent base pairs apart, causing an even greater error in the template function of DNA by shifting the reading frame.

Destruction of Target Molecules

In addition to adduct formation, toxicants alter the primary structure of endogenous molecules by means of cross-linking and fragmentation. Bifunctional electrophiles, such as 2,5-hexanedione, carbon disulfide, acrolein, 4-oxonon-2-enal, 4-hydroxynon-2-enal, and nitrogen mustard alkylating agents, cross-link cytoskeletal proteins, DNA, or DNA with proteins. HOOH and hydroxyl radicals can also induce cross-linking by converting proteins into either reactive electrophiles (eg, protein sulfenic acids and protein carbonyls, respectively), which react with a nucleophilic group (eg, thiol, amine) in another macromolecule. Radicals (eg, those shown in Fig. 3-4) may induce cross-linking of macromolecules by converting them into radicals, which react with each other by radical recombination. Cross-linking imposes both structural and functional constraints on the linked molecules.

Some target molecules are susceptible to spontaneous degradation after chemical attack. Free radicals such as Cl₃COO^• and HO^• can initiate peroxidative degradation of lipids by hydrogen abstraction from fatty acids (Recknagel et al., 1989). The lipid radical (L^•) formed is converted successively to lipid peroxyl radical (LOO^•) by oxygen fixation, lipid hydroperoxide (LOOH) by hydrogen abstraction, and lipid alkoxyl radical (LO^•) by the Fe²⁺-catalyzed Fenton reaction. Subsequent fragmentation gives rise to hydrocarbons such as ethane and reactive aldehydes such as 4-hydroxynon-2-enal and malondialdehyde (Fig. 3-9). Thus, lipid peroxidation not only destroys lipids in cellular membranes but also generates endogenous toxicants, both free radicals (eg, LOO^•, LO^•) and electrophiles (eg, 4-oxonon-2-enal, 4-hydroxynon-2-enal). These substances can readily react with adjacent molecules, such as membrane proteins, or diffuse to more distant molecules such as DNA. F₂-isoprostanes are stable peroxidation products of arachidonic acid; these are not only sensitive markers of lipid peroxidation but also potent pulmonary and renal vasoconstrictors (Basu, 2004).

Apart from hydrolytic degradation by toxins and radiolysis, toxicant-induced fragmentation of proteins is not well documented. There are, however, examples for destruction of the prosthetic group in enzymes. For instance, cytochrome P450 converts allyl isopropyl acetamide into a reactive metabolite, which alkylates the heme moiety of the enzyme. This leads to loss of the altered heme and to porphyria (De Matteis, 1987). Arsine (AsH₃) acutely induces heme release from oxyhemoglobin, which may underlie its hemolytic effect. Aconitase is attacked by ONOO⁻ at its [4Fe-4S]²⁺ cluster, in which one of the Fe atoms is genuinely labile (as is complexed to inorganic sulfur and not to enzyme-bound cysteines like the others). As a result of the oxidant action of ONOO⁻, the labile Fe is lost, inactivating the enzyme (Castro et al., 1994) and compromising the citric acid cycle where aconitase functions.

Several forms of DNA fragmentation are caused by toxicants. For instance, attack of DNA bases by HO^• can result in the formation of imidazole ring–opened purines or imidazole ring–contracted pyrimidines, which block DNA replication. Formation of a bulky adduct at guanine N-7 destabilizes the N-glycosylic bond, inducing depurination. Depurination results in apurinic sites that are mutagenic. Single-strand breaks (SSB) typically are caused by hydroxyl radicals via abstraction of H from desoxyribose in DNA yielding the C-4′ radical, followed by addition, Criegee rearrangement, and cleavage of the phosphodiester bond (Breen and Murphy, 1995). Multiple hydroxyl radical attacks on a short length of DNA, which occur after ionizing radiation, cause double-strand breaks (DSB) that are typically lethal to the affected cell.

Neoantigen Formation

Whereas the covalent binding of xenobiotics or their metabolites is often inconsequential with respect to the function of the immune system, in some individuals these altered proteins, which carry the xenobiotic adduct as a hapten, evoke an immune response. Some chemicals (eg, dinitrochlorobenzene, penicillin, and nickel ion) are sufficiently reactive to bind to proteins spontaneously. Others may obtain reactivity by auto-oxidation to quinones (eg, urushiols, the allergens in poison ivy) or by enzymatic biotransformation (Park et al., 2005). For example, CYP2E1 biotransforms halothane to an electrophile, trifluoroacetyl chloride, which binds as a hapten to microsomal and cell surface proteins in the liver, inducing a hepatitis-like immune reaction in sensitive patients.

Haptenized proteins released from cells may evoke antibody-mediated (humoral) and/or T-cell-mediated (cellular) immune response. In the humoral response, B cells play the leading role: they bind the complete antigen through their B-cell receptors and associate with T-helper cells (CD4⁺). Antigen binding (as signal #1) and cell surface costimulatory molecules on T-helper cells (as signal #2) induce differentiation of B cells into plasma B cells that manufacture and secrete antibody. By binding to the antigen, the antibody assists in destruction of the antigen by phagocytosis; however, harmful consequences may also result. For example, when penicillin-bound proteins as antigens react with IgE-type antibodies on the surface of mast cells, the reaction triggers release of mast cell mediators (eg, histamine, leukotrienes), which in turn may cause bronchoconstriction (asthma), vasodilatation, and plasma exudation (wheal, anaphylactic shock).

In cellular immune response, T cells are the main mediators. In the sensitization phase, T-helper cells become activated by two signals coming from antigen-presenting cells, such as macrophages in internal tissues and dendritic cells in tissues that are in contact with external environment (ie, skin and mucosal lining of the respiratory and GI tract). The APC phagocytoses the haptenized protein and migrates to lymph nodes where most T cells are located. Meanwhile, the APC processes the antigen into appropriate peptides, externalizes, and transfers the processed antigen to the major histocompatibility complex (MHC) on its surface. On reaching the lymph node, the APC presents the MHC-bound peptide to the T-cell receptor (TCR) of T-helper cells (signal #1). In addition, cell surface coactivator molecules (eg, CD80 and CD86) on APC bind to their counterparts (eg, CD28) on T cells (signal #2). Without signal #2 (which verifies that the antigen detected is foreign) the T cell becomes functionally inert (anergic). On receiving both signals, the helper T cell becomes specific to one particular antigen, expresses the T-cell growth factor IL-2 and its receptor, proliferates, differentiates, and is released into the circulation. On reexposure to the allergen, the elicitation phase commences. Antigen-specific T cells enter the exposure site (eg, the skin) and using IL-2 and other cytokines, they can activate cytotoxic T cells (CD8⁺) and induce expression of adhesion molecules at the site of allergen contact. Whereas this response is protective against virus-infected cells and tumor cells, it causes inflammation and cell injury in cells containing xenobiotic neoantigens. This model describes the response to contact allergens, such as nickel and urushiols, and is the basis of detecting contact allergens in mice with the local lymph node assay.

Besides the above-described classical mechanism of neoantigen-initiated APC-mediated T-cell activation, xenobiotics may cause T-cell activation by more direct ways. For example, Ni ions may not only form coordinate covalent bonds with proteins, making them neoantigens, but also apparently cross-link MHC of APC with the TCR of T cells, activating the latter. Moreover, some drugs (eg, sulfamethoxazole), which can be biotransformed into adduct forming metabolites (eg, nitroso-sulfamethoxazole) that triggers T-cell activation by the classical mechanism, purportedly may also stimulate the TCR of T cells directly, without involvement of APC.

Regarding contact allergens, a new concept has emerged: to elicit allergic contact dermatitis chemicals should not only initiate formation of antigen-specific T cells (as described above) but also evoke a less specific inflammatory reaction (referred to as innate immune signal). For example, nickel alone cannot give a positive response in the local lymph node assay in mice; however, it can when coadministered with lipopolysaccharide (LPS), a bacterial cell wall component. LPS acts through Toll-like receptor 4 (TLR4) on resident skin cells (macrophages, dendritic cells, fibroblasts) to trigger inflammatory gene expression via signaling pathways that involves NF-κB. However, nickel alone can evoke contact dermatitis in sensitive humans, apparently because it activates the human TLR4 that, unlike the mouse receptor, contains 2 nonconserved histidine residues (Schmidt and Goebeler, 2011). The need for an innate immune signal may account for the observation that contact allergy may be facilitated by an injury evoking inflammatory response, such as toxic cell injury by an irritant chemical or microbial infection.

Drug-induced lupus and possibly many cases of drug-induced agranulocytosis are thought to be mediated by immune reactions triggered by drug–protein adducts. The causative chemicals are typically nucleophiles, such as aromatic amines (eg, aminopyrine, clozapine, procainamide, and sulfonamides), hydrazines (eg, hydralazine and isoniazid), and thiols (eg, propylthiouracil, methimazole, and captopril). These substances can be oxidized by myeloperoxidase of granulocytes and monocytes (the precursors of APC) or by the ROS/RNS such cells produce (HO^•, ONOO⁻, hypochlorous acid [HOCl], see Fig. 3-26) to reactive metabolites that bind to the surface proteins of these cells, making them antigens (Uetrecht, 1992). The reactive metabolites may also activate monocytes and thus promote the immune reaction. Activation of CD4⁺ T cells by inhibition of promoter methylation of specific genes by procainamide and hydralazine as a possible lupus-inducing mechanism is discussed in the section “Dysregulation of Transcription.”

Toxicity Not Initiated by Reaction with Target Molecules

Some xenobiotics not only interact with a specific endogenous target molecule to induce toxicity but also, instead, alter the biological microenvironment (see step 2b in Fig. 3-1). Included here are (1) chemicals that alter H⁺ ion concentrations in the aqueous biophase, such as (a) acids and substances biotransformed to acids, such as methanol and ethylene glycol, (b) protonophoric uncouplers such as 2,4-dinitrophenol and pentachlorophenol, which dissociate their phenolic protons in the mitochondrial matrix, thus dissipating the proton gradient that drives ATP synthesis, and (c) cationic amphiphilic drugs (eg, chloroquine and amiodarone) that diffuse into lysosomes and accumulate there after being protonated at their amino groups; (2) solvents and detergents that physicochemically alter the lipid phase of cell membranes and destroy transmembrane solute gradients that are essential to cell functions; and (3) other xenobiotics that cause harm merely by occupying a site or space. For example, some chemicals (eg, the ethylene glycol-derived Ca-oxalate, methotrexate, and acyclovir) form water-insoluble precipitates in renal tubules. The poorly water-soluble complex produced in renal tubules from melamine and its derivative cyanuric acid caused the renal injury observed in cats and dogs fed adulterated pet food (Dobson et al., 2008) and is probably also responsible for a similar disorder that occurred in children fed melamine-contaminated infant formula. By occupying bilirubin binding sites on albumin, compounds such as the sulfonamides induce bilirubin toxicity (kernicterus) in neonates. Carbon dioxide displaces oxygen in the pulmonary alveolar space and causes asphyxiation.

The reaction of toxicants with a target molecule may result in impaired cellular function as the third step in the development of toxicity (Fig. 3-1). Each cell in a multicellular organism carries out defined programs. Certain programs determine the destiny of cells—that is, whether they undergo division, differentiation (ie, express proteins for specialized functions), or apoptosis. Other programs control the ongoing (momentary) activity of differentiated cells, determining whether they secrete more or less of a substance, whether they contract or relax, and whether they transport and metabolize nutrients at higher or lower rates. For regulation of these cellular programs, cells possess signaling networks (such as those shown in Figs. 3-12 and 3-15) that can be activated and inactivated by external signaling molecules. To execute the programs, cells are equipped with synthetic, metabolic, kinetic, transport, and energy-producing systems as well as structural elements, organized into macromolecular complexes, cell membranes, and organelles, by which they maintain their own integrity (internal functions) and support the maintenance of other cells (external functions).

As outlined in Fig. 3-10, the nature of the primary cellular dysfunction caused by toxicants, but not necessarily the ultimate outcome, depends on the role of the target molecule affected. If the target molecule is involved in cellular regulation (signaling), dysregulation of gene expression and/or dysregulation of momentary cellular function occur primarily. However, if the target molecule is involved predominantly in the cell’s internal maintenance, the resultant dysfunction can ultimately compromise the survival of the cell. The reaction of a toxicant with targets serving external functions can influence the operation of other cells and integrated organ systems. The following discussion deals with these consequences.

Toxicant-Induced Cellular Dysregulation

Cells are regulated by signaling molecules that activate specific cellular receptors linked to signal transducing networks that transmit the signals to the regulatory regions of genes and/or to functional proteins. Receptor activation may ultimately lead to (1) altered gene expression that increases or decreases the quantity of specific proteins and/or (2) a chemical modification of specific proteins, typically by phosphorylation, which activates or inhibits proteins. Programs controlling the destiny of cells primarily affect gene expression, whereas those regulating the ongoing activities primarily influence the activity of functional proteins; however, one signal often evokes both responses because of branching and interconnection of signaling networks.

Dysregulation of Gene Expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. The genetic information may be transcribed from DNA into messenger RNA (mRNA), which in turn is translated into a protein product. The nonprotein-coding genes may be transcribed into other RNAs, among them the recently discovered small silencing RNA or microRNA (miRNA), which can repress translation of mRNA into proteins, thereby regulating protein synthesis posttranscriptionally (Fig. 3-11). Chemicals may dysregulate the expression of genes encoding mRNA, miRNA, or both, in either way causing altered synthesis of specific proteins. Dysregulation of gene expression may occur at elements that are directly responsible for transcription, at components of the intracellular signal transduction pathway, and at the synthesis, storage, or release of the extracellular signaling molecules. Dysregulation of gene expression initiated by chemicals at these 3 levels is discussed below.

Dysregulation of Transcription

Transcription of genetic information from DNA to mRNA and miRNA is controlled largely by interplay between TFs and the regulatory or promoter region of genes coding for mRNA and miRNA (Fig. 3-11). By binding to distinctive nucleotide sequences in this region, ligand- and signal-activated TFs facilitate or impede the formation of the preinitiation complex, thereby promoting or repressing transcription of the adjacent gene. Xenobiotics may interact with the TFs (or other components of the preinitiation complex) and/or may alter the promoter region of the gene.

Dysregulation of Transcription by Chemicals Acting Through Ligand-Activated Transcription Factors

Several endogenous compounds, such as hormones (eg, steroids, thyroid hormones) and vitamins (retinoids and vitamin D), influence gene expression by binding to and activating TFs or intracellular receptors (Table 3-4). Xenobiotics may mimic the natural ligands. For example, fibric acid–type lipid-lowering drugs, phthalate esters, and compound Wy-14,643 substitute for polyunsaturated fatty acids as ligands for the peroxisome proliferator–activated receptor (PPARα) (Poellinger et al., 1992), and xenoestrogens (eg, DES, DDT, methoxychlor) substitute estradiol, an endogenous ligand for estrogen receptors (ER).

Acting through ligand-activated TFs, natural or xenobiotic ligands when administered at extreme doses or at critical periods during ontogenesis may cause toxicity by inappropriately influencing the fate of cells and inducing cell death or mitosis (Table 3-4). Glucocorticoids such as dexamethasone induce apoptosis of lymphoid cells. Whereas desirable in the treatment of lymphoid malignancies, this is an unwanted response in many other conditions. TCDD, a ligand of the AhR, produces thymic atrophy by causing apoptosis of thymocytes. Estrogens exert mitogenic effects in cells that express the ER, such as those found in female reproductive organs, mammary gland, and liver. ER-mediated proliferation appears to be responsible, at least in part, for formation of tumors in these organs during prolonged estrogen exposure (Green, 1992), as well as for induction of the rare vaginal adenocarcinoma in late puberty after transplacental exposure to the synthetic estrogen DES. Stimulation of these receptors during a critical prenatal period of differentiation reprograms the estrogen target tissue (ie, Mullerian duct), resulting in benign and malignant abnormalities in the reproductive tract later in life (Newbold, 2004). Mice exposed neonatally to DES served to model DES-induced late carcinogenesis in the reproductive tract. Compared with the wild-type animals, mice overexpressing estrogen receptor-α (ERα) were exceedingly sensitive to DES-induced uterine adenocarcinoma, whereas ERα-knockout mice were resistant, indicating the role of ERα in tumorigenesis. It has been speculated that environmental xenoestrogens such as DDT, polychlorinated biphenyls, bisphenol A, and atrazine contribute to an increased incidence of breast cancer. Zearalenone, a mycoestrogen feed contaminant, causes vulval prolapse in swine, an example of an ER-mediated proliferative lesion. The mitogenic and hepatic tumor-promoting effects of peroxisome proliferators in rodents are also receptor-mediated, because they are not observed in PPARα-null mice (Peters et al., 1998). Humans do not respond with hepatocellular and peroxisomal proliferation to fibrates and express PPARα at low levels and often in nonfunctional forms. Humanized mice (ie, PPARα-null mice transfected with human PPARα) also fail to respond with hepatocellular and peroxisomal proliferation to fibrates, indicating that the human receptor cannot signal for proliferation (Gonzalez and Yu, 2006). In addition, pathways downstream of the receptor are also inappropriate to produce such a response in humans, because transfection of rat PPARα into human hepatocytes failed to make the peroxisomes inducible by PPARα ligands in the human cells. Chemicals that act on ligand-activated TFs, such as glucocorticoids, TCDD, and retinoids, induce fetal malformations that may be regarded as inappropriate gene expression. Candidate target genes are the homeobox genes that determine the body plan during early ontogenesis.

In addition to altering the fate of specific cells, compounds that act on ligand-activated TFs can also evoke changes in the metabolism of endobiotics and xenobiotics by inducing overexpression of relevant enzymes. For example, the PPARα-ligand fibrates induce lipoprotein lipase and enzymes involved in fatty acid oxidation. Unlike the hepatic proliferative effects of PPARα agonists, these effects occur not only in rodents but also in humans, and form the basis of the use of fibrates as lipid-lowering drugs. TCDD, phenobarbital, and pregnenolone 16α-carbonitrile (PCN) activate AhR, the constitutive androstane receptor (CAR), and the pregnane X receptor (PXR), respectively (Table 3-4), thereby exerting their well-known cytochrome P450-inducing effects. However, rodent, rabbit, and human PXRs and CARs exhibit low degree of sequence conservation in their ligand-binding domains. This accounts for marked species differences in the induction potential of their ligands, such as PCN, rifampicin, and TCPOBOP (Gonzalez and Yu, 2006). Genes of several xenobiotic-metabolizing enzymes may be activated by these chemicals. For example, TCDD increases the expression of cytochrome P450 lAl, UDP-glucuronosyltransferase-1, and several subunits of mouse and rat glutathione S-transferase in an AhR-dependent manner. In AhR-null mice, TCDD induces neither these enzymes nor the adverse effects listed in Table 3-4 (Gonzalez and Fernandez-Salguero, 1998). CYP1A1 becomes also strongly expressed in the epithelial cells of the dioxin-induced cystic lesions in the human skin and thus CYP1A1 induction (which is detectable immunohistochemically in these structures) may serve as a biomarker of human dioxin exposure (Saurat et al., 2012). Chloracne, as this multiple dermal lesion has been called inappropriately, is now identified as hamartoma. Dioxin-induced hamartomas appear in the epidermis and are characterized by (1) nonmalignant proliferation of columnar epithelial cells enclosing cysts with inflammatory signs and (2) involution of sebaceous glands. Whole genome microarray analysis of TCDD-induced hamartomas revealed severe dysregulation of gene expression, with marked overexpression of some genes, such as those coding for CYP1A1, inflammatory proteins, and gremlin 2 (a cytokine that inhibits bone morphogenic protein, a transforming growth factor-β [TGF-β]–like growth factor), and repression of many others, such as those encoding several sebaceous lipid synthesis enzymes and solute carriers (Saurat et al., 2012). Mechanistic interpretation of many of these TCDD-induced alterations in gene expression and their role in development of dioxin-induced dermal hamartomas is still uncertain.

The above-mentioned alterations in cell fate and function caused by endobiotics and xenobiotics that act on ligand-activated TFs may in part be mediated by transcriptional upregulation or downregulation of protein-coding genes, that is, genes that are transcribed into mRNA, which in turn is translated into protein (Fig. 3-11). For example, the drug-metabolizing enzymes are induced by TCDD (see above) because the promoter regions of their genes contain a dioxin (or xenobiotic) response element (DRE or XRE). The TCDD-activated AhR complexed with its nuclear translocator protein Arnt (also called HIF-1β) binds to this sequence and promotes the transcription of these genes and secondarily the synthesis of enzyme proteins. The cyclooxygenase-2 (COX-2) gene, whose product is involved in the synthesis of inflammatory prostaglandins, also contains DRE/XRE permitting its transactivation by TCDD-activated AhR. Similarly, the ligand-activated PPARα, after dimerizing with RXR, upregulates the transcription of genes encoding enzymes involved in fatty acid transport and β-oxidation by interacting with the peroxisome proliferator response element (PPRE) located in the promoter of these genes.

As discovered recently, the effects of endobiotics and xenobiotics that act on ligand-activated TFs may also be mediated by transcriptional upregulation or downregulation of genes coding for miRNAs. Because miRNAs repress the translation of target mRNAs into proteins, upregulation of miRNA transcription results in increased repression of protein translation, whereas downregulation of miRNA causes derepression of protein translation from the target mRNAs, and in turn increases the synthesis of specific proteins (Fig. 3-11). The mechanism of hepatocellular proliferation in mice exposed to the peroxisome proliferator Wy-14,643 involves transcriptional downregulation by the Wy-14,643-activated mouse PPARα of the gene coding for let-7c, a miRNA (Gonzalez and Shah, 2008). Let-7c is known to target c-Myc mRNA and represses its translation into c-Myc protein. Therefore, declining levels of let-7c in response to Wy-14,643 result in derepression of c-Myc mRNA and a consequential surge in c-Myc protein synthesis. As c-Myc is a TF signaling for mitosis (see Figs. 3-12 and 3-32), its increased abundance in the liver induces hepatocellular proliferation. Interestingly, the proliferative action of c-Myc also involves miRNA gene regulation: c-Myc upregulates miR-17-92, a cluster of miRNAs, which in turn repress the translation of p21, a cyclin-dependent kinase inhibitor with antimitotic action (see Fig. 3-32), and Bim, a proapoptotic protein (see Fig. 3-15), thereby permitting mitosis and opposing apoptosis. In contrast to normal mice, PPARα-humanized mice (expressing the human PPARα) exhibit neither downregulation of let-7c gene expression nor hepatocellular proliferation on Wy-14,643 treatment, supporting a causative relationship between these events. The mode of action of some other ligand-activated TFs also involves downregulation of miRNA gene transcription with subsequent derepression of the target mRNAs, causing their increased translation into specific proteins. For example, dexamethasone downregulates the expression of the miRNA cluster miR-17-92, which results in elevated levels of Bim, a proapoptotic member of the Bcl family of proteins (see Fig. 3-15). In addition, glucocorticoids can also upregulate the transcription of the Bim gene. Overexpression of the Bim protein, at both transcriptional and translational levels, may be the principal mechanism of glucocorticoid-induced apoptosis in lymphoid cells (Molitoris et al., 2011). Glucocorticoids also cause skeletal muscle wasting, partly because they stimulate the production of myostatin by the muscle. Acting on the muscle in a paracrine manner, this TGF-β-like signaling protein negatively regulates muscle mass (see Fig. 3-30). Glucocorticoids decrease the expression of miR-27a and b (which normally repress the translation of myostatin mRNA, as this mRNA contains a recognition sequence for miR-27a and b in its 3′-untranslated region), thereby increasing the synthesis of myostatin protein. Whereas TCDD, acting through the AhR and the XRE in the COX-2 gene promoter, can transcriptionally upregulate COX-2 expression as mentioned above, studies on mice indicate that TCDD can induce COX-2 protein also by downregulating of miR-101a gene, because one of the targets for miR-101a is the mRNA that translates into the COX-2 enzyme (Yoshioka et al., 2011). It appears that COX-2 induction plays a role in TCDD-induced liver injury in mice. Furthermore, the expression of drug-metabolizing enzymes is regulated not only by TFs (see above and in Table 3-4) but also by miRNAs. For example, the translation of CYP3A4 (which forms the genotoxic metabolites of aflatoxin B₁ and benzo[a]pyrene) and CYP2E1 (which contributes to toxification of acetaminophen, carbon tetrachloride, and dimethylnitrosamine) is repressed by miR-27b and miR-378, respectively (Yokoi and Nakajima, 2011). The complexity of protein expression regulation is underlined by the recent recognition that while TFs can control the expression of miRNAs at their transcription, miRNAs can control the expression of TFs at their translation (Yokoi and Nakajima, 2011).

Finally, ligand-activated TFs do not act only by regulating gene expression through binding to their cognate response element but also by interacting with other proteins, including other nuclear receptors or their associates. Such interactions underlie, for example, the antiestrogenic effect of TCDD and dioxin-like chemicals, which involves at least two mechanisms. First, the TCDD-ligated AhR can associate with the ER, facilitating its nuclear export and proteasomal degradation. In addition, dimerization of the TCDD-ligated AhR with its partner Arnt reduces the availability of this protein for ER that employs Arnt as a transcriptional coactivator (Denison et al., 2011).

Dysregulation of Transcription by Chemicals Altering the Regulatory Region of Genes

Xenobiotics may dysregulate transcription by also altering the regulatory gene regions through direct chemical interaction or by changing their methylation pattern. It has been hypothesized that thalidomide (or its hydrolysis product) exerts teratogenic effect in the embryo by intercalating to GGGCGG sequences (also called GC boxes) that are binding sites for Sp1 and Egr-1 TFs (Stephens et al., 2000). Thalidomide would thus impair insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF2) signaling pathways necessary for angiogenesis and limb formation, because both pathways encompass multiple proteins whose genes contain GC boxes in the regulatory region but lack TATA and CCAAT boxes (eg, specific integrins, FGF2, IGF-1, and IGF-1 receptor substrate-1). Another chemical feature of thalidomide that may account for impaired Sp1-mediated transcriptional activation is that the hydrolysis product of this drug is a glutamine analogue and thus mimics the glutamine-rich activation domain of Sp1 protein.

As described in more detail in the section “Carcinogenesis,” methylation of cytosines in CpG islands located in the promoter of genes is a major epigenetic mechanism, which together with coupled histone modifications (eg, acetylation, methylation) influences the transcriptional activity of the adjacent gene. Increased promoter methylation (hypermethylation) silences genes, whereas decreased methylation (hypomethylation) permits their activation. Importantly, when DNA replication occurs, the methylation pattern is copied from the parent strand to the daughter strand by DNA methyltransferase-1 (DNMT1), making the pattern heritable. Nevertheless, promoter methylation is subject to environmental influences, which can thus cause heritable changes in gene regulation. Altered promoter methylation has been implicated in chemically induced systemic lupus erythemathosus (SLE), in developmental deficiencies manifested postnatally or even in subsequent generations, and in cancer. The antiarrhythmic drug procainamide and the antihypertensive hydralazine often induce SLE, an autoimmune inflammatory disease. Both drugs inhibit DNA methylation and induce global DNA hypomethylation in CD4⁺ T lymphocytes with concomitant overexpression of proteins important in inflammation, such as CD11a (an integrin subunit), CD70 (a B-cell costimulatory molecule), and perforin (a cell-killing molecule) (Ballestar et al., 2006). Idiopathic lupus also involves global DNA hypomethylation in T cells with overexpression of these proinflammatory proteins. Therefore, promoter hypomethylation and overexpression of critical genes that convert T cells into aggressive inflammatory cells appears fundamental in the pathogenesis of both idiopathic and drug-induced SLE.

DNA methylation is an important process also in genomic imprinting that takes place during formation of germ cells. The vast majority of genes in both germ cells (ie, the sperm and the egg) similarly undergo or avoid epigenetic alterations (eg, methylation and related histone modifications); therefore, the offspring carries equally expressed 2 copies (ie, maternal and paternal alleles) of most genes. However, a relatively few (100–200) genes (called the imprinted genes) are epigenetically altered (generally methylated and thus typically silenced) only in the male or female germ cells; therefore, either the paternal or the maternal gene copy is expressed in the offspring, whereas the other allele is not expressed. For example, the insulin-like growth factor-2 gene, which encodes an autocrine growth factor important in fetal growth, is expressed only from the paternal allele. Because the genomically imprinted genes have only a single active copy, they are especially susceptible for epigenetic dysregulation. Dysregulation of imprinting can range from loss of imprinting, which results in biallelic expression and double amounts of the gene product, to silencing of both alleles. Although prenatal exposure to chemicals, such as TCDD and ethanol, influences genomic imprinting (Wu et al., 2004; Stouder et al., 2011), mechanistic interpretation of the role of such epigenetic alterations in chemically induced embryonic, early postnatal, or transgenerational defects in somatic or germ cell development is still uncertain due to the complexity of gene regulation.

The genome is especially susceptible to epigenetic alterations during early development, as in the preimplantation mammalian embryo, the genome (except for the imprinted genes) undergoes extensive demethylation, and appropriate patterns of cytosine methylation are reestablished after implantation. Developing mammalian germ cells are also subject to such changes: primordial germ cells undergo genomic demethylation while they migrate into the early gonad, and then they undergo remethylation during sex determination in the gonad, for example, testis development. During this period (embryonic 11–15 days in the rat), the androgen receptor and ER-β are expressed in the developing testis, and ligands of these nuclear receptors can influence the methylation process. Indeed, transient exposure of gestating female rats during the period of gonadal sex determination to vinclozolin (an agricultural fungicide with antiandrogenic activity) or methoxychlor (a DDT-type insecticide with estrogenic activity) decreased the spermatogenic capacity (with reduced sperm number and viability) in the adult male offspring and increased the incidence of male infertility (Anway et al., 2005). Importantly, these effects were transferred through the male germ line to nearly all males of the subsequent 3 generations. These adverse transgenerational effects on reproduction correlate with altered DNA methylation patterns in the germ line. Thus, endocrine disruptors have the potential to epigenetically reprogram the germ line and promote transgenerational disease state, although the relevance of these findings under human exposure conditions is still uncertain.

Transcription is also severely dysregulated in cancer cells by altered promoter methylation (ie, global DNA hypomethylation and tumor suppressor gene hypermethylation). DNA methylation, including its biochemistry, its influence on histone modifications (acetylation and methylation), and its role in chemical carcinogenesis, is discussed in this chapter in the section “Epigenetic Mechanisms in Carcinogenesis: Inappropriate Activation or Responsiveness of the Regulatory Region of Critical Genes.”

Dysregulation of Signal Transduction

Extracellular signaling molecules (such as growth factors, cytokines, hormones, neurotransmitters, and some secreted proteins) engage cell surface receptors and intracellular signal transducing networks and ultimately activate TFs. Fig. 3-12 depicts a simplified scheme for some of these networks and identifies some signal-activated TFs that are turned on by these systems and that in turn control the transcriptional activity of genes that determine the fate of cells by influencing mitosis and/or apoptosis. Among these TFs are c-Fos and c-Jun proteins, which bind in dimeric combinations (called AP-1) to the tetradecanoylphorbol acetate (TPA) response element (TRE), for example, in the promoter of the cyclin D gene whose product promotes the cell division cycle (see Figs. 3-25 and 3-32). Other signal-activated TFs that upregulate the transcription of cyclin D gene by binding to their cognate nucleotide sequences include c-Myc, E2F, NF-κB, CREB, and STAT. Mitogenic signaling molecules thus induce cell proliferation. In contrast, TGF-β activates SMADs and induces the expression of cyclin-dependent kinase inhibitor proteins (eg, p15, p21, and p57), which mediate its antimitotic effect (Johnson and Walker, 1999) (see Fig. 3-32), as well as the proapoptotic protein Bim (see Fig. 3-19), which conveys its apoptotic action.

In addition to the proliferatory signaling pathways illustrated in Fig. 3-12, Figs. 3-13 and 3-14 depict 2 other pathways, the activation of which can also promote cell division. These are set in motion by secreted glycoproteins, the Wnt and Hedgehog (Hh) ligands, which act on specific cell surface receptors, ultimately activating the transcriptional coactivator β-catenin and the TF Gli, respectively, which in turn can also activate the transcription of cyclin D gene, among many others. The Wnt–β-catenin and Hh–Gli systems are especially important in certain proliferative processes, such as embryogenesis, development of adult stem cells as well as progenitor cells, tissue regeneration after injury, and oncogenesis.

The signal from the cell surface receptors to the TFs is relayed mainly by successive protein–protein interactions and protein phosphorylations at specific serine, threonine, or tyrosine hydroxyl groups. Cell surface growth factor receptors (item 4 in Fig. 3-12) are in fact phosphorylating enzymes (ie, receptor protein tyrosine kinases). Their ligands induce them to phosphorylate themselves, which, in turn, enable these receptors to bind adapter proteins. Through these, the growth factor receptors can initiate proliferative signaling via two important pathways: (1) by activating Ras, they set in motion the mitogen-activated protein kinase (MAPK) cascade (Qi and Elion, 2005), and (2) by activating PI3K, they start the PI3K–Akt pathway. A crucial step in the latter is the PI3K-catalyzed phosphorylation of a plasma membrane lipid to generate phosphatidylinositol-3,4,5-triphosphate (PIP₃) that recruits protein kinase Akt to the membrane for activation. Because Akt (also called protein kinase B) has numerous substrates, the PI3K–Akt pathway branches (Morgensztern and McLeod, 2005). Fig. 3-12 shows the branch that leads to activation of the TF NF-κB, a prolife pathway. In addition, Akt may also indirectly activate the mammalian target of rapamycin (mTOR), a ubiquitous protein kinase, whose phosphorylated products promote the trophic state of cells by boosting protein synthesis and restraining autophagy (see Fig. 3-29). Furthermore, Akt catalyzes the inactivating phosphorylation of two important signaling proteins. One is glycogen synthase kinase 3 (GSK), a ubiquitous multifunctional constitutionally active protein kinase, whose inactivation by Akt (or by some other Ser/Thr kinases) causes numerous effects ranging from promotion of glycogen synthesis from UDP-glucose to suppression of apoptosis (see later), and whose incapacitation by Wnt and Hh signaling permits β-catenin and Gli, respectively, to escape degradation and act as transcription activators (see Figs. 3-13 and 3-14). The other is the FoxO family of TFs, whose inactivation by Akt-catalyzed phosphorylation prevents protein degradation in the skeletal muscle and promotes proliferation, thereby countering muscle atrophy (see Fig. 3-30).

The intracellular signal transducer proteins are typically but not always activated by phosphorylation, which is catalyzed by protein kinases, and are usually inactivated by dephosphorylation, which is carried out by protein phosphatases. Interestingly, growth factor receptors can amplify their signals by inducing formation of HOOH. As discussed earlier, by attacking protein thiolate groups and converting them into sulfenic acid groups, HOOH reversibly inactivates PTP and PTEN, a lipid phosphatase that eliminates PIP₃ (see Fig. 3-12; Rhee et al., 2005). The source of HOOH is produced by the enzyme complex Nox, which is activated by growth factor receptors via the PI3K → PIP₃ → Rac (a G-protein that associates with Nox) pathway. Even if once thought to be restricted to phagocytes, Nox occurs in the plasma membrane of many other cells, such as endothelial cells and vascular smooth muscle cells (Bokoch and Knaus, 2003). Although Nox in these cells is much less efficient to produce than Nox in phagocytes, the nonphagocytotic Nox releases into the cytoplasm, whereas the phagocytotic Nox delivers it to the external face of the plasma membrane (El-Benna et al., 2005).

Chemicals may cause aberrant signal transduction in a number of ways, most often by altering protein phosphorylation, occasionally by interfering with the GTPase activity of G proteins (eg, Ras), by disrupting normal protein–protein interactions or by establishing abnormal ones, or by altering the synthesis or degradation of signaling proteins. Such interventions may ultimately influence cell cycle progression.

Chemically Altered Signal Transduction with Proliferative Effect

Xenobiotics that facilitate phosphorylation of signal transducers often promote mitosis and tumor formation. Such are the phorbol esters and fumonisin B that activate PKC. These chemicals functionally mimic diacylglycerol (DAG), one of the physiologic activators of PKC (Fig. 3-12). The other physiologic PKC activator Ca²⁺ is mimicked by Pb²⁺, whose effect on PKCα is concentration-dependent: stimulatory at picomolar concentration, when Pb²⁺ occupies only high-affinity binding sites on PKC, and inhibitory at micromolar concentration, where the low-affinity sites are also occupied (Sun et al., 1999). Lead salts do induce marked hepatocellular proliferation in rats. The activated PKC promotes mitogenic signaling at least in two ways: (1) by phosphorylating Raf, the first protein kinase in the MAPK pathway (Fig. 3-12), and (2) by phosphorylating a protein phosphatase that dephosphorylates the TF c-Jun at specific sites (Thr 231, Ser 234, and Ser 249), thereby permitting its binding to DNA. Protein kinases may also be activated by interacting proteins that had been altered by a xenobiotic. For example, the TCDD-liganded AhR binds to MAPK. This so-called nongenomic effect of TCDD may contribute to overexpression of cyclins and cyclin-dependent kinases in guinea pig liver (Ma and Babish, 1999).

Hyperphosphorylation of proteins may result not only from increased phosphorylation by kinases but also from decreased dephosphorylation by phosphatases. Inhibition of phosphatases, including the lipid phosphatase PTEN (see Fig. 3-12), appears to be the underlying mechanism of the mitogenic effect of various chemicals, oxidative stress, and ultraviolet (UV) irradiation (Herrlich et al., 1999). PTP and dual-specificity phosphatases (ie, enzymes that remove phosphate from phosphorylated tyrosine as well as serine and threonine residues) as well as PTEN contain a catalytically active cysteine, and are susceptible to inactivation by HOOH via sulfenic acid formation and by covalent reaction with other SH-reactive chemicals (Forman et al., 2004; Rhee et al., 2005). Indeed, xenobiotics such as the SH-reactive iodoacetamide, the organometal compound tributyltin, arsenite, as well as HOOH cause phosphorylation of the epidermal growth factor (EGF) receptor (item 4 in Fig. 3-12) by interfering with the PTP that would dephosphorylate and thus “silence” this receptor (Herrlich et al., 1999; Chen et al., 1998). Arsenite-induced oxidative stress (Lynn et al., 2000; Smith et al., 2001) may in fact result from activation of growth factor receptors. These signal via the PI3K → PIP₃ → Rac pathway to the neighboring Nox, which also resides in the plasma membrane. Overproduction of by the activated Nox in endothelial cells and vascular smooth muscle cells might contribute to the vasculotoxic effects of arsenic. Arsenite may also inactivate the dual-specificity phosphatase that dephosphorylates and “silences” certain MAPKs (c-jun N-terminal kinase [JNK], p38), whereas methylmethane sulfonate (MMS) appears to inhibit a protein phosphatase that inactivates Src, a protein tyrosine kinase (Herrlich et al., 1999). The thiol-oxidizing agent diamide (which increases phosphorylation of MAPKs) and phenolic antioxidants (which form phenoxyl radicals and increase c-Fos and c-Jun expression) (Dalton et al., 1999) may also act by incapacitating PTP. Protein phosphatase 2A (PP2A) is the major soluble Ser/Thr phosphatase in cells and is likely responsible, at least in part, for reversing the growth factor–induced stimulation of MAPK, thereby keeping the extent and duration of MAPK activation under control (Goldberg, 1999). PP2A also removes an activating phosphate from a mitosis-triggering protein kinase (p34^cdc2). Several natural toxins are extremely potent inhibitors of PP2A, including the blue-green algae poison microcystin-LR and the dinoflagellate-derived okadaic acid (Toivola and Eriksson, 1999), which are tumor promoters in experimental animals subjected to prolonged low-dose exposure. It is to be noted, however, that acute high-dose exposure to microcystin induces severe liver injury, whereas such exposure to okadaic acid is the underlying cause of the diarrhetic shellfish poisoning. In these conditions, hyperphosphorylation of proteins other than those involved in proliferative signaling (eg, hepatocellular microfilaments in microcystin poisoning) may be primarily responsible for the pathogenesis.

Apart from phosphatases, there are also inhibitory binding proteins that can keep signaling under control. Such is IκB, which binds to NF-κB, preventing its transfer into the nucleus and its function as a TF. On phosphorylation by its designated IκB kinase (IKK), IκB is degraded by the proteasome and NF-κB is set free. IKK can be phosphorylated (activated) by other protein kinases, such as Raf (a member of the MAPK cascade) and Akt (a member of the PI3K–Akt pathway) (Fig. 3-12). NF-κB is an important contributor to proliferative and prolife signaling, because via the above pathways, growth factor receptor stimulation can cause IκB phosphorylation and thus the release of NF-κB, and because the released NF-κB transactivates genes whose products accelerate the cell division cycle (eg, cyclin D1 and c-Myc) and inhibit apoptosis (eg, antiapoptotic Bcl proteins and the caspase inhibitor of apoptosis proteins [IAP]) (Karin, 2006). NF-κB activation, via the Akt-mediated IKK phosphorylation, is involved in the proliferative (and possibly carcinogenic) effect of arsenite, nicotine, and the NNK. In addition, because NF-κB also targets the genes of several cytokines (eg, TNF, IL-1β), chemokines (eg, monocyte chemotactic protein-1 [MCP-1], cytokine-induced neutrophil chemoattractant [CINC]-1), cell adhesion molecules (eg, ICAM-1, E- and P-selectins), enzymes producing inflammatory lipid mediators (eg, PLA2, COX-2), and acute-phase proteins (eg, C-reactive protein, α1-acid glycoprotein), and because such cytokines acting on their receptors (items 2 and 3 in Fig. 3-12) activate NF-κB, this TF plays a leading role also in inflammatory and acute-phase reactions, as well as in cancer caused by chronic inflammation (Karin, 2006; Waddick and Uckun, 1999). Conversely, inactivation of a subtype of NF-κB protein (RelA/p65) by binding to the TCDD-ligated AhR may contribute to the immunosuppressive effect of TCDD (Denison et al., 2011). The proliferative Wnt signaling is also restrained by a set of binding proteins and protein kinases, the so-called β-catenin destruction complex that includes axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK), which destroy the transcriptional coactivator β-catenin by phosphorylation and ubiquitination (see Fig. 3-13). Impaired function of this complex (eg, by inactivating mutation of APC) can lead to unleashing the transcriptional coactivator β-catenin and in turn to colon cancer.

Another site from which aberrant mitogenic signals may originate is the GTP/GDP-binding protein Ras, which is active in GTP-bound form but inactive in GDP-bound form. The activity of Ras is normally terminated via stimulation of its own GTPase activity by a GTPase-activating protein (GAP) (Fig. 3-12) that returns Ras into its inactive GDP-bound state. Fatty acids, which may accumulate, for example, in response to phospholipase A activation and exposure to peroxisome proliferators (Rose et al., 1999), inhibit GAP and can delay the turning off of Ras. As discussed in more detail later in the chapter, genotoxic carcinogens may mutate Ras, and if the mutation leads to a loss of its GTPase activity, this would result in permanent signaling for the MAPK pathway—a condition that contributes to malignant transformation of the affected cell population.

Finally, increased mobilization of a mitogenic TF from a “silent” intracellular pool may also be a mechanism for chemically altered signal transduction with proliferative effect. This may underlie, at least in part, the nephrocarcinogenicity of cadmium and the colon tumor–promoting action of secondary bile acids, such as deoxycholic acid. By disrupting the E-cadherin/β-catenin complex in the adherens junctions (AJ) of renal and colonic epithelial cells, respectively, these chemicals mobilize β-catenin from AJ (where this protein serves a structural function) into the cytoplasm and the nucleus. There β-catenin functions as a TF of Wnt signaling (Fig. 3-13) and activates the transcription of several genes, including cyclin D1 that promotes the G1/S transition of cells in the cell division cycle.

Chemically Altered Signal Transduction with Antiproliferative Effect

Turning off the increased proliferative signaling after cell injury may compromise replacement of injured cells. This prediction has been made from a study on cultured Hepa 1–6 cells that exhibited the following, seemingly consequential alterations on exposure to acetaminophen (follow the path in Fig. 3-12): inhibition of Raf → diminished degradation of IκB → diminished binding of NF-κB to DNA → diminished expression of c-Myc mRNA (Boulares et al., 1999). Downregulation of a normal mitogenic signal is a step away from survival and toward apoptosis. Indeed, inhibitors of PKC (staurosporin), PI3K (wortmannin), and IκB degradation (gliotoxin) (Waddick and Uckun, 1999) are apoptosis inducers. TGF-β and glucocorticoids increase IκB synthesis and, in turn, decrease NF-κB activation and c-Myc expression (Waddick and Uckun, 1999). These mechanisms may contribute to the apoptotic effect of TGF-β and glucocorticoids, the latter in lymphoid cells.

Offsetting the Hh–Gli pathway (Fig. 3-14), a key mitogenic signaling pathway controlling embryonic development, is the mechanism of the teratogenic action of cyclopamine (ie, cyclopia). This severe cephalic malformation was observed in the offspring of sheep grazing in a field where Veratrum californicum grew. This plant contains the steroidal alkaloid cyclopamine, which is an inhibitor of Smoothened, a positive regulator of Hh signaling (Chen et al., 2002).

Dysregulation of Extracellular Signal Production

Hormones of the anterior pituitary exert mitogenic effects on endocrine glands in the periphery by acting on cell surface receptors. Pituitary hormone production is under negative feedback control by hormones of the peripheral glands. Perturbation of this circuit adversely affects pituitary hormone secretion and, in turn, the peripheral gland. For example, xenobiotics that inhibit thyroid hormone production (eg, the herbicide amitrole and the fungicide metabolite ethylenethiourea) or enhance thyroid hormone elimination (eg, phenobarbital) reduce thyroid hormone levels and increase the secretion of thyroid-stimulating hormone (TSH) because of the reduced feedback inhibition. The increased TSH secretion stimulates cell division in the thyroid gland, which is responsible for the goiters or thyroid tumors caused by such toxicants (see Chap. 21). Decreased secretion of pituitary hormone produces the opposite adverse effect, with apoptosis followed by involution of the peripheral target gland. For example, estrogens produce testicular atrophy in males by means of feedback inhibition of gonadotropin secretion. The low sperm count in workers intoxicated with the xenoestrogen chlordecone probably results from such a mechanism.

Dysregulation of Ongoing Cellular Activity

Ongoing control of specialized cells is exerted by signaling molecules acting on membrane receptors that transduce the signal by regulating Ca²⁺ entry into the cytoplasm or stimulating the enzymatic formation of intracellular second messengers. The Ca²⁺ or other second messengers ultimately alter the phosphorylation of functional proteins, changing their activity and, in turn, cellular functions almost instantly. Toxicants can adversely affect ongoing cellular activity by disrupting any step in signal coupling.

Dysregulation of Electrically Excitable Cells

Many xenobiotics influence cellular activity in excitable cells, such as neurons, skeletal, cardiac, and smooth muscle cells. Cellular functions such as the release of neurotransmitters and muscle contraction are controlled by transmitters and modulators synthesized and released by adjacent neurons. The major mechanisms that control such cells are shown schematically in Fig. 3-15, and chemicals that interfere with these mechanisms are listed in Table 3-5.

Altered regulation of neural and/or muscle activity is the basic mechanism of action of many drugs and is responsible for toxicities associated with drug overdosage, pesticides, and microbial, plant, and animal toxins (Herken and Hucho, 1992). As neurons are signal-transducing cells, the influence of chemicals on neurons is seen not only on the neuron affected by the toxicant but also on downstream cells influenced by the primary target. Thus, tetrodotoxin, which blocks voltage-gated Na⁺ channels (item 7 in Fig. 3-15) in motor neurons, causes skeletal muscle paralysis. In contrast, cyclodiene insecticides, which block GABA receptors (item 3 in Fig. 3-15) in the central nervous system, induce neuronal excitation and convulsions (Narahashi, 1991).

Perturbation of ongoing cellular activity by chemicals may be due to an alteration in (1) the concentration of neurotransmitters, (2) receptor function, (3) intracellular signal transduction, or (4) the signal-terminating processes.

Alteration in Neurotransmitter Levels

Chemicals may alter synaptic levels of neurotransmitters by interfering with their synthesis, storage, release, or removal from the vicinity of the receptor. The convulsive effect of hydrazine and hydrazides (eg, isoniazid) is due to their ability to decrease the synthesis of the inhibitory neurotransmitter GABA (Gale, 1992). Reserpine causes its several adverse effects by inhibiting the neuronal storage of norepinephrine, 5-hydroxytryptamine, and dopamine, thereby depleting these transmitters. Skeletal muscle paralysis caused by botulinum toxin is due to inhibition of acetylcholine release from motor neurons and the lacking stimulation of the acetylcholine receptors at the neuromuscular junction (receptor 1 in Fig. 3-15). In contrast, inhibition of acetylcholinesterase by organophosphate or carbamate insecticides or chemical warfare agents (eg, soman) prevents the hydrolysis of acetylcholine, resulting in massive stimulation of cholinergic receptors (receptors 1, 5, and 11 in Fig. 3-15) and a cholinergic crisis (Table 3-5). Inhibition of the neuronal reuptake of norepinephrine by cocaine or tricyclic antidepressants is responsible for overexcitation of α₁-adrenergic receptors on vascular smooth muscles, resulting in nasal mucosal ulceration and myocardial infarction in heavy cocaine abusers, whereas overstimulation of β₁-adrenergic receptors contributes to life-threatening arrhythmias. Similar cardiac complications may result from amphetamine abuse, because amphetamine enhances the release of norepinephrine from adrenergic neurons and competitively inhibits neuronal reuptake of this transmitter. A hypertensive crisis can occur with the combined use of tricyclic antidepressants and monoamine oxidase inhibitors, drugs that block different mechanisms of norepinephrine elimination (Hardman et al., 1995). Concomitant use of drugs that increase the level of serotonin (5-HT) by enhancing its neuronal release and decreasing its neuronal reuptake (eg, fluoxetine) or its biotransformation (eg, monoamine oxidase inhibitors) induce the serotonin syndrome with cognitive and behavioral changes, autonomic dysfunction, and neuromuscular abnormalities. It is thought that cytotoxic antineoplastic drugs (eg, cisplatin) and radiation cause nausea and emesis, a disturbing reaction to chemotherapy and radiotherapy, by inducing release of 5-HT from enterochromaffin cells of the intestinal mucosa, which stimulates the 5-HT₃ receptors (5-HT-gated cation channel, functionally similar to item 1 in Fig. 3-15) on the adjacent vagal afferent neurons, thereby evoking the vomiting reflex (Endo et al., 2000). Intestinal release of 5-HT and stimulation of vagal afferent neurons are also involved in the emetic effect of ipecac syrup and its alkaloids (cephaelin and emetine). The α₂-adrenergic receptor agonist clonidine induces release in the brain of β-endorphin, an endogenous peptide that stimulates opioid receptors (item 6 in Fig. 3-15). This explains why clonidine intoxication mimics several symptoms of morphine poisoning, including depressed respiration and pinpoint pupils.

Toxicant–Neurotransmitter Receptor Interactions

Some chemicals interact directly with neurotransmitter receptors, including (1) agonists that associate with the ligand-binding site on the receptor and mimic the natural ligand, (2) antagonists that occupy the ligand-binding site but cannot activate the receptor, (3) activators, and (4) inhibitors that bind to a site on the receptor that is not directly involved in ligand binding. In the absence of other actions, agonists and activators mimic, whereas antagonists and inhibitors block, the physiologic responses characteristic of endogenous ligands. For example, muscimol, a mushroom poison, is an agonist at the inhibitory GABA_A receptor (item 3 in Fig. 3-15), whereas barbiturates, benzodiazepines, general anesthetics, and alcohols are activators (Narahashi, 1991). Thus, all these chemicals cause inhibition of central nervous system activity, resulting in sedation, general anesthesia, coma, and ultimately blockade of the medullary respiratory center, depending on the dose administered. There are also similarities in the responses evoked by agonist/activators on excitatory receptors and those elicited by antagonists/inhibitors on inhibitory sites. Thus, glutamate receptor agonists and muscarinic receptor agonists cause neuronal hyperactivity in the brain and ultimately convulsions, as do inhibitors of GABA_A receptor. It is also apparent that chemicals acting as agonists/activators on inhibitory receptors and those acting as antagonists/inhibitors on excitatory receptors may exert similar effects. Moreover, general anesthetic solvents induce general anesthesia not only by activating the inhibitory ligand-gated chloride-ion channels (ie, GABA_A and glycine receptors; see items 3 and 4, respectively, in Fig. 3-15) but also by inhibiting the excitatory ligand-gated cation channels (ie, neuronal nicotinic acetylcholine receptor and glutamate receptors; see items 1 and 2, respectively, in Fig. 3-15) (Franks and Lieb, 1998; Perouansky et al., 1998). Because there are multiple types of receptors for each neurotransmitter, these receptors may be affected differentially by toxicants. For example, the neuronal nicotinic acetylcholine receptor is extremely sensitive to inhibition by lead ions, whereas the muscular nicotinic receptor subtype is not (Oortgiesen et al., 1993). Other chemicals that produce neurotransmitter receptor–mediated toxicity are listed in Table 3-5.

Some sensory neurons have membrane receptors that are stimulated by noxious chemicals and operate as ligand-gated cation channels. Such are the transient receptor potential (TRP) channels, whose opening cause influx of Na⁺ and Ca²⁺ that depolarize and activate the neuron. The TRPV1 receptor is excited by capsaicin, the pungent ingredient of red peppers, and mediates the burning sensation of the tongue and reflex stimulation of the lacrimal gland associated with exposure to pepper spray (also called OC tear gas). The TRPA1 receptor, however, is activated by thiol-reactive and oxidant chemicals, such as the lacrimator compounds in tear gas (eg, chloroacetophenone, chlorobenzalmalonitrile, and dibenzoxazepine), acrolein, methyl isocyanate, corrosive gases (eg, phosgene, chloropicrin, and chlorine) and other irritants. These chemicals evoke sensations (irritation and pain), secretory and respiratory reflexes (lacrimation, bronchial secretion, sneezing, coughing, and bronchospasm), and neurogenic inflammation through the TRPA1 receptor (Bessac and Jordt, 2010).

Toxicant–Signal Transducer Interactions

Many chemicals alter neuronal and/or muscle activity by acting on signal transduction processes. Voltage-gated Na⁺ channels (item 7 in Fig. 3-15), which transduce and amplify excitatory signals generated by ligand-gated cation channels (receptors 1 and 2 in Fig. 3-15), are activated by a number of toxins derived from plants and animals (Table 3-5) as well as by synthetic chemicals such as DDT, resulting in overexcitation (Narahashi, 1992). In contrast, chemicals that block voltage-gated Na⁺ channels (such as tetrodotoxin and saxitoxin) cause paralysis. The Na⁺ channels are also important in signal transduction in sensory neurons; therefore, Na⁺-channel activators evoke sensations and reflexes, whereas Na⁺-channel inhibitors induce anesthesia. This explains the reflex bradycardia and burning sensation in the mouth that follow the ingestion of monkshood, which contains the Na⁺-channel activator aconitine, as well as the use of Na⁺-channel inhibitors such as procaine and lidocaine for local anesthesia.

Toxicant–Signal Terminator Interactions

The cellular signal generated by cation influx is terminated by removal of the cations through channels or by transporters (Fig. 3-15). Inhibition of cation efflux may prolong excitation, as occurs with the blockade of Ca²⁺-activated K⁺ channels (item 9 in Fig. 3-15) by Ba²⁺, which is accompanied by potentially lethal neuroexcitatory and spasmogenic effects. The arachidonic acid metabolite 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) also blocks Ca²⁺-activated K⁺ channels, causing vasoconstriction. Induction by cyclosporine of 20-HETE production in renal proximal tubular cells and the resultant decrease in renal cortical blood flow may underlie the nephrotoxic effect of cyclosporine (Seki et al., 2005). Blockade of specific voltage-gated K⁺ channels (called hERG channels; item 9 in Fig. 3-15) in the heart delays cardiac repolarization (as indicated by prolonged QT time in ECG) and may evoke polymorphic ventricular tachycardia (termed torsade de pointes), which may cause sudden death if degenerates into ventricular fibrillation (Sanguinetti and Mitcheson, 2005). Several drugs with such an effect (eg, astemizole, cisapride, grepafloxacin, terfenadine) have been withdrawn from clinical use. Glycosides from digitalis and other plants inhibit Na⁺,K⁺-ATPase (item 10 in Fig. 3-15) and thus increase the intracellular Na⁺ concentration, which, in turn, decreases Ca²⁺ export by Ca²⁺/Na⁺ exchange (Fig. 3-15). The resultant rise in the intracellular concentration of Ca²⁺ enhances the contractility and excitability of cardiac muscle. Inhibition of brain Na⁺,K⁺-ATPase by chlordecone may be responsible for the tremor observed in chlordecone-exposed workers (Desaiah, 1982). Lithium salts, although used therapeutically, have the potential to produce hyperreflexia, tremor, convulsions, diarrhea, and cardiac arrhythmias (Hardman et al., 1995). Lithium also markedly potentiates cholinergic-mediated seizures. Besides inhibition of Li⁺-sensitive enzymes (eg, inositol monophosphatase, bisphosphate 3′-nucleotidase, and GSK), another possible reason for these toxic effects is inefficient repolarization of neurons and muscle cells in the presence of Li⁺. Whereas Li⁺ readily enters these cells through Na⁺ channels, contributing to the signal-induced depolarization, it is not a substrate for the Na⁺,K⁺ pump. Therefore, the cells fail to repolarize properly if a fraction of intracellular Na⁺ is replaced by Li⁺.

Failure of the Na⁺,K⁺ pump is also believed to contribute to the neuronal damage resulting from hypoxia, hypoglycemia, and cyanide intoxication. Inasmuch as 70% of the ATP produced in neurons is used to drive the Na⁺,K⁺ pump, cessation of ATP synthesis causes a cell to become or remain depolarized. The depolarization-induced release of neurotransmitters such as glutamate from such neurons is thought to be responsible for the hypoxic seizures and further amplification of neuronal injury by the neurotoxic actions of glutamate (Patel et al., 1993).

Dysregulation of the Activity of Other Cells

While many signaling mechanisms also operate in nonexcitable cells, disturbance of these processes is usually less consequential. For example, rat liver cells possess α₁-adrenergic receptors (item 12 in Fig. 3-15) whose activation evokes metabolic changes, such as increased glycogenolysis and glutathione export, through elevation of intracellular Ca²⁺, which may have toxicological significance.

Many exocrine secretory cells are controlled by muscarinic acetylcholine receptors (item 11 in Fig. 3-15). Salivation, lacrimation, and bronchial hypersecretion after organophosphate insecticide poisoning are due to stimulation of these receptors. In contrast, blockade of these receptors contributes to the hyperthermia characteristic of atropine poisoning. Kupffer cells, resident macrophages in the liver, secrete inflammatory mediators (see Fig. 3-26) that can harm neighboring cells. Because Kupffer cells possess glycine receptors, that is, glycine-gated Cl⁻ channels (item 4 in Fig. 3-15), the secretory function of these macrophages (eg, secretion of inflammatory mediators) can be blocked by administration of glycine, which induces hyperpolarization via influx of Cl⁻. Such intervention alleviates ethanol-induced liver injury (Yin et al., 1998).

The discovery that some sulfonamides produce hypoglycemia in experimental animals led to the development of oral hypoglycemic agents for diabetic patients. These drugs inhibit K⁺ channels in pancreatic β cells, inducing sequential depolarization, Ca²⁺ influx through voltage-gated Ca²⁺ channels, and exocytosis of insulin (Hardman et al., 1995). The antihypertensive diazoxide acts in the opposite fashion on K⁺ channels and impairs insulin secretion. Whereas this effect is generally undesirable, it is exploited in the treatment of inoperable insulin-secreting pancreatic tumors.

Toxic Alteration of Cellular Maintenance

Numerous toxicants interfere with cellular maintenance functions. In a multicellular organism, cells must maintain their own structural and functional integrity as well as provide supportive functions for other cells. Execution of these functions may be disrupted by chemicals, resulting in a toxic response.

Impairment of Internal Cellular Maintenance: Mechanisms of Toxic Cell Death

For survival, all cells must synthesize endogenous molecules; assemble macromolecular complexes, membranes, and cell organelles; maintain the intracellular environment; and produce energy for operation. Chemicals that disrupt these functions, especially the energy-producing function of mitochondria and protein synthesis controlling function of the genome, jeopardize survival and may cause toxic cell death.

There are three critical biochemical disorders that chemicals inflicting cell death may initiate, namely, ATP depletion, sustained rise in intracellular Ca²⁺, and overproduction of ROS and RNS. In the following discussion, these events and the chemicals that may cause them are individually characterized. Then it is pointed out how their concerted action may induce a bioenergetic catastrophe, culminating in necrosis. Finally, there follows a discussion of the circumstances under which the cell can avoid this disordered decay and how it can execute death by activating catabolic processes that bring about an ordered disassembly and removal of the cell, called apoptosis.

Primary Metabolic Disorders Jeopardizing Cell Survival: ATP Depletion, Ca2+ Accumulation, ROS/RNS Generation

Depletion of ATP

ATP plays a central role in cellular maintenance both as a chemical for biosynthesis and as the major source of energy. It is utilized in numerous biosynthetic reactions, activating endogenous compounds by phosphorylation and adenylation, and is incorporated into cofactors as well as nucleic acids. It is required for muscle contraction and polymerization of the cytoskeleton, fueling cellular motility, cell division, vesicular transport, and the maintenance of cell morphology. ATP drives ion transporters, such as the Na⁺,K⁺-ATPase in the plasma membrane, the Ca²⁺-ATPase in the plasma and the ER membranes, and H⁺-ATPase in the membrane of lysosomes and neurotransmitter-containing vesicles. These pumps maintain conditions essential for various cell functions. For example, the Na⁺ concentration gradient across the plasma membrane generated by the Na⁺,K⁺ pump drives Na⁺–glucose and Na⁺–amino acid cotransporters as well as the Na⁺/Ca²⁺ antiporter, facilitating the entry of these nutrients and the removal of Ca²⁺.

Chemical energy is released by hydrolysis of ATP to ADP or AMP. The ADP is rephosphorylated in the mitochondria by ATP synthase (Fig. 3-16). Coupled to oxidation of hydrogen to water, this process is termed oxidative phosphorylation. In addition to ATP synthase, oxidative phosphorylation requires the (1) delivery of hydrogen in the form of NADH to the initial electron transport complex; (2) delivery of oxygen to the terminal electron transport complex; (3) delivery of ADP and inorganic phosphate to ATP synthase; (4) flux of electrons along the electron transport chain to O₂, accompanied by ejection of protons from the matrix space across the inner membrane; and (5) return of protons across the inner membrane into the matrix space down an electrochemical gradient to drive ATP synthase (Fig. 3-16).

Several chemicals impede these processes, interfering with mitochondrial ATP synthesis (Commandeur and Vermeulen, 1990; Wallace and Starkov, 2000; Wallace, 2008). These chemicals are divided into 5 groups (Table 3-6). Substances in class A interfere with the delivery of hydrogen to the electron transport chain. For example, fluoroacetate inhibits the citric acid cycle and the production of reduced cofactors. Class B chemicals such as rotenone and cyanide inhibit the transfer of electrons along the electron transport chain to oxygen. Class C agents interfere with oxygen delivery to the terminal electron transporter, cytochrome oxidase. All chemicals that cause hypoxia ultimately act at this site. Chemicals in class D inhibit the activity of ATP synthase, the key enzyme for oxidative phosphorylation. At this site, the synthesis of ATP may be inhibited in one of four ways: (1) direct inhibition of ATP synthase, (2) interference with ADP delivery, (3) interference with inorganic phosphate delivery, and (4) deprivation of ATP synthase from its driving force, the controlled influx of protons into the matrix space. Protonophoric chemicals (uncouplers) such as 2,4-dinitrophenol and pentachlorophenol import protons into the mitochondrial matrix, dissipating the proton gradient that drives the controlled influx of protons into the matrix, which, in turn, drives ATP synthase. Finally, chemicals causing mitochondrial DNA injury, and thereby impairing synthesis of specific proteins encoded by the mitochondrial genome (eg, subunits of complex I and ATP synthase), are listed in group E. These include the dideoxynucleoside antiviral drugs used against AIDS, such as zidovudine. Table 3-6 lists other chemicals that impair ATP synthesis.

ATP synthase is also present at sites in addition to the mitochondria, such as the outer segment disk of the retinal rods as well as the plasma membrane of certain cells and the concentric membranes that form the myelin sheath around the axon of neurons. Unlike its mitochondrial counterpart, the ATP synthase in plasma and myelin membranes forms ATP on the external membrane face and is called ecto-ATP synthase. It is hypothesized that ATP produced by the myelinic ATP synthase (supported by the colocalized respiratory chain complexes and O₂ well dissolved in the myelin lipids) is delivered into the axon through gap junctions that traverse the myelin membranes and the axolemma (Morelli et al., 2011). Myelinic ATP supply to axons, in which mitochondria are sparse, may explain why demyelinated axons die and why the white matter (composed of myelinated axons) is sensitive to hypoxia. Oxidative phosphorylation of ADP in isolated myelin vesicles (which are devoid of mitochondria) is sensitive to chemicals listed in Table 3-6 as mitochondrial electron transport chain inhibitors (eg, antimycin and cyanide), ATP synthase inhibitors (eg, oligomycin), and uncouplers (eg, CCCP and valinomycin). If the trophic role of myelin in axonal maintenance under in vivo conditions becomes validated, it will be necessary to evaluate whether the ATP-synthesizing machinery of myelin is a relevant target for known neurotoxicants, such as lead, methyl mercury, hexachlorophene, and the methanol-derived formic acid.

Impairment of oxidative phosphorylation is detrimental to cells because failure of ADP rephosphorylation results in the accumulation of ADP and its breakdown products as well as depletion of ATP. Accordingly, hepatocytes exposed to KCN and iodoacetate exhibit a rapid rise in cytosolic H⁺ and Mg²⁺ as a result of the hydrolysis of adenosine diphosphates and triphosphates (existing as Mg salts) and the release of phosphoric acid and Mg²⁺ (Herman et al., 1990). The increased conversion of pyruvate to lactate also may contribute to the acidosis. The lack of ATP compromises the operation of ATP-requiring ion pumps, leading to the loss of ionic and volume-regulatory controls (Buja et al., 1993). Shortly after intracellular acidosis and hypermagnesemia, liver cells exposed to KCN and iodoacetate exhibit a rise in intracellular Na⁺, probably as a result of the failing Na⁺ pump, after which plasma membrane blebs appear. The intracellular phosphoric acidosis is beneficial for the cells presumably because the released phosphoric acid forms insoluble calcium phosphate, preventing the rise of cytosolic Ca²⁺, with its deleterious consequences (see below). In addition, a low pH also directly decreases the activity of phospholipases and inhibits mitochondrial permeability transition (MPT; see later). Terminally, the intracellular pH rises, increasing phospholipase activity, and this contributes to irreversible membrane damage (ie, rupture of the blebs) not only by degrading phospholipids but also by generating endogenous detergents such as lysophospholipids and free fatty acids. The lack of ATP aggravates this condition because the reacylation of lysophospholipids with fatty acids is impaired.

Sustained Rise of Intracellular Ca2+

Intracellular Ca²⁺ levels are highly regulated (Fig. 3-17). The 10,000-fold difference between extracellular and cytosolic Ca²⁺ concentration is maintained by the impermeability of the plasma membrane to Ca²⁺ and by transport mechanisms that remove Ca²⁺ from the cytoplasm (Richter and Kass, 1991). Ca²⁺ is actively pumped from the cytosol across the plasma membrane and is sequestered in the ER and mitochondria (Fig. 3-17). Because mitochondria are equipped with a low-affinity Ca²⁺ transporter, they play a significant role in Ca²⁺ sequestration only when the cytoplasmic levels rise into the micromolar range. Under such conditions, a large amount of Ca²⁺ accumulates in the mitochondria, where it is deposited as calcium phosphate.

Toxicants induce elevation of cytoplasmic Ca²⁺ levels by promoting Ca²⁺ influx into or inhibiting Ca²⁺ efflux from the cytoplasm (Table 3-7). Opening of the ligand- or voltage-gated Ca²⁺ channels or damage to the plasma membrane causes Ca²⁺ to move down its concentration gradient from extracellular fluid to the cytoplasm. Toxicants also may increase cytosolic Ca²⁺ by inducing its leakage from the mitochondria or the EPR. They also may diminish Ca²⁺ efflux through inhibition of Ca²⁺ transporters or depletion of their driving forces. Several chemicals that can cause a sustained rise in cytoplasmic Ca²⁺ levels are listed in Table 3-7.

Sustained elevation of intracellular Ca²⁺ is harmful because it can result in (1) depletion of energy reserves, (2) dysfunction of microfilaments, (3) activation of hydrolytic enzymes, and (4) generation of ROS and RNS. There are at least 3 mechanisms by which sustained elevations in intracellular Ca²⁺ unfavorably influence the cellular energy balance. First, high cytoplasmic Ca²⁺ concentrations cause increased mitochondrial Ca²⁺ uptake by the Ca²⁺ “uniporter,” which, like ATP synthase, utilizes the inside negative mitochondrial membrane potential (ΔΨm) as the driving force. Consequently, mitochondrial Ca²⁺ uptake dissipates ΔΨm and inhibits the synthesis of ATP. Moreover, chemicals that oxidize mitochondrial NADH activate a transporter that extrudes Ca²⁺ from the matrix space (Richter and Kass, 1991). The ensuing continuous Ca²⁺ uptake and export (“Ca²⁺ cycling”) by the mitochondria further compromise oxidative phosphorylation. Second, Ca²⁺ may also impair ATP synthesis by causing oxidative injury to the inner membrane by mechanisms described later. Third, a sustained rise in cytoplasmic Ca²⁺ not only impairs ATP synthesis but also increases ATP consumption by the Ca²⁺-ATPases working to eliminate the excess Ca²⁺.

A second mechanism by which an uncontrolled rise in cytoplasmic Ca²⁺ causes cell injury is microfilamental dissociation (Nicotera et al., 1992; Leist and Nicotera, 1997). The cell-wide network of actin filaments maintains cellular morphology by attachment of the filaments to actin-binding proteins in the plasma membrane. An increase of cytoplasmic Ca²⁺ causes dissociation of actin filaments from α-actinin and fodrin, proteins involved in anchoring the filaments to the plasma membrane. This represents a mechanism leading to plasma membrane blebbing, a condition that predisposes the membrane to rupture.

A third event whereby high Ca²⁺ concentrations are deleterious to cells is activation of hydrolytic enzymes that degrade proteins, phospholipids, and nucleic acids (Nicotera et al., 1992; Leist and Nicotera, 1997). Many integral membrane proteins are targets for Ca²⁺-activated neutral proteases, or calpains (Liu et al., 2004). Calpain-mediated hydrolysis of actin-binding proteins may also cause membrane blebbing. Indiscriminate activation of phospholipases by Ca²⁺ causes membrane breakdown directly and by the generation of detergents. Activation of a Ca²⁺-Mg²⁺-dependent endonuclease causes fragmentation of chromatin. Elevated levels of Ca²⁺ can lock topoisomerase II in a form that cleaves but does not religate DNA. In summary, intracellular hypercalcemia activates several processes that interfere with the ability of cells to maintain their structural and functional integrity. The relative importance of these processes in vivo requires further definition.

Overproduction of ROS and RNS

There are a number of xenobiotics that can directly generate ROS and RNS, such as the redox cyclers (Fig. 3-3) and the transition metals (Fig. 3-4). In addition, overproduction of ROS and RNS can be secondary to the intracellular hypercalcemia, as Ca²⁺ activates enzymes that generate ROS and/or RNS in the following ways:

1. Activation of the dehydrogenases in the citric acid cycle by Ca²⁺ accelerates the hydrogen output from the citrate cycle and, in turn, the flux of electrons along the electron transport chain (see Fig. 3-16). This, together with the suppressed ATP-synthase activity (owing to the Ca²⁺-induced uncoupling), increases the formation of by the mitochondrial electron transport chain.

2. Ca²⁺-activated proteases proteolytically convert xanthine dehydrogenase into xanthine oxidase, whose by-products Display Formula$O2•¯$ are and HOOH (Harrison, 2002).

3. Neurons and endothelial cells constitutively express NOS that is activated by Ca²⁺. Given the extremely high reactivity of ^•NO with Display Formula$O2•¯$, coproduction of these radicals will inevitably lead to formation of ONOO⁻, a highly reactive oxidant (Murphy, 1999) (Fig. 3-4). Moreover, ONOO⁻ can increase its own formation by incapacitating the highly sensitive Mn-SOD, which would eliminate Display Formula$O2•¯$, a precursor of ONOO⁻.

Interplay between the Primary Metabolic Disorders Spells Cellular Disaster

The primary derailments in cellular biochemistry discussed above do not remain isolated but interact and amplify each other in a number of ways (Fig. 3-18):

1. Depletion of cellular ATP reserves deprives the endoplasmic and plasma membrane Ca²⁺ pumps of fuel, causing elevation of Ca²⁺ in the cytoplasm. With the influx of Ca²⁺ into the mitochondria, ΔΨm declines, hindering ATP synthase.

2. As stated above, intracellular hypercalcemia facilitates formation of ROS and RNS, which oxidatively inactivate the thiol-dependent Ca²⁺ pump, which, in turn, aggravates the hypercalcemia.

3. The ROS and RNS can also drain the ATP reserves. ^•NO is a reversible inhibitor of cytochrome oxidase; NO⁺ (nitrosonium cation, a product of ^•NO) S-nitrosylates and thus inactivates glyceraldehyde 3-phosphate dehydrogenase, impairing glycolysis, whereas ONOO⁻ irreversibly inactivates respiratory chain complexes I, II, III, and aconitase (by reacting with their Fe-S center) (Murphy, 1999). Therefore, ^•NO and ONOO⁻ inhibit cellular ATP synthesis.

4. Furthermore, ONOO⁻ can induce DNA SSB, which activates poly(ADP-ribose) polymerase (PARP) (Szabó, 1996). As part of the repair strategy, activated PARP transfers multiple ADP-ribose moieties from NAD⁺ to nuclear proteins and PARP itself (D’Amours et al., 1999). Consumption of NAD⁺ severely compromises ATP synthesis (see Fig. 3-16), whereas resynthesis of NAD⁺ consumes ATP. Hence, a major consequence of DNA damage by ONOO⁻ is a cellular energy deficit (Murphy, 1999).

The chain of events and their contribution to the worsening metabolic conditions are somewhat cell- and toxicant-specific. For example, cyanide toxicity in neurons is associated with depolarization and glutamate release (Patel et al., 1993), followed by Ca²⁺ influx through voltage-gated as well as glutamate-gated channels (see items 8 and 2, respectively, in Fig. 3-15). As they express Ca²⁺-activated NOS, neurons are also prone to generate “nitrosative stress,” which affects not only themselves but also perhaps more significantly the neighboring astrocytes (Szabó, 1996). In contrast, in cyanide- and iodoacetate-poisoned liver cells, the increase in cytoplasmic Ca²⁺ is not an early event (Herman et al., 1990). A nitrosative stress critically contributes to propagation of acetaminophen-induced hepatocellular injury, in which the initiating event is covalent binding of N-acetyl-p-benzoquinoneimine (NAPBQI) to mitochondrial proteins (Jaeschke and Bajt, 2006). This purportedly causes a surge in mitochondrial (Display Formula$O2•¯$) generation, followed by in situ formation of ONOO⁻. Owing to depletion of the protective glutathione by NAPBQI, ONOO⁻ readily nitrates mitochondrial proteins. Concomitantly, covalent binding incapacitates the plasma membrane Ca²⁺ pump and the resultant hypercalcemia further deteriorates mitochondrial function and ATP production. It appears that in this and many other cytotoxicities the interplay of ATP depletion, intracellular hypercalcemia, and overproduction of ROS and RNS, involving multiple vicious cycles (Fig. 3-18), can progressively aggravate the biochemical disorder until it becomes a disaster.

Mitochondrial Permeability Transition and the Worst Outcome: Necrosis

Mitochondrial Ca²⁺ uptake, decreased ΔΨm, generation of ROS and RNS, depletion of ATP, and consequences of the primary metabolic disorders (eg, accumulation of inorganic phosphate, free fatty acids, and lysophosphatides) are all considered as causative factors of an abrupt increase in the mitochondrial inner-membrane permeability, termed MPT. This is thought to be caused by misfolded proteins from the inner and outer membranes, which aggregate and open a proteinaceous pore (“megachannel”) that spans both mitochondrial membranes (Kroemer et al., 1998; Kim et al., 2003; Rodriguez-Enriquez et al., 2004). As this pore is permeable to solutes of size <1500 Da, its opening permits free influx of protons into the matrix space, causing rapid and complete dissipation of ΔΨm and cessation of ATP synthesis as well as osmotic influx of water, resulting in mitochondrial swelling. Ca²⁺ that accumulates in the matrix space effluxes through the pore, flooding the cytoplasm. Such mitochondria not only are incapable of synthesizing ATP but also even waste the remaining resources because depolarization of the inner membrane forces the ATP synthase to operate in the reverse mode, as an ATPase, hydrolyzing ATP. Then even glycolysis may become compromised by the insufficient ATP supply to the ATP-requiring glycolytic enzymes (hexokinase, phosphofructokinase). A complete bioenergetic catastrophe ensues in the cell if the metabolic disorders evoked by the toxic chemical (such as ones listed in Tables 3-6 and 3-7) are so extensive that most or all mitochondria in the cell undergo MPT, causing depletion of cellular ATP (see Fig. 3-20). Degradative processes already outlined (eg, oxidative and hydrolytic degradation of macromolecules and membranes as well as disintegration of intracellular solute and volume homeostasis) will go to completion, causing a complete failure in maintenance of cellular structure and functions and culminating in cell lysis or necrosis.

An Alternative Outcome of MPT: Apoptosis

Chemicals that adversely affect cellular energy metabolism, Ca²⁺ homeostasis, and redox state and then ultimately cause necrosis may also induce apoptosis, another form of cell demise. Whereas the necrotic cell swells and lyses, the apoptotic cell shrinks; its nuclear and cytoplasmic materials condense, and then it breaks into membrane-bound fragments (apoptotic bodies) that are phagocytosed (Wyllie, 1997).

As discussed above, the multiple metabolic defects that a cell suffers in its way to necrosis are causal yet rather random in sequence. In contrast, the routes to apoptosis are ordered, involving cascade-like activation of catabolic processes that finally disassemble the cell. A scheme of the apoptotic pathways is presented in Fig. 3-19. Pathways initiated by EPR stress will be dealt with in the section “Mechanisms of Adaptation.”

It appears that most if not all chemical-induced cell deaths will involve the mitochondria, and the resulting mitochondrial dysfunction (such as Ca²⁺ accumulation, dissipation of ΔΨm, and overproduction of ROS/RNS) may ultimately trigger either necrosis or apoptosis, and that MPT can be a crucial event in both. A related event is release into the cytoplasm of cytochrome c (cyt c), a small positively charged heme protein that normally resides in the mitochondrial intermembrane space attached electrostatically to cardiolipin, a specific inner membrane phospholipid with excess negative charge. Peroxidation of cardiolipin by HOOH, a process catalyzed by cyt c, results in detachment of cyt c from the lipid, which may be a critical first step of cyt c release into the cytoplasm (Orrenius et al., 2011).

The significance of cyt c release is two-fold (Cai et al., 1998): (1) as cyt c is the penultimate link in the mitochondrial electron transport chain, its loss will block ATP synthesis, increase formation of Display Formula$O2•¯$ (instead of as shown in Fig. 3-16), and potentially thrust the cell toward necrosis. (2) Simultaneously, the unleashed cyt c (and some other proteins set free from the mitochondria) represents a signal or an initial link in the chain of events directing the cell to the apoptotic path (Cain, 2003) (Fig. 3-19). Cyt c together with dATP/ATP induces the cytoplasmic adapter protein apoptotic protease activating factor (Apaf-1) to oligomerize and bind the latent procaspase-9 (forming a complex called apoptosome), facilitating its conversion to active caspase-9.

Caspases are cysteine proteases (ie, they possess a catalytically active cysteine) that cleave proteins after specific aspartate residues. They reside mostly in the cytoplasm in inactive forms, as procaspases, which are activated by either dimerization (initiators) or proteolytic cleavage (effectors) (Boatright and Salvesen, 2003). Perhaps in order to guarantee that inadvertent activation of caspases should not occur, IAPs, such as XIAP and cIAP, reside in the cytoplasm and physically capture caspases that might have become activated (Cain, 2003). Therefore, cyt c alone probably could not induce sufficiently strong caspase activation if it were not for proteins that physically remove the IAPs from the caspases just being activated. These proteins, named Smac and Omi (or Diablo and HtrA2, respectively), are not only the helpers of cyt c but also residents of the mitochondrial intermembrane space, from where they are jointly mobilized to promote the caspase cascade. Some caspases on the “top” of the cascade (eg, 8 and 9) cleave and activate procaspases. Thereby these initiator caspases carry the activation wave to the so-called effector caspases (eg, 3, 6, and 7), which cleave specific cellular proteins, activating or inactivating them. It is the hydrolysis of these specific proteins that accounts directly or indirectly for the morphological and biochemical alterations in apoptotic cells. For example, proteolytic inactivation of PARP prevents futile DNA repair and wasting of ATP; caspase-mediated cleavage of Beclin 1 aborts autophagy, hydrolysis of the inhibitor of caspase-activated DNase (ICAD) permits caspase-activated DNase (CAD) to translocate to the nucleus and cleave internucleosomal DNA; cleavage of structural proteins (α-fodrin, actin, lamins) aids in disassembly of the cell; incapacitation of focal adhesion kinase (see Fig. 3-12) permits detachment of the cell from the extracellular matrix (ECM); and hydrolytic activation of sterol regulatory element–binding proteins may contribute to accumulation of sterols and externalization of phosphatidylserine in the plasma membrane that identify the apoptotic cell to phagocytes (and the latter also enables experimental detection of apoptotic cells with fluorescently labeled annexin-V that binds to phosphatidylserine residues on the cell surface). Besides caspases, DNA fragmentation can be induced by apoptosis-inducing factor (AIF) and endonuclease G that are also released from the mitochondrial intermembrane space and may contribute to DNA fragmentation during apoptosis and acetaminophen-induced caspase-independent hepatocellular necrosis (Jaeschke and Bajt, 2006). Prerequisite for the release of AIF is its hydrolysis by the Ca²⁺-activated protease calpain localized to the intermembrane space. Oxidative modification of AIF by mitochondrial ROS increases its affinity to calpain (Orrenius et al., 2011).

The decisive mitochondrial event of cell death, that is, release of cyt c and other proapoptotic proteins (eg, Smac, Omi, AIF), is controlled by the Bcl-2 family of proteins, which includes members that facilitate (eg, Bax, Bak, Bad, Bid, Bim, BNIP3, Puma, Noxa) and those that inhibit (eg, Bcl-2, Bcl-XL) these processes. The death-promoting members can oligomerize and form pores in the mitochondrial outer membrane (MOM). By doing so, they may facilitate release of cyt c and other intermembrane proapoptotic proteins via MPT induced by toxic insult of the mitochondria; however, mitochondrial outer membrane permeabilization (MOMP) alone by Bax and its congeners is sufficient to evoke egress of cyt c from the mitochondria. MOMP induced by Bax, Bak, and/or Bid is responsible for cyt c release in apoptosis initiated at extramitochondrial targets, including death receptors and the nuclear DNA (see below). The death suppressor Bcl-2 and Bcl-XL can dimerize with the death-inducing counterparts and neutralize them. Thus, the relative amount of these antagonistic proteins functions as a regulatory switch between cell survival and death (Reed et al., 1998).

The proapoptotic Bax and Bid proteins, which can induce MOMP, represent links whereby death programs initiated extramitochondrially, for example, by DNA damage in the nucleus or by stimulation of the so-called death receptors (Fas receptor and TNF receptor-1 [TNR1]) at the cell surface, can engage the mitochondria into the apoptotic process (Green, 1998) (Fig. 3-19). DNA damage evoked by ionizing and UV radiations, alkylating chemicals, and topoisomerase II inhibitors, such as doxorubicin (Adriamycin), induces stabilization and activation of p53 protein that can promote apoptosis at dual cellular locations and by dual mechanisms. In the nucleus, p53 acts as a TF, which increases expression of proapoptotic members of the Bcl-family of proteins, such as Bax, Puma, and Noxa (Liu and Chen, 2006) (see also Fig. 3-33). Cytoplasmic p53 participates in protein–protein interactions. In such a manner, p53 abrogates the function of antiapoptotic proteins (eg, Bcl-X_L) and activates the MOMP-forming function of proapoptotic Bax and Bak (Green and Kroemer, 2009). As discussed further on, DNA damage is potentially mutagenic and carcinogenic; therefore, apoptosis of cells with damaged DNA is an important self-defense of the body against oncogenesis. Furthermore, the antitumor drugs targeting the nuclear DNA exert their desirable toxic effects against tumor cells (and also their undesirable cytotoxic effects against rapidly dividing normal cells such as hematopoietic cells and small intestinal mucosal cells) by inducing apoptosis primarily via a p53-dependent mechanism.

Stimulation of the Fas receptor (also known as CD95 or Apo-1), TNR1, or TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5, not shown) by their ligands induces receptor oligomerization and recruitment of adapter proteins. The thus formed death-inducing protein complex (DISC) activates caspase-8, an initiator caspase that sets the caspase cascade in motion (Fig. 3-19). In addition, death receptor activation can also engage the mitochondria into the apoptosis program via caspase-8-mediated cleavage of Bid to its active form. The Fas system is involved in cell-mediated cytotoxicity, as cytotoxic T lymphocytes express the Fas ligand that activates the Fas receptor in the membrane of potential target cells, such as those of the liver, heart, and lung. Cholestatic liver injury involves apoptosis induced by the retained hydrophobic bile acids mediated partly through their mitochondrial effect (ie, MPT) and through the death receptors (Higuchi and Gores, 2003). By activating PKC, toxic bile acids promote trafficking of Fas receptor and TRAIL receptor-2 (also known as DR5) from the Golgi complex into the plasma membrane of liver cells, where the increased receptor density induces spontaneous (ie, ligand-independent) receptor oligomerization and caspase activation. Increased expression of soluble FasL and Fas receptor is thought to play a causative role in the apoptosis of pulmonary alveolar epithelial cells underlying acute lung injury (or acute respiratory distress syndrome) (Martin et al., 2005), a potentially lethal outcome of various pathologies and heroin intoxication. The Fas system also mediates germ cell apoptosis in the testes of rodents exposed to mono-(2-ethylhexyl)phthalate or 2,5-hexanedione, the ultimate toxicant formed from hexane. These chemicals damage the microtubules in the Sertoli cells that normally nurse the germ cells. Unable to support the germ cells, Sertoli cells overexpress the Fas ligand to limit the number of germ cells (which upregulate their Fas receptor) by deleting them via apoptosis (Cohen et al., 1997; Lee et al., 1997).

Thus, apoptosis can be executed via multiple pathways, most involving caspase activation. The route preferred will depend, among others, on the initial insult (Fig. 3-19) as well as on the type and state of the cell. For example, T lymphocytes lacking the Bax gene can still undergo p53-dependent death in response to ionizing radiation, probably by increasing Fas expression (Fig. 3-19), whereas Bax-null fibroblasts cannot. AIF appears to play a critical role in neuronal cell death (Orrenius et al., 2011), whereas TRAIL is an important death inducer in various tumor cells. In contrast, keratinocytes and endothelial cells are resistant to death ligand-mediated apoptosis, as they strongly express cFLIP, an inhibitor of caspase-8.

What Determines the Form of Cell Death?

For reasons discussed later, it is not inconsequential for the surrounding tissue whether a cell dies by apoptosis or necrosis. Therefore, considerable research has focused on what determines the form of cell death. Interestingly, there are some common features in the process of apoptosis and necrosis. Both forms of cell death caused by cytotoxic agents may involve similar metabolic disturbances and MPT (Kim et al., 2003; Kroemer et al., 1998; Qian et al., 1999), and blockers of the latter (eg, cyclosporin A, Bcl-2 overexpression) prevent both apoptosis and necrosis in different settings. Furthermore, many xenobiotics—such as the hepatotoxicant 1,1-dichloroethylene, thioacetamide, and cadmium as well as the nephrotoxicant ochratoxin—can cause both apoptosis and necrosis (Corcoran et al., 1994). However, toxicants tend to induce apoptosis at low exposure levels or early after exposure at high levels, whereas they cause necrosis later at high exposure levels. This indicates that the severity of the insult determines the mode of cell death. Based on experimental evidence, it appears that a larger toxic insult causes necrotic cell death rather than apoptosis because it incapacitates the cell to undergo apoptosis. This incapacitation may result from three causatively related cellular events, that is, increasing number of mitochondria undergoing MPT, depletion of ATP, and failed activation of caspases or their inactivation.

Lemasters and coworkers (Kim et al., 2003; Rodriguez-Enriquez et al., 2004) used confocal microscopy to visualize mitochondria in cells exposed to an apoptogenic stimulus and found that MPT does not occur uniformly in all mitochondria. They proposed a model in which the number of mitochondria undergoing MPT (which probably depends on the degree of chemical exposure) determines the fate of the cell. According to this model, when only a few mitochondria develop MPT, they are removed by selective autophagy (mitophagy; see the section “Cellular Repair”) and the cell survives. When more mitochondria suffer MPT, the autophagic mechanism becomes overwhelmed, and the released proapoptotic factors (eg, cyt c, Smac, and Omi) initiate caspase activation (Fig. 3-19). By hydrolyzing Beclin 1, a component of the autophagic mechanism, caspase-8 aborts mitophagy (Li et al., 2011), and by hydrolyzing other cellular proteins (see above) and DNA, caspases and AIF, respectively, promote apoptosis. When eventually MPT involves virtually all mitochondria, cytolysis occurs. Fig. 3-20 illustrates an expanded version of this model.

For reasons discussed above, ATP is bound to become severely depleted in cells in which most mitochondria suffer MPT. The degree of ATP depletion may decide the fate of the cell. In experimental models as distinct as Ca²⁺-exposed hepatocytes, Fas-stimulated T lymphocytes, and HOOH-exposed endothelial cells, necrosis occurs instead of apoptosis when cells are depleted of ATP, whereas apoptosis takes place rather than necrosis when ATP depletion is alleviated by providing substrates for ATP generation (Leist et al., 1997; Kim et al., 2003; Lelli et al., 1998). Lack of ATP can prevent execution of the apoptotic program, because apoptosis involves ATP-requiring steps, such as activation of procaspase-9 in the apoptosome complex (Fig. 3-19).

Failure of caspase activation can also result from direct action of reactive toxicants on these enzymes. The active site of caspases is composed of a pentapeptide (QACXG) with a reactive cysteine in it. At high concentrations, soft electrophiles, disulfides (eg, glutathione disulfide), and oxidants (eg, ROS), can react with this cysteine, causing caspase inactivation and ablation of the apoptotic program, with necrosis rather than apoptosis as the final outcome. Such a scenario has been demonstrated for cell death evoked by tributyltin, pyrrolidine dithiocarbamate, and arsenic trioxide (Orrenius, 2004) and might also underlie the acetaminophen-induced hepatocellular necrosis, which involves cyt c release with no caspase activation or apoptosis (Jaeschke and Bajt, 2006).

Induction of Cell Death by Unknown Mechanisms

In addition to chemicals that ultimately injure mitochondria by disrupting oxidative phosphorylation and/or intracellular Ca²⁺ homeostasis, there are toxicants that cause cell death by affecting other functions or structures primarily. Included here are (1) chemicals that directly damage the plasma membrane, such as lipid solvents, detergents, and venom-derived hydrolytic enzymes; (2) xenobiotics that damage the lysosomal membrane, evoking release of lysosomal hydrolases and Fe²⁺, such as aminoglycoside antibiotics and hydrocarbons binding to α_2u-globulin that injure renal tubular cells; (3) toxins that destroy the cytoskeleton, such as the microfilament toxins (eg, phalloidin and cytochalasins) and the microtubule toxins (eg, colchicine and 2,5-hexanedione); (4) the protein phosphatase inhibitor and hepatotoxicant microcystin, which causes hyperphosphorylation of microfilaments and other cellular proteins (Toivola and Eriksson, 1999); (5) toxins that disrupt protein synthesis, such as α-amanitin and ricin; and (6) cholesterol-lowering drugs (statins) that inhibit HMG-coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway, and rarely cause myotoxicity.

The events leading to cell death after exposure to these chemicals are generally unknown. It is likely that cell death caused by these chemicals is ultimately mediated by impairment of oxidative phosphorylation, sustained elevation of intracellular Ca²⁺, and/or overproduction of ROS/RNS, and that it takes the form of necrosis if these processes are abrupt, but apoptosis if they are protracted. For example, direct injury of the plasma membrane would lead rapidly to increased intracellular Ca²⁺ levels. Neurofilamental toxins that block axonal transport cause energy depletion in the distal axonal segment. HMG-coenzyme A reductase inhibitors not only diminish the synthesis of cholesterol but may also compromise formation of other products of the mevalonate pathway. Such are the mitochondrial electron-transporting molecule ubiquinone (also called coenzyme Q) and isopentenyl pyrophosphate, which is utilized for isopentenylation of selenocysteinyl-tRNA, an obligatory step for synthesis of selenoproteins, including the antioxidant glutathione peroxidase (see Fig. 3-5) and thioredoxin reductase (see Fig. 3-22). These alterations may contribute to statin-induced myopathy and rhabdomyolysis.

Impairment of External Cellular Maintenance

Toxicants may also interfere with cells that are specialized to provide support to other cells, tissues, or the whole organism. Chemicals acting on the liver illustrate this type of toxicity. Hepatocytes produce and release into the circulation a number of proteins and nutrients. They remove cholesterol and bilirubin from the circulation, converting them into bile acids and bilirubin glucuronides, respectively, for subsequent excretion into bile. Interruption of these processes may be harmful to the organism, the liver, or both. For example, inhibition of hepatic synthesis of coagulation factors by coumarins does not harm the liver, but may cause death by hemorrhage (Hardman et al., 1995). This is the mechanism of the rodenticidal action of warfarin. In the fasting state, inhibitors of hepatic gluconeogenesis, such as hypoglycin (the ackee fruit-derived causative agent of Jamaican vomiting sickness; methylene cyclopropyl alanine), may be lethal by limiting the supply of glucose to the brain. Similarly, Reye syndrome, which is viewed as a hepatic mitochondrial injury caused by a combination of a viral disease (which may induce hepatic NOS) and intake of salicylate (which provokes MPT) (Fromenty and Pessayre, 1997; Kim et al., 2003), causes not only hepatocellular injury but also severe metabolic disturbances (hypoglycemia, hyperammonemia) that affect other organs as well. Chemical interference with the β-oxidation of fatty acids or the synthesis, assembly, and secretion of lipoproteins overloads the hepatocytes with lipids, causing hepatic dysfunction (Fromenty and Pessayre, 1997). α-Naphthylisothiocyanate (ANIT) causes separation of the intercellular tight junctions that seal bile canaliculi (Krell et al., 1987), impairing biliary secretion and leading to the retention of bile acids and bilirubin; this adversely affects the liver as well as the entire organism.

The fourth step in the development of toxicity is inappropriate repair and adaptation (Fig. 3-1). As noted previously, many toxicants alter macromolecules, which eventually cause damage at higher levels of the biological hierarchy in the organism. Progression of toxic lesions can be intercepted by repair mechanisms operating at molecular, cellular, and tissue levels (Fig. 3-21). Another strategy whereby the organism can resist the noxious chemical is by increasing its own readiness to cope with it and with its harmful effects. This phenomenon is called adaptation. Because the capacity of the organism to repair itself and adapt to the toxic exposure and effects is so important in determining the outcome of chemical exposure, the mechanisms of repair and adaptation will be discussed below.

Mechanisms of Repair

Molecular Repair

Damaged molecules may be repaired in different ways. Some chemical alterations, such as oxidation of protein thiols and methylation of DNA, are simply reversed. Hydrolytic removal of the molecule’s damaged unit or units and insertion of a newly synthesized unit or units often occur with chemically altered DNA and peroxidized lipids. In some instances, the damaged molecule is totally degraded and resynthesized. This process is time-consuming but unavoidable in cases such as the regeneration of cholinesterase after organophosphate intoxication.

Repair of Proteins

Thiol groups are essential for the function of numerous proteins, such as receptors, enzymes, cytoskeletal proteins, and TFs. Oxidation of protein thiols (Prot-SHs) to protein disulfides (Prot-SS, Prot₁-SS-Prot₂), protein–glutathione mixed disulfides (Prot-SSG), and protein sulfenic acids (Prot-SOH) can be reversed by reduction (Watson et al., 2004; Gravina and Mieyal, 1993) (Fig. 3-22). The endogenous reductants are thioredoxins and glutaredoxins, small, ubiquitous proteins with two redox-active cysteines in their active centers (Holmgren et al., 2005). These proteins as well as thioredoxin reductase have two isoenzymes; those labeled 1 are located in the cytosol, whereas those labeled 2 are mitochondrial. Because the catalytic thiol groups in these proteins become oxidized, they are reduced by NADPH, which is generated by NADP⁺-dependent isocitrate dehydrogenase localized in various cell compartments (cytosol, mitochondria, peroxisomes), as well as by the cytosolic glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway. ROS may oxidize methionines in proteins into sulfoxides (protein-Met-S=O), forming both the S and R epimers, which can be reduced by methionine sulfoxide reductase (Msr) A and B enzymes, respectively (Moskovitz, 2005). Msr enzymes can reverse this modification at the expense of oxidation of their catalytic cysteine to sulfenic acid (Msr-Cys-S-OH). This then reacts with a neighboring thiol (in MsrA), forming an intramolecular disulfide, or with glutathione (in some other Msr proteins), forming a protein–glutathione disulfide. Finally the disulfide enzyme is reduced by thioredoxin or glutaredoxin, respectively, with subsequent steps depicted in Fig. 3-22. Reduction of methionine sulfoxides in lens proteins (eg, α-crystallin) is especially critical for maintenance of the transparency of the eye lens. MsrA knockout mice develop cataract on repeated exposure to hyperbaric oxygen (Kantorow et al., 2010). Repair of oxidized hemoglobin (methemoglobin) occurs by means of electron transfer from cytochrome b₅, which is then regenerated by a NADH-dependent cytochrome b₅ reductase (also called methemoglobin reductase).

Soluble intracellular proteins such as cytosolic enzymes are typically folded into a globular form with their hydrophobic amino acid residues hidden inside, whereas the hydrophilic residues are located externally together with a hydrophobic cleft that constitutes the ligand (substrate) binding site. Physical or chemical insults may evoke an unduly large opening of this cleft that may lead to unfolding of the protein (denaturation) and its aggregation. Molecular chaperones such as the heat-shock proteins (Hsp, eg, Hsp90, Hsp70, and Hsp40) can prevent protein unfolding by “clamping down” onto the exposed hydrophobic region of their client protein, using the energy of ATP hydrolysis to execute their conformational change that entails this maneuver. According to a recent model (Pratt et al., 2010), Hsp90 plays the leading role in stabilizing the cleft and impeding further unfolding, which would eventually lead to degradation of the protein by the ubiquitin–proteasome system (UPS) as described below. Indeed, the benzoquinone ansamycin Hsp90 inhibitors (eg, geldanamycin and herbimycin) promote degradation of Hsp90 client proteins by the UPS.

When unfolding progresses beyond a limit, Hsp90 dissociates. This may occur in response to chemical-induced protein damage, such as that inflicted by mechanism-based enzyme inactivators that are converted into reactive metabolites that bind covalently to the catalytic site of the enzyme. Dissociation of Hsp90 from the unfolding protein allows Hsp70 and its co-chaperone Hsp40 to recruit the Hsp70-dependent E3 ubiquitin ligases (eg, CHIP and Parkin) that in turn direct ubiquitin-charged E2 enzymes to the Hsp70-bound client protein to tag it with a polyubiquitin chain as the final step in a process presented in Fig. 3-23 (Bedford et al., 2011). The protein tagged with a Lys48-linked polyubiquitin chain is then recognized and degraded in the proteasomes. The 26S proteasome is large barrel-shaped multiprotein complex that binds the ubiquitinated protein, deubiquitinates and unfolds it using ATP, and finally hydrolyzes the protein with its threonine-containing protease active sites into small peptides. The UPS controls the cellular level of numerous regulatory proteins (eg, p53, IκB, Nrf2, β-catenin, cyclins) and also has a prominent role in eliminating oxidized or otherwise damaged and unfolded intracellular proteins (Poppek and Grune, 2006). NOS exposed to its mechanism-based inactivator guanabenz that causes loss of tetrahydrobiopterin from the substrate-binding cleft will suffer this fate (Pratt et al., 2010). CYP2E1, in which the heme becomes cross-linked to the protein as a consequence of reductive dechlorination of CCl₄ to the trichloromethyl free radical by this enzyme, is also eliminated by proteasomal degradation.

Oligomerization and aggregation of damaged and unfolded proteins preclude the proteasome from degrading them; such substrates can even trap proteasomes, rendering them nonfunctional. After ubiquitination, protein aggregates can be eliminated by autophagy, a process described in more detail in the section “Cellular Repair.” Ubiquitin on the modified proteins is recognized and bound by autophagic adaptor proteins, such as p62 or Nbr1, which interact with LC3 to deliver them to autophagosomes, newly formed vesicles that encapsulate their protein cargo. After fusing of autophagosomes with lysosomes, the damaged protein is hydrolyzed by proteases. Lysosomal proteases degrade, for example, the immunogenic trifluoroacetylated proteins that are formed in the liver during halothane anesthesia (Cohen et al., 1997). Erythrocytes have ATP-independent, nonlysosomal proteolytic enzymes that rapidly and selectively degrade proteins denatured by HO^• (Davies, 1987). Red blood cells containing protein aggregates (Heinz body) after exposure to methemoglobin-forming chemicals are removed via phagocytosis by macrophages in the spleen.

Repair of Lipids

Phospholipids containing fatty acid hydroperoxides are preferentially hydrolyzed by phospholipase A2, with the peroxidized fatty acids replaced by normal fatty acids (van Kuijk et al., 1987). Peroxidized lipids (eg, fatty acid hydroperoxides and phospholipid-associated hydroperoxides) may be reduced by the glutathione peroxidase–glutathione–glutathione reductase system (see Fig. 3-24) or by the peroxiredoxin–thioredoxin–thioredoxin reductase system (shown in part in Fig. 3-22). Again, NADPH is needed to “repair” the reductants that are oxidized in the process.

Repair of DNA

Despite its high reactivity with electrophiles and free radicals, nuclear DNA is remarkably stable, in part because it is packaged in chromatin and because several repair mechanisms are available to correct alterations. The mitochondrial DNA, however, lacks histones and efficient repair mechanisms and therefore is more prone to damage. Different types of damages are corrected by specialized mechanisms, each employing a different set of repair proteins (Christmann et al., 2003).

Direct Repair

Certain covalent DNA modifications are directly reversed by enzymes such as DNA photolyase, which cleaves adjacent pyrimidines dimerized by UV light. Inasmuch as this chromophore-equipped enzyme uses the energy of visible light to correct damage, its function is restricted to light-exposed cells.

Minor adducts, such as methyl groups, attached to DNA bases by alkylating agents (eg, MMS) may be removed by special enzymes (Christmann et al., 2003). Such groups attached to the O⁶ position of guanine are cleaved off by O⁶-methyguanine-DNA-methyltransferase (MGMT). While repairing the DNA, this alkyltransferase sacrifices itself, transferring the adduct onto one of its cysteine residues. This results in its inactivation, ubiquitination, and proteasomal degradation. Thus, like glutathione, which is depleted during detoxication of electrophiles, MGMT is consumed during the repair of DNA. Methyl groups attached to N¹ of adenine and guanine, and N³ of thymine and cytosine are removed by oxidative demethylation catalyzed by DNA dioxygenases (ABH2 and ABH3). These peculiar O₂, Fe²⁺, ascorbate, and 2-oxoglutarate-dependent enzymes oxygenate the methyl group adduct, which in turn leaves as formaldehyde, whereas 2-oxoglutarate is oxidatively decarboxylated to succinate.

Excision Repair

Base excision and nucleotide excision are two mechanisms for removing damaged bases from DNA (see Chaps. 8 and 9). Lesions that do not cause major distortion of the helix typically are removed by base excision, in which the altered base is recognized by a relatively substrate-specific DNA glycosylase that hydrolyzes the N-glycosidic bond, releasing the modified base and creating an apurinic or apyrimidinic (AP) site in the DNA. For example, 8-OH-Gua, a major mutagenic product of oxidative stress, is removed from the DNA by a specific 8-OH-Gua DNA glycosylase. The AP site is recognized by the AP endonuclease, which hydrolyzes the phosphodiester bond adjacent to the abasic site. After its removal, the abasic sugar is replaced with the correct nucleotide by a DNA polymerase and is sealed in place by a DNA ligase. Interestingly, AP endonuclease is a bifunctional protein and is also called redox factor-1 (Ref-1). In concert with thioredoxin and thioredoxin reductase, it maintains TFs with sensitive thiol groups in their DNA-binding domain (Fos, Jun, NF-κB) in an active reduced state (Hansen et al., 2006).

Bulky lesions such as adducts produced by aflatoxins or aminofluorene derivatives and dimers caused by UV radiation are removed by nucleotide excision repair system, which consists of approximately 30 proteins (Christmann et al., 2003). Lesions in the nontranscribed strands or the nontranscribed regions of the genome are corrected by the global genomic repair system. This involves proteins that recognize the distorted double helix at the lesion, unwind the DNA, and excise a number of intact nucleotides on both sides of the lesion together with the one containing the adduct. The excised section of the strand is restored by insertion of nucleotides into the gap by DNA polymerase and ligase, using the complementary strand as a template. Lesions in the transcribed DNA strand blocking the RNA polymerase in the actively transcribed genes are removed by another variation of nucleotide excision repair, the transcription-coupled repair system. This involves assembly of repair proteins to remove the stalled RNA polymerase before excision of the damage and filling the gap. Resynthesis of the removed section of the strand is designated “unscheduled DNA synthesis” and can be detected by the appearance of altered deoxynucleosides in urine. Excision repair has a remarkably low error rate of less than one mistake in 10⁹ bases repaired.

PARP appears to be an important contributor to excision repair. On base damage or SSB, PARP binds to the injured DNA and becomes activated. The active PARP cleaves NAD⁺ to use the ADP-ribose moiety of this cofactor for attaching long chains of polymeric ADP-ribose to nuclear proteins, such as histones. Because one ADP-ribose unit contains two negative charges, the poly(ADP-ribosyl)ated proteins accrue negativity and the resultant electrorepulsive force between the negatively charged proteins and DNA causes decondensation of the chromatin structure. It is hypothesized that PARP-mediated opening of the tightly packed chromatin allows the repair enzymes to access the broken DNA and fix it. Thereafter, poly(ADP-ribose) glycohydrolase gains access to the nucleus from its perinuclear localization and reverses the PARP-mediated modification of nuclear proteins (D’Amours et al., 1999). Other features of PARP that are relevant in toxicity—such as destruction of PARP by caspases during apoptosis as well as the significance of NAD⁺ (and consequently ATP) wasting by PARP in necrosis—have been discussed earlier in this chapter.

Nonhomologous End Joining

This process repairs DSB that may be formed when two SSB occur in close proximity, or when DNA with SSB undergoes replication. This repair system directly ligates the broken ends without the need for a homologous template. DSBs are recognized by the Ku protein (a heterodimer of Ku70 and Ku80) that binds to the DNA end. Ku then binds and activates DNA-dependent protein kinase catalytic subunit (DNA-PKcs), leading to recruitment and activation of end-processing enzymes, DNA polymerases, and DNA ligase. Although the nonhomologous end joining is error-prone, it can operate in any phase of the cell cycle and is the mechanism for DSB repair in nondividing terminally differentiated cells (eg, neurons). By contrast, the other and more faithful mechanism for DSB repair, homologous recombination, can function only after replication (in S and G2 phases), when sister chromatid sequences are available for use as templates (see below), and thus is not an option in nondividing cells.

Recombinational (or Postreplication) Repair

Homologous recombination can be used to fix postreplication gaps and DSBs (Li and Heyer, 2008). The former defect may result when excision of a bulky adduct or an intrastrand pyrimidine dimer fails to occur before DNA replication begins. At replication, such a lesion prevents DNA polymerase from polymerizing a daughter strand along a sizable stretch of the parent strand that carries the damage. Then the replication results in two homologous (“sister”) yet dissimilar DNA duplexes; one has a large postreplication gap in its daughter strand and an intact duplex synthesized at the opposite leg of the replication fork. This intact sister duplex is utilized to complete the postreplication gap in the damaged sister duplex. This is accomplished by recombination (“crossover”) of the appropriate strands of the two homologous duplexes. After separation, the sister duplex that originally contained the gap carries in its daughter strand a section originating from the parent strand of the intact sister, which in turn carries in its parent strand a section originating from the daughter strand of the damaged sister. This strand recombination explains the phenomenon of “sister chromatid exchange,” which is indicative of DNA damage corrected by recombinational repair. After completing the postreplication gap by recombination, the damage still present in the parent strand may be removed by nucleotide excision.

In addition to eliminating postreplication gaps, homologous recombination can also repair DSB in a complex process employing numerous proteins. These include, for example, the MRN complex (the trimer of Mre11, Rad50, and Nbs1 proteins, which binds to the DSB and is involved in end processing by generation of single-stranded DNA), Rad51, Rad54, and the breast cancer susceptibility proteins BRCA1 and BRCA2 (which assist the single-stranded DNA in invading the undamaged template in an ATP-dependent process), as well as polymerases, nucleases, helicases, and other components, which mediate DNA ligation and substrate resolution. A combination of excision and recombinational repairs occurs in restoration of DNA with interstrand cross-links caused by bifunctional electrophiles, such as nitrogen mustard-type drugs and cisplatin.

Cellular Repair

Autophagic removal of damaged cell organelles may be viewed as a universal mechanism of cellular repair, whereas clearance and regeneration of damaged axons is a mechanism specific for nerve cells.

Autophagy of Damaged Cell Organelles

Cells suffering mild injury may repair themselves by removing and degrading damaged components, such as organelles and protein aggregates, in a process called autophagy, meaning eating of self (Rabinowitz and White, 2010). While autophagy serves the purpose of cell restitution in all cells, it is particularly important in terminally differentiated cells, such as neurons and myocytes, where cell renewal by cell replication is not possible. In addition, autophagy of normal cell constituents is an ultimate means of nutrient generation (see the section “Adaptation to Energy Depletion—The Energy Stress Response”).

In autophagy, a so-called isolation membrane (or phagophore) emerges possibly from the EPR, which engulfs the cytoplasmic material and then encapsulates it in a double-membrane vesicle (called autophagosome). This vesicle moves along microtubules, driven by dynein motors, to the lysosome and on fusing with the lysosome generates an autolysosome. The cargo as well as the inner membrane of the original autophagosome is then degraded by lysosomal hydrolytic enzymes, such as proteases, lipases, nucleases, and glycosidases (which work optimally at the acidic pH in the lysosomes) to amino acids, lipids, nucleosides, and carbohydrates. These breakdown products are then released by lysosomal permeases and transporters into the cytosol, where they may be further metabolized or used for synthesis of new macromolecules.

Autophagic removal of damaged cell organelles is a method of their quality control, disposal and recycling. This is well exemplified by deletion of depolarized mitochondria through selective autophagy or mitophagy in cells exposed to mitochondrial poisons, such as the protonophoric uncoupler CCCP, valinomycin, and paraquat (see Table 3-6). Several proteins mediate this process (Mehrpour et al., 2010; Youle and Narendra, 2011), including (1) Pink1 (a protein kinase) localized in the MOM that accumulates there on dissipation of the mitochondrial membrane potential, (2) the cytosolic Parkin (an E3 ubiquitin ligase) that is recruited to the mitochondria by Pink1 and that then polyubiquitinates itself and other proteins, among them (3) the MOM protein mitofusins (Mfn1, Mfn2) and voltage-dependent anion channel (VDAC), (4) p62 (an autophagic adaptor protein) that binds to both ubiquitinated proteins and to (5) the cytosolic LC3 (microtubule-associated protein light chain 3), which, after cleavage to LC3-I, becomes conjugated with phosphatidylethanolamine by (6) a ubiquitin-like conjugation system that requires Atg7 (E1-like), Atg3 (E2-like), and the Atg12-Atg5:Atg16L complex (E3-like), (7) the phosphatidylinositol 3-kinase vesicular protein sorting 34 (Vps34), which is activated by interacting with (8) Beclin 1 (Bcl-2-interacting protein-1). The lipidated LC-3 (called LC3-II) can integrate into the membrane of autophagosome and can recruit the ubiqutin-tagged mitochondria into this structure. The latter protein (which later decomposes in the lysosome) is a marker for autophagosomes.

Removal of damaged mitochondria by autophagy is important not only for maintaining a functional mitochondrial pool but also for limiting oxidative cell damage and preventing apoptosis because injured mitochondria may be sources of ROS and apoptotic factors, such as cyt c, Smac, and AIF (see Fig. 3-20). Indeed, overexpression of Parkin suppresses cell death (Tanaka, 2010). In contrast, loss-of-function mutations in the genes encoding Pink1 and Parkin cause early onset monogenic forms of Parkinson disease probably through defective mitophagy of damaged mitochondria in the dopaminergic nigrostriatal neurons (Youle and Narendra, 2011). Autophagy also contributes to structural restitution of liver cells. For example, leftover peroxisomes after exposure to peroxisome proliferators (eg, fibrate esters) and lipid droplets in hepatic steatosis are also cleared by autophagy.

Regeneration of Damaged Axons

Peripheral neurons with axonal damage can regenerate their axons with the assistance of macrophages and Schwann cells. Macrophages remove debris by phagocytosis and produce cytokines and growth factors, which activate Schwann cells to proliferate and transdifferentiate from myelinating operation mode into a growth-supporting mode. Distal to the injury, Schwann cells play an indispensable role in promoting axonal regeneration by increasing their synthesis of cell adhesion molecules (eg, N-CAM), by elaborating ECM proteins for base membrane construction, and by producing an array of neurotrophic factors (eg, brain-derived neurotrophic factor, glial cell line–derived neurotrophic factor, and nerve growth factor) and their receptors (Boyd and Gordon, 2003). While comigrating with the regrowing axon, Schwann cells physically guide as well as chemically lure the axon to reinnervate the target cell.

In the mammalian central nervous system, axonal regrowth is prevented partly by the nonpermissive external environment. At the site of injury, the oligodendrocytes produce growth inhibitory myelin-associated proteins (eg, Nogo, NI 35, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, ephrin, and semaphorin), which act through specific receptors, whereas the astrocytes produce chondroitin sulfate proteoglycans and fibrotic scar (whose components include laminin, fibronectin, and collagen IV) (Johnson, 1993) that hinder axonal regrowth. Signaling by TGF-β through Smad2 TF (see pathway #8 in Fig. 3-12) plays a leading role in overexpression of these extrinsic growth inhibitory factors. In addition, CNS neurons lose their intrinsic growth capacity postnatally, which occurs simultaneously with increased expression of some neuronal growth inhibitory Kröppel-like TFs, such as KLF4. For these reasons, damage to central neurons is irreversible but is compensated for in part by the large number of reserve nerve cells that can take over the functions of lost neurons. For example, in Parkinson disease, symptoms are not observed until there is at least an 80% loss of nigrostriatal neurons.

Tissue Repair

In tissues with cells capable of multiplying, damage is reversed by deletion of the injured cells and regeneration of the tissue by proliferation. The damaged cells are eliminated by apoptosis or necrosis.

Apoptosis: An Active Deletion of Damaged Cells

Apoptosis initiated by cell injury can serve as tissue repair for two reasons: first, because it may intercept the process leading to necrosis, as discussed earlier (see Fig. 3-20). Necrosis is a more harmful sequel than apoptosis for the tissue in which the injured cell resides. A cell destined for apoptosis shrinks; its nuclear and cytoplasmic materials condense, and then it breaks into membrane-bound fragments (apoptotic bodies) that are phagocytosed (Bursch et al., 1992). During necrosis, cells and intracellular organelles swell and disintegrate with membrane lysis. Whereas apoptosis is orderly, necrosis is a disorderly process that ends with cell debris in the extracellular environment. The constituents of the necrotic cells attract aggressive inflammatory cells, and the ensuing inflammation amplifies cell injury (see further on). With apoptosis, dead cells are removed without inflammation. Second, apoptosis may intercept the process leading to neoplasia by eliminating the cells with potentially mutagenic DNA damage. This function of apoptosis is discussed in more detail in the final section of this chapter.

It must be emphasized, however, that apoptosis of damaged cells may serve tissue restoration only for tissues that are made up of constantly renewing cells (eg, the bone marrow, the respiratory and GI epithelium, and the epidermis of the skin), or of conditionally dividing cells (eg, hepatic and renal parenchymal cells), because in these tissues the apoptotic cells are readily replaced. The role of apoptosis as a tissue repair strategy is markedly lessened in organs containing nonreplicating and nonreplaceable cells, such as the neurons, cardiac muscle cells, and female germ cells, because deletion of such cells, if extensive, can cause a deficit in the organ’s function. Apoptosis may also be harmful when it occurs at a critical location. For example, in the pulmonary alveolar epithelium, an extremely tight barrier, apoptosis could cause flooding of the alveolar space with interstitial fluid, a potentially lethal outcome.

Proliferation: Regeneration of Tissue

Tissues are composed of various cells and the ECM. Tissue elements are anchored to each other by transmembrane proteins. Cadherins allow adjacent cells to adhere to one another, whereas connexins associate into tubular structures and connect neighboring cells internally (gap junctions). Integrins link cells to the ECM. Therefore, repair of injured tissues involves not only regeneration of lost cells and the ECM but also reintegration of the newly formed elements. In parenchymal organs such as liver, kidney, and lung, various types of cells are involved in the process of tissue restoration. Nonparenchymal cells of mesenchymal origin residing in the tissue, such as resident macrophages and endothelial cells, and those migrating to the site of injury, such as blood monocytes, produce factors that stimulate parenchymal cells to divide and stimulate some specialized cells (eg, the stellate cells in the liver) to synthesize ECM molecules.

Replacement of Lost Cells by Mitosis

Soon after injury, cells adjacent to the damaged area enter the cell division cycle (Fig. 3-25). Enhanced DNA synthesis is detected experimentally as an increase in the labeling index (which is the proportion of cells that incorporate administered ³H-thymidine or bromodeoxyuridine into their nuclear DNA during the S phase of the cycle), or by increased expression of proliferating cell nuclear antigen (PCNA, a trimeric protein that functions as a sliding clamp to hold DNA polymerase-δ to the template DNA strand during DNA replication). Also, mitotic cells can be observed microscopically. As early as 2 to 4 hours after administration of a low dose of carbon tetrachloride to rats, the mitotic index in the liver increases dramatically, indicating that cells already in the G₂ phase progress rapidly to the M phase. The mitotic activity of the hepatocytes culminates at 36 to 48 hours, after a full transit through the cycle, indicating that quiescent cells residing in G₀ enter and progress to mitosis (M). Peak mitosis of nonparenchymal cells occurs later, after activation and replication of parenchymal cells. In severe toxic liver injury, when hepatocyte replication is impaired (eg, in rats dosed with galactosamine or acetylaminofluorene, and in humans intoxicated with acetaminophen), restoration of the liver may depend on stem cell–derived cells, called oval cells, which are located in the terminal bile ductules. These cells proliferate and differentiate into both hepatocytes and biliary epithelial cells (Fausto et al., 2006; Vessey and Hall, 2001). As oval cells produce α-fetoprotein, the increase in serum α-fetoprotein levels indicates an improved outcome of acetaminophen-induced injury. Hepatic sinusoidal endothelial cells originate from bone marrow progenitors and when they are chemically injured (eg, by monocrotaline) bone marrow–derived precursor cells move into the sinusoids and contribute to their replacement, as demonstrated in a rat model of hepatic sinusoidal obstruction syndrome (Harb et al., 2009). Accordingly, bone marrow infusion ameliorates, and myelosuppression aggravates, such liver injury. In an ozone-exposed lung, the nonciliated Clara cells and type II pneumocytes undergo mitosis and terminal differentiation to replace, respectively, the damaged ciliated bronchial epithelial cells and type I pneumocytes (Mustafa, 1990). In some tissues, such as intestinal mucosa and bone marrow, stem cells first divide to provide self-renewal and then differentiate to replace more mature cells lost through injury.

Sequential changes in gene expression occur in cells that are destined to divide. In rats subjected to partial hepatectomy to study regeneration of the liver, oligonucleotide microarray analysis revealed that more than 150 genes are involved in the early gene response with upregulation or downregulation (Su et al., 2002). The overexpressed genes include those that code for TFs important in proliferative signaling, such as c-fos, c-jun, Egr1, and c-myc (see Fig. 3-12), the genes of the antiapoptotic protein Bcl-X_L (see Fig. 3-19), and that of mdm2 that constrains p53, a proapoptotic and cell cycle inhibitory TF (see Fig. 3-32). Interestingly, some genes whose products decelerate the cell cycle also become temporarily overexpressed (eg, the cyclin-dependent kinase inhibitor p21 and gadd45; see Fig. 3-32), suggesting that this duality keeps tissue regeneration precisely regulated. Nevertheless, the genetic expression is apparently reprogrammed so that DNA synthesis and mitosis gain priority over specialized cellular activities. For example, as a result of dedifferentiation, regenerating hepatocytes underexpress cytochrome P450, N-acetyltransferase-2, as well as PPARα, and hepatic stellate cells cease to accumulate fat and vitamin A.

It has been speculated that the regenerative process is initiated by the release of chemical mediators from damaged cells. The nonparenchymal cells, such as resident macrophages and endothelial cells, are receptive to these chemical signals and produce a host of secondary signaling molecules, cytokines, and growth factors that promote and propagate the regenerative process (Fig. 3-26). In rodents subjected to partial hepatectomy, the initial or priming phase of liver regeneration is controlled by the cytokines TNF and IL-6, whose hepatic mRNA and serum levels increase. TNF originates from the Kupffer cells. This cytokine acts on these macrophages in an autocrine manner, activating its receptor (item 2 in Fig. 3-12) and the coupled signal transducing network. This in turn causes activation of NF-κB, which increases IL-6 expression. The secreted IL-6 then acts on the hepatocytes and through its receptor (item 1 in Fig. 3-12) activates Janus kinase (JAK) and induces TFs (eg, Stat3, C/EBPβ), which activate several target genes. This cytokine network promotes transition of the quiescent liver cells (G₀) into cell cycle (G₁) and makes them receptive to growth factors (“priming”). Growth factors, especially the hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α), and heparin-binding epidermal growth factor–like growth factor (HB-EGF), initiate the progression of the “primed” cells in the cycle toward mitosis (Costa et al., 2003; Fausto et al., 2006). Despite its name, neither the formation nor the action of HGF is restricted to the liver. It is produced by resident macrophages and endothelial cells of various organs—including liver, lung, and kidney—and in a paracrine manner activates receptors on neighboring parenchymal cells (Fig. 3-26). In rats intoxicated with carbon tetrachloride, the synthesis of HGF in hepatic and renal nonparenchymal cells increases markedly (Noji et al., 1990) and HGF levels in blood rise rapidly (Lindroos et al., 1991). The communication between parenchymal and nonparenchymal cells during tissue repair is mutual. For example, TGF-α, a potent mitogen produced by regenerating hepatocytes, acts both as an autocrine and a paracrine mediator on liver cells as well as on adjacent nonparenchymal cells (Fig. 3-26). By activating their receptors (item 4 in Fig. 3-12), these growth factors initiate signaling through the MAPK pathway and the PI3K–Akt pathway (Fig. 3-12), thereby mediating activation of TFs (c-Jun, c-Fos, c-Myc, FoxM1B, NF-κB, Stat3). These, among others, induce cyclins and the protein phosphatase cdc25, two groups of short-lived regulatory proteins. Then cyclins activate Cdks by associating with them (Fig. 3-25), whereas cdc25 activates Cdk1 and Cdk2 by dephosphorylating their 2 amino acid residues (Thr14 and Tyr15). The activated Cdks accelerate the cell cycle mainly by phosphorylation of pRb. This in turn releases the TF E2F, which induces enzymes and regulatory proteins needed for cell cycle progression (Fig. 3-32). The growth factor signaling also activates mTOR that upregulates mRNA translation, thereby meeting the demand for increased protein synthesis (see Fig. 3-29). Similar signaling appears to mediate regeneration of the S-(1,2-dichlorovinyl)-l-cysteine-injured kidney that exhibits increased expression of the cytokine IL-6, the growth factors TGF-α and HB-EGF, the growth factor receptors EGFR (also receptor for TGF-α) and IGF-1R, and the MAP kinase isoform Erk1 (Vaidya et al., 2003).

Although the cytotokine- and growth factor–controlled mitotic cell replacement is likely an essential part in the repair of most tissues built up of cells with proliferative capacity, there are also tissue-specific features of tissue repair. For example in the liver, bile acids also stimulate hepatocyte proliferation through their nuclear receptor, FXR (Huang et al., 2006). In mice subjected to partial hepatectomy, liver regrowth is hastened by bile acid feeding, but is markedly delayed when FXR is deleted or recirculation of bile acids from the intestine to the liver is prevented. Epithelia composed of a single cell layer form important barriers; therefore, replacement of mortally injured epithelial cells, which become detached from the basement membrane, is an urgent need. This can be achieved more rapidly by cell migration than by mitotic cell replacement. For example, in the damaged mucosa of the GI tract, cells of the residual epithelium rapidly migrate to the site of injury as well as elongate and become thin to reestablish the continuity of the surface even before this could be achieved by cell replication. Cell movement involves orderly dissociation of cadherin-mediated cell–cell contacts (involving β-catenin phosphorylation) and integrin-mediated cell–ECM contacts at focal adhesions (involving FAK phosphorylation), assembly of actin stress fibers, and formation of lamellopodia (cell projection filled with F-actin meshwork). Mucosal repair is dictated not only by growth factors and cytokines operative in tissue repair elsewhere but also by trefoil factors (TFFs). TFFs are small (7–12 kDa) protease-resistant proteins that are abundantly secreted from specific mucosal cells (eg, intestinal goblet cells) and are associated with the mucous layer of the GI tract (Taupin and Podolsky, 2003). TFF expression is rapidly upregulated at the margins of mucosal injury by cytokine and growth factor signaling. Whereas growth factors (eg, HGF and EGF) exert both motogenic (motility-increasing) and mitogenic effects on enterocytes, TFFs are potent motogens, but are not mitogens. Although TFFs do not act alone, they are the only peptides shown to be essential for restitution of the injured intestinal mucosa. Normal mice exposed to dextran sodium sulfate in the drinking water develop diffuse colonic mucosal injury, some exhibiting bloody diarrhea. In contrast, the majority of TFF3-null mice develop frank bloody diarrhea and die in response to dextran sodium sulfate. The immediate target molecule (eg, receptor) for TFFs remains unknown. Migration of the surviving cells also precedes mitotic cell replacement in the tubular epithelium of the injured kidney. It appears that ECM components, such as collagen IV, beneath the tubular epithelial cells aid in the restitution of the injured epithelium and reestablishment of its polarity (Nony and Schnellmann, 2003).

Replacement of the Extracellular Matrix

The ECM is composed of proteins, glycosaminoglycans, and the glycoprotein and proteoglycan glycoconjugates (Gressner, 1992). In liver, these molecules are synthesized by stellate or fat-storing cells located in the space of Disse, between the hepatic sinusoid and the hepatocytes (Fig. 3-26). The stellate cells become activated during liver regeneration, undergoing mitosis and major phenotypic changes. The latter changes include not only increased synthesis and secretion of ECM constituents but also expression of α-smooth muscle actin as well as the myogenic TF MyoD and loss of fat, vitamin A, and PPARγ content. Thus, resting stellate cells become transdifferentiated into myofibroblast-like contractile and secretory cells. Activation of stellate cells is mediated chiefly by 2 growth factors—platelet-derived growth factor (PDGF) and TGF-β (Fig. 3-26). Both may be released from platelets (which accumulate and degranulate at sites of injury) and later from the activated stellate cells themselves. The main sources of TGF-β, however, are the neighboring tissue macrophages residing in the hepatic sinusoids (Gressner, 1992). A dramatic increase in TGF-β mRNA levels in Kupffer cells is observed with in situ hybridization after carbon tetrachloride–induced hepatic necrosis. Proliferation of stellate cells is induced by the potent mitogen PDGF. Through its receptor (like item 4 in Fig. 3-12), PDGF activates the PI3K–Akt pathway, which not only signals for proliferation but also inactivates the FoxO1 TF, which would halt the cell cycle in G1 phase by increasing the expression of p27, a cyclin-dependent kinase inhibitor. TGF-β acts on the stellate cells to induce their transdifferentiation and to stimulate the synthesis of ECM components, including collagens, fibronectin, tenascin, and proteoglycans. TGF-β acts through its Ser/Thr kinase receptor (item 8 in Fig. 3-12), which phosphorylates the TFs Smad2 and 3 (Flanders, 2004). TGF-β also plays a central role in ECM formation in other tissues. In kidney and lung, for example, TGF-β targets the mesangial cells and the septal fibroblasts, respectively. Remodeling of the ECM is aided by matrix metalloproteinases, which hydrolyze specific components of the matrix, as well as by tissue inhibitors of matrix metalloproteinases. The former group of these proteins originates from various types of nonparenchymal cells, including inflammatory cells; however, their inhibitors are mainly produced by stellate cells (Arthur et al., 1999).

The way in which tissue regeneration is terminated after repair is unclear, but the gradual dominance of TGF-β, which is a potent antimitogen and apoptogen, over mitogens is a contributing factor in the termination of cell proliferation. ECM production may be halted by an intracellular negative feedback mechanism in the ECM-producing cells, that is, by induction of Smad7 (an inhibitory Smad), which competitively inhibits phosphorylation of the receptor-activated Smads (Smad2 and 3). In addition, extracellular products of the proliferative response, such as the proteoglycan decorin and the positive acute-phase protein α₂-macroglobulin, can bind and inactivate TGF-β; thereby they may contribute to its silencing (Gressner, 1992).

Side Reactions to Tissue Injury

In addition to mediators that aid in the replacement of lost cells and the ECM, resident macrophages and endothelial cells activated by cell injury also produce other mediators that induce ancillary reactions with uncertain benefit or harm to tissues (Fig. 3-26). Such reactions include inflammation, altered production of acute-phase proteins, and generalized reactions such as fever.

Inflammation—Leukocyte Invasion

Alteration of the microcirculation and accumulation of inflammatory cells are the hallmarks of inflammation. These processes are largely initiated by resident macrophages secreting cytokines, such as TNF and interleukin-1 (IL-1), in response to tissue damage (Baumann and Gauldie, 1994) (Fig. 3-26). These cytokines, in turn, stimulate neighboring stromal cells, such as the endothelial cells and fibroblasts, to release mediators that induce dilation of the local microvasculature and cause permeabilization of capillaries. Activated endothelial cells also facilitate the egress of circulating leukocytes into the injured tissue by releasing chemoattractants and expressing cell adhesion molecules, which are cell surface glycoproteins (Jaeschke, 1997). One group of cell adhesion molecules, called selectins, located on the membrane of endothelial cells, interact with their ligands on the surface of leukocytes, thereby slowing down the flow of these cells and causing them to “roll” on the capillary surface. Subsequently a stronger interaction (adhesion) is established between the endothelial cells and leukocytes with participation of intercellular adhesion molecules (eg, ICAM-1) expressed on the endothelial cell membrane and integrins expressed on the membrane of leukocytes. This interaction is also essential for the subsequent transendothelial migration of leukocytes. This is facilitated by gradients of chemoattractants that induce expression of leukocyte integrins. Chemoattractants originate from various stromal cells and include chemotactic cytokines (or chemokines), such as the MCP-1 and IL-8 (whose rat homologue is the CINC-1), as well as lipid-derived compounds, such as platelet-activating factor (PAF) and leukotriene B₄ (LTB₄). Ultimately all types of cells in the vicinity of injury express ICAM-1, thus promoting leukocyte invasion; the invading leukocytes also synthesize mediators, thus propagating the inflammatory response. Production of most inflammatory mediators is induced by signaling, turned on by TNF and IL-1, which results in activation of TFs, notably NF-κB and C/EBP (Poli, 1998) (see Fig. 3-12). Genes of many of the proteins mentioned above (eg, selectins, ICAM-1, MCP-1, IL-8) and below (eg, inducible NOS, acute-phase proteins) as well as the genes of TNF and IL-1 themselves contain binding sites for the NF-κB (Lee and Burckart, 1998).

Inflammation—ROS and RNS Production

Macrophages, as well as leukocytes, recruited to the site of injury undergo a respiratory burst, discharging free radicals and enzymes (Weiss and LoBuglio, 1982) (Fig. 3-26). Free radicals are produced in the inflamed tissue in three ways, each of which involves a specific enzyme: Nox, NOS, or myeloperoxidase.

Nox is an electron-transporting protein complex composed of two transmembrane proteins (one of them the FAD- and heme-containing catalytic subunit) and 4 cytoplasmic proteins (including the G-protein Rac) (El-Benna et al., 2005). In resting cells Nox is dormant; however, in activated cells the cytoplasmic subunits become extensively phosphorylated and move to the membrane to assemble the Nox complex. Constituents of microorganisms, such as the bacterial LPS (the active endotoxin component of gram-negative bacteria), acting through the cell surface Toll-like receptors (TLR), and PKC activators, such as phorbol miristate acetate (PMA), may evoke Nox activation. Unlike in nonphagocytic cells, in macrophages and granulocytes activation causes a sudden and rapid electron transfer from NADPH through the FAD and heme in Nox to the molecular oxygen, releasing the thus formed superoxide anion radical (Display Formula$O2•¯$) in a burst (“respiratory burst”) at the external membrane surface into the phagocytic vacuole:

The Display Formula$O2•¯$ can give rise to the hydroxyl radical (HO^•) in two sequential steps: the first is spontaneous or is catalyzed by SOD, and the second, the Fenton reaction, is catalyzed by transition metal ions (see also Fig. 3-4):

Macrophages, but not granulocytes, generate another cytotoxic free radical, nitric oxide (^•NO). This radical is produced from arginine by NOS (Wang et al., 1993), which is inducible in macrophages by bacterial endotoxin and the cytokines IL-1 and TNF:

l-Arginine + O₂ → l-citrulline + ^•NO.

Subsequently, Display Formula$O2•¯$ and ^•NO, both of which are products of activated macrophages, can react with each other, yielding peroxynitrite anion; on reaction with carbon dioxide, this decays into two radicals, nitrogen dioxide and carbonate anion radical (Fig. 3-4):

Granulocytes, but not macrophages, discharge the lysosomal enzyme myeloperoxidase into engulfed extracellular spaces, the phagocytic vacuoles (Wang et al., 1993). Myeloperoxidase catalyzes the formation of HOCl, a powerful oxidizing agent, from HOOH and chloride ion:

HOOH + H⁺ + Cl⁻ → HOH + HOCl.

Like HOOH, HOCl can form HO^• as a result of electron transfer from Display Formula$O2•¯$ Fe²⁺ or from to HOCl:

All these reactive chemicals, as well as the discharged lysosomal proteases, are destructive products of inflammatory cells. Although these chemicals exert antimicrobial activity at the site of microbial invasion, at the site of toxic injury they can damage the adjacent healthy tissues and thus contribute to propagation of tissue injury (see the section “Tissue Necrosis”). Moreover, in some chemically induced injuries, inflammation plays the leading role. For example, ANIT, a cholestatic chemical, causes neutrophil-dependent hepatocellular damage. ANIT apparently acts on bile duct epithelial cells, causing them to release chemoattractants for neutrophil cells, which on invading the liver, injure hepatocytes (Hill et al., 1999). Kupffer cell activation, TNF release, and subsequent inflammation are also prominent and causative events in galactosamine-induced liver injury in rats (Stachlewitz et al., 1999).

Whereas it is well recognized that chemical-inflicted tissue injury can induce inflammation as a side reaction of tissue repair, it is becoming clear that inflammation (even if harmless alone) can precipitate an overt tissue injury on chemical exposure that is noninjurious alone. For example, a small harmless quantity of the macrophage activator LPS converts the nontoxic doses of monocrotaline, aflatoxin B₁, and allyl alcohol into markedly hepatotoxic doses (Roth et al., 2003). Moreover, in rats pretreated with LPS, unlike in untreated animals, chlorpromazine, ranitidine, and trovafloxacin caused liver injury. These drugs (and many others) when given to patients can induce rare, unexpected, and not obviously dose-related liver injury. Therefore, it is hypothesized that such idiosyncratic drug reactions develop when some endotoxin exposure decreases the threshold for drug toxicity by priming the Kupffer cells that produce ROS and inflammatory mediators discussed above. Manifest or subclinical infection, GI disturbance, or alcohol consumption (which greatly increases the intestinal permeability for endotoxin) may be the source of endotoxin. It is not surprising that most idiosyncratic drug reactions affect the liver, because this organ contains 80% to 90% of the body’s fixed macrophages (ie, Kupffer cells), because the liver is the first organ to be exposed to LPS translocating from the intestinal lumen, and because the Kupffer cells not only are activated by LPS but also remove it from the circulation, thereby protecting other organs from its inflammatory effects (Roth et al., 2003). However, the causative relationship between inflammation and idiosyncrasy needs further substantiation.

Altered Protein Synthesis: Acute-Phase Proteins

Cytokines released from macrophages and endothelial cells of injured tissues also alter protein synthesis, predominantly in the liver (Baumann and Gauldie, 1994) (Fig. 3-21). Mainly IL-6 but also IL-1 and TNF act on cell surface receptors and increase or decrease the transcriptional activity of genes encoding certain proteins called positive and negative acute-phase proteins, respectively, utilizing primarily the TFs NF-κB, C/EBP, and STAT (Poli, 1998; see Fig. 3-15). Many of the hepatic acute-phase proteins, such as C-reactive protein and hepcidin, are secreted into the circulation, and their elevated levels in serum are diagnostic of tissue injury, inflammation, or neoplasm. Increased sedimentation of red blood cells, which is also indicative of these conditions, is due to enrichment of blood plasma with positive acute-phase proteins such as fibrinogen.

Apart from their diagnostic value, positive acute-phase proteins may play roles in minimizing tissue injury and facilitating repair. For example, many of them, such as α₂-macroglobulin and α₁-antiprotease, inhibit lysosomal proteases released from the injured cells and recruited leukocytes. Haptoglobin binds hemoglobin in blood, MT complexes metals in the cells, heme oxygenase oxidizes heme to biliverdin, and opsonins facilitate phagocytosis. Thus, these positive acute-phase proteins may be involved in the clearance of substances released on tissue injury. Induction of hepcidin, which triggers degradation of the iron exit channel ferroportin (see the section “Mechanisms of Adaptation”) and thus decreases the duodenal Fe²⁺ absorption and Fe²⁺ export from macrophages, may be protective by limiting iron availability in the circulation for pathogens and by minimizing the Fe²⁺-catalyzed Fenton reaction (Fig. 3-4), the source of reactive hydroxyl radicals that may cause tissue injury.

Negative acute-phase proteins include some plasma proteins, such as albumin, transthyretin, and transferrin, as well as hepatic enzymes (eg, several forms of cytochrome P450 and glutathione S-transferase), ligand-activated TFs (eg, PPARα and the bile acid receptor FXR), and transporters, such as bile acid transporters at the sinusoidal and canalicular membrane of hepatocytes (NTCP and bile salt export pump [BSEP], respectively) and the bile canalicular export pump Mrp2. Because the latter enzymes and transporters play important roles in the toxication, detoxication, and excretion of endobiotics and xenobiotics, the disposition and toxicity of bile acids and toxicants may be altered markedly during the acute phase of tissue injury.

Although the acute-phase response is phylogenetically preserved, some of the acute-phase proteins are somewhat species-specific. For example, during the acute phase of tissue injury or inflammation, C-reactive protein and serum amyloid A levels dramatically increase in humans but not in rats, whereas the concentrations of α₁-acid glycoprotein and α₂-macroglobulin increase markedly in rats but only moderately in humans.

Generalized Reactions

Cytokines released from activated macrophages and endothelial cells at the site of injury also may evoke neurohumoral responses. Thus, IL-1, TNF, and IL-6 alter the temperature set point of the hypothalamus, triggering fever. IL-1 possibly also mediates other generalized reactions to tissue injury, such as hypophagia, sleep, and “sickness behavior” (Rothwell, 1991). In addition, IL-1 and IL-6 act on the pituitary to induce the release of ACTH, which in turn stimulates the secretion of cortisol from the adrenals. This represents a negative feedback loop because corticosteroids inhibit cytokine gene expression.

Mechanisms of Adaptation

Adaptation may be defined as a harm-induced capability of the organism for increased tolerance to the harm itself. It involves responses acting to preserve or regain the biological homeostasis in the face of increased harm. Theoretically, adaptation to toxicity may result from biological changes causing (1) diminished delivery of the causative chemical(s) to the target, (2) decreased size or susceptibility of the target, (3) increased capacity of the organism to repair itself, and (4) strengthened mechanisms to compensate the toxicant-inflicted dysfunction. Mechanistically, adaptation involves sensing the noxious chemical and/or the initial damage or dysfunction, and a response that typically occurs through altered gene expression. Such mechanisms will be briefly overviewed below.

Adaptation by Decreasing Delivery to the Target

The first step in the development of toxicity is delivery of the ultimate toxicant (a xenobiotic, its metabolite or xenobiotic-generated ROS and RNS) to the target (Fig. 3-2). Certain chemicals induce adaptive changes that lessen their delivery by diminishing the absorption, increasing their sequestration by intracellular binding proteins, enhancing their detoxication, or promoting their cellular export.

Repression of Iron Absorption

Adaptive mechanisms triggered by iron itself adjust the intestinal absorption of this essential yet potentially harmful metal ion, a catalyst of hydroxyl radical formation by the Fenton reaction (Fig. 3-4), to its demand. Fe²⁺ is taken up from the intestinal lumen into the enterocyte by the proton-coupled divalent metal transporter 1 (DMT1) localized in the luminal membrane and is exported across the basolateral membrane of these cells through the ferroportin iron channel. High iron intake diminishes the expression of both DMT1 and ferroportin in the enterocytes, whereas low intake has the opposite effect.

The expression of DMT1 is regulated at the translational level by the intracellular iron-regulatory proteins (IRP) that contain a [4Fe-4S] cluster. Iron supply influences the Fe content of this cluster and in turn affects the capacity of IRP to bind to mRNAs containing iron response element (IRE) in their untranslated regions and to alter translation. In iron deficiency, loss of Fe from the [4Fe-4S] cluster allows the thus formed IRP1 apoprotein to bind to and protect DMT1 mRNA from enzymatic degradation. On becoming more abundant, DMT1 mRNA translates into more DMT1 protein. Conversely, iron overload saturates IRP1 with Fe, making it incapable of binding to and stabilizing the DMT1 mRNA. The consequential loss of DMT1 mRNA and its translation into less DMT1 protein then limits Fe²⁺ absorption. This physiologically important adaptive mechanism may have toxicological corollary. Since DMT1 also mediates the transport of Cd²⁺, DMT1 overexpression in iron deficiency increases intestinal absorption of this highly toxic metal ion (Park et al., 2002).

Ferroportin allows exit of iron not only from enterocytes but also from macrophages (which obtain iron from phagocytosed senescent or damaged erythrocytes) and hepatocytes (which store iron). The abundance of ferroportin in the plasma membrane of these cells is regulated directly by hepcidin, a peptide hormone secreted by the hepatocytes of the liver (Ganz, 2011). By binding to ferroportin, hepcidin triggers the internalization and lysosomal hydrolysis of ferroportin. Hepatic production of hepcidin is sensitive to iron supply. Iron deficiency decreases hepcidin secretion, precluding ferroportin degradation and thus increasing the abundance of this iron exporter in the basolateral membrane of enterocytes. This (together with the upregulated DMT1 in the apical membrane of these cells) permits increased iron absorption from the duodenum. In contrast, iron overload increases the secretion of hepcidin, which in turn induces degradation of ferroportin, thereby preventing iron export from the enterocytes. Thus, decreased synthesis of DMT1 (see above) and degradation of ferroportin act in concert to shut down iron absorption from the intestine and protect against iron excess from the diet, from lysed red blood cells, or from ferritin. The toxicological significance of this mechanism is exemplified by findings with diquat, a redox cycling hepatotoxic herbicide that (like paraquat, see Fig. 3-3) can accept an electron, forming diquat monocation radical (DQ^•+), which in turn transfers the electron to O₂, producing superoxide anion radical (Display Formula$O2•¯$). Soon after diquat injection to rats, hepatic free iron increased dramatically (purportedly because both DQ^•+ and had reductively released Fe²⁺ from ferritin) followed by a rise in hepatic hepcidin mRNA first, and later by a decrease in plasma iron (Higuchi et al., 2011), a seemingly appropriate protective response. Inappropriately elevated hepcidin, however, may cause iron deficiency. For example, one might speculate that elevated plasma levels of prohepcidin observed in chronic lead intoxication contribute to lead-induced anemia.

Hepcidin production by hepatocytes is transcriptionally regulated by Smad4 and STAT3 TFs through their cognate response elements in the hepcidin gene (Ganz, 2011). Two pathways converge on Smad4: one signals the amount of intracellular iron, whereas the other signals the quantity of plasma iron. Increase in intracellular iron in the liver cells is detected by unidentified sensors that induce secretion of bone morphogenetic protein (BMP6, a TGF-β-like signaling molecule) acting in an autocrine or paracrine manner on its cell surface BMP receptor that activates Smad4 by phosphorylation (like TGF-β does in pathway 8 shown in Fig. 3-12). Increase in plasma iron is detected by the transferrin receptor (TFR) of hepatocytes when activated by transferrin saturated with iron. Then TFR relays the signal through adapter proteins to the BMP receptor, which thus becomes increasingly sensitive to its ligand, BMP6. The pathway descending to STAT3 is initiated by IL-6 (see Fig. 3-12, item 1), a cytokine that triggers the acute-phase response, explaining why hepcidin is also a positive acute-phase protein (see above). In summary, the pathways initiated by transferrin and BMP receptors upregulate the hepatic production of hepcidin that reduces iron absorption in response to iron overload, whereas the pathway triggered by IL-6 has a similar effect in response to inflammation, tissue injury, or cancer, conditions with increased IL-6 production. The latter disorders may cause anemia by this mechanism.

Induction of Ferritin and Metallothionein

Adaptive cellular accumulation of the binding proteins ferritin and MT is protective against their respective ligands, iron and cadmium ions. Interestingly, upregulation of ferritin by iron overload, like downregulation of DMT1, is also translationally mediated by IRP1. However, the apoIRP1 (which is formed in iron deficiency) acts oppositely on ferritin mRNA and blocks its translation. Iron overload relieves this blockade, as it yields IPR1 with the [4Fe-4S] cluster, which does not bind to the IRE in ferritin mRNA, thus causing a surge in ferritin translation. Ferritin is protective as it removes Fe²⁺ from the Fenton reaction (Fig. 3-4) and incorporates it as Fe3+ through its ferroxidase activity.

MT is greatly induced by cadmium and elevated levels of MT protect the liver by restricting distribution of this toxic metal ion to sensitive intracellular targets (Klaassen et al., 1999). Induction of MT by Cd²⁺ is likely indirect, mediated by Zn (displaced from intracellular binding sites), which activates the metal-responsive transcription factor 1 (MTF-1) that in turn augments transcription of the MT gene by binding to the metal-responsive elements in its promoter (Lichtlen and Schaffner, 2001).

Induction of Detoxication—The Electrophile Stress Response, Part 1

Adaptive increases in detoxication (ie, elimination of xenobiotics, their reactive metabolites, harmful endobiotics, or ROS and RNS by biotransformation) and cellular export have a major role in limiting toxicity. Such adaptation is typically induced by compounds with thiol reactivity (ie, soft electrophiles, oxidants, and those generating oxidative stress), which are sensed by the cytosolic Keap1–Nrf2 protein complex. The response is initiated by the TF Nrf2, which activates genes with electrophile response element (EpRE) in their regulatory region (Fig. 3-27; Dinkova-Kostova et al., 2005). Normally Nrf2 is retained in the cytoplasm by Keap1, a cysteine-rich homodimeric protein. Keap1 keeps Nrf2 inactive and at low intracellular levels by anchoring it to the cytoskeleton and also by linking it to cullin 3, a component in certain E3 ubiquitin ligase complexes, thereby initiating ubiquitination of Nrf2 for subsequent proteasomal degradation. On disruption of the Keap1–Nrf2 complex, the active Nrf2 escapes rapid degradation and accumulates in the cell. Electrophiles, such as quinones (eg, t-butylquinone), quinoneimines (eg, NAPBQI derived from acetaminophen), quinone methides (eg, metabolite of butylated hydroxytoluene), α,β-unsaturated aldehydes and ketones (eg, the lipid peroxidation products 4-oxonon-2-enal, 4-hydroxynon-2-enal, and 15-A_2t-isoprostane), isothiocyanates (eg, ANIT), thiol-reactive metal ions (eg, Cd²⁺), and trivalent arsenicals, as well as direct and indirect oxidants (eg, HOOH, diquat, quinones) may attack Keap1 at its reactive cysteine thiol groups by binding to them covalently or oxidizing them, thereby forcing Keap1 to release Nrf2. After being released from Keap1, Nrf2 translocates into the nucleus, forms a heterodimer with small Maf proteins, and activates genes through binding to EpREs.

There are many genes with EpRE motifs that encode proteins known to be important in detoxication and export (Fig. 3-27). These include genes that code for (1) enzymes that detoxify xenobiotics (eg, NQO1, NQO2, AR, EH-1, CES1e1, CES2a6, GST, and UGT), (2) enzymes that eliminate (ie, SOD1) and HOOH (eg, GPX2, catalase, and Srx1; the latter reduces the overoxidized peroxiredoxins that in turn can reduce not only HOOH [see Fig. 3-5] but also lipid hydroperoxides and peroxinitrite), (3) proteins that detoxify heme (HO-1) and Fe²⁺ (ferritin), (4) enzymes involved in the synthesis of GSH and its regeneration from GSSG (eg, GCL and the NADPH-forming G6PDH), and (5) transporters that pump xenobiotics and their metabolites out of cells (eg, Mrp2, 3, and 4. Other Nrf2-induced proteins of known toxicological relevance will be mentioned later.

As demonstrated in Fig. 3-28 through the example of t-butylhydroquinone (tBHQ), these Nrf2-mediated adaptive changes facilitate elimination and detoxication of electrophilic chemicals, as well as ROS that may be generated by them, and assist in repairing or removing damaged proteins (to be discussed later); therefore, Nrf2 conveys protection against a wide range of toxicants. Indeed, Nrf2 knockout mice are more sensitive to the hepatotoxicity of acetaminophen, pentachlorophenol, and carbon tetrachloride, the pulmonary toxicity of butylated hydroxytoluene, hyperoxia, or cigarette smoke, the neurotoxicity of 3-nitropropionic acid, and the carcinogenicity of benzo[a]pyrene (Klaassen and Reisman, 2010). Conversely, liver-specific deletion of Keap1 constitutively activates Nrf2 in hepatocytes, causing them to overexpress many detoxifying enzymes and become resistant to acetaminophen-induced hepatotoxicity.

Nrf2 can be activated by treatment with several chemicals of low toxicity that are thiol-reactive as soft electrophiles or oxidants. These include tBHQ, butylated hydroxyanisole (BHA), sulforaphane (a compound in broccoli with an electrophilic isothiocyanate group), curcumin (a compound in turmeric containing 2 α,β-unsaturated ketone groups), resveratrol (a hydroquinone-type compound in red grapes that may undergo oxidation to a quinone and redox cycling, like shown for tBHQ in Fig. 3-28), derivatives of oleanolic acid, such as 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, which contains 2 electrophilic enone groups), and oltipraz (a dithiolenethione compound whose metabolites can form mixed disulfides with thiols, probably also with those on Keap1). These chemicals induce Nrf2 target genes (Fig. 3-27) and protect from toxicant-induced tissue injury and cancer. For example, treatment with oleanolic acid or CDDO ameliorates acetaminophen-induced hepatotoxicity and aflatoxin-induced hepatocarcinogenesis (Klaassen and Reisman, 2010). The superpotent Nrf2 agonist CDDO is now tested clinically for use in chronic kidney diseases. Of these chemopreventive agents, some (eg, tBHQ, BHA, and resveratrol) are antioxidants; hence the inappropriate names “antioxidant response” and “antioxidant response element” (ARE) for the Nrf2-mediated adaptive alterations and the cognate DNA-binding site for Nrf2, respectively. It became apparent only later that the electrophilic quinone metabolites of these chemicals are the inducers and not the antioxidants.

Adaptation by Decreasing the Target Density or Responsiveness

Decreasing the density and sensitivity of the xenobiotic target is an adaptation mechanism for several cell surface receptors. Such alterations underlie the tolerance induced by opioids, abused drugs of considerable clinical toxicological interests.

Induction of Opioid Tolerance

The main target of opioids (eg, morphine, heroine, methadone) is the μ-opioid receptor. Stimulation of this G_i protein–coupled inhibitory receptor by an agonist results in adenyl cyclase inhibition (causing decline in cyclic AMP levels and PKA activity) and K⁺ channel opening (causing hyperpolarization) (Fig. 3-15) in neurons with opioid receptors, such as those in the midbrain periaqueductal gray. Even brief stimulation induces adaptive alterations: the receptor is desensitized by G-protein receptor kinase–mediated phosphorylation and β-arrestin binding, and then becomes uncoupled from the G protein and internalized via a clathrin-dependent pathway. Whereas some receptors are recycled to the cell membrane, others are degraded in the lysosomes, causing receptor downregulation (Bailey and Connor, 2005). On prolonged stimulation, adenyl cyclase signaling undergoes a compensatory increase. Tolerance to opioids, though far from being clarified mechanistically, may result from downregulation of the receptors and upregulation of adenyl cyclase signaling. These changes would require increasing doses of agonist to produce an effect (ie, inhibition of adenyl cyclase signaling) as intensive as after its first application. These adaptive changes could also explain the withdrawal reaction, that is, appearance of clinical symptoms (dysphoria, excitement, pain sensation), contrasting with the pharmacologic effects of opioids (euphoria, sedation, analgesia), on abrupt termination of drug treatment, because withdrawal of the opioid would disinhibit the reinforced signaling it had inhibited. Nevertheless, mechanistic relationships between tolerance and the withdrawal reaction remain controversial (Bailey and Connor, 2005). An important clinical feature of opioid tolerance is that the tolerance to the respiratory depressive effect is short-lived and sensitivity returns after some abstinence. Therefore, abusers often kill themselves with a dose tolerated earlier.

Adaptation by Increasing Repair

There are several repair mechanisms that can be induced after toxicant exposure. Some of these may aid in repairing damaged molecules, proteins, and DNA, others in regenerating the injured tissue.

Induction of Enzymes Repairing Oxidized Proteins—The Electrophile Stress Response, Part 2

After sublethal exposure to chemicals, such as tBHQ, 4-hydroxynon-2-enal, and Cd²⁺, not only enzymes functioning in xenobiotic detoxication but also some of those mediating protein repair become overexpressed as part of the above-described electrophile response. The induced proteins include thioredoxin 1 (Trx1) and thioredoxin reductase 1 (TR1), which can reduce oxidized proteins (protein disulfides, -sulfenic acids, and -methionine sulfoxides) (Fig. 3-22), and several subunits of the proteasome complex, which hydrolyzes damaged proteins. These repair proteins are transcribed from genes containing EpRE, and their transcription is controlled by Nrf2 (Fig. 3-27). As Trx1 and TR1 are reduction partners for ribonucleotide reductase, they support this enzyme in forming deoxyribonucleotides for DNA synthesis. Thus, induction of Trx1 and TR1 also assists DNA repair.

Induction of Chaperones Repairing Misfolded Proteins—The Heat-Shock Response

The cellular abundance of many molecular chaperones, which can disaggregate and refold denatured proteins, also increases after physical and chemical stresses (eg, heat, ionizing radiation, oxidants, electrophile reactants, and metal ions). Two adaptive reactions involving overexpression of chaperones are known; they are the heat-shock response and the EPR stress response.

Although first observed as a result of hyperthermia, the heat-shock response is an adaptive mechanism also triggered by various pathologic conditions (eg, trauma, tissue ischemia) and by virtually all reactive chemicals and/or their metabolites (eg, electrophiles, oxidants, lipid peroxidation products, metal ions, arsenite) that denature proteins. Thus, it takes place simultaneously with the electrophile response discussed above. This reaction, however, is governed by heat-shock transcription factors (HSF), mainly HSF1, which transactivate genes that encode Hsp through heat-shock response elements (HSE) within the promoter region. HSF1, like Nrf2, normally resides in the cytoplasm, where it associates with Hsp90, Hsp70, and Hsp40. On heat- or chemical-induced protein damage, these Hsps are purportedly sequestered by damaged proteins, allowing HSF1 to be released. HSF1 then migrates into the nucleus, trimerizes, undergoes phosphorylation, and stimulates the transcription of Hsp genes. As described in the section “Repair of Proteins,” the chaperones Hsp90 and Hsp70, together with co-chaperone proteins, are especially important in maintaining the integrity of hundreds of proteins (Pratt et al., 2010). The client proteins include not only those carrying out housekeeping functions but also those involved in signaling and apoptosis. Therefore, induction of Hsps has pleiotropic effects besides increased protection from cytotoxity. However, when the proteotoxic stress induces excessive protein unfolding, Hsp70 and Hsp40 recruit the ubiquitin system (Fig. 3-23) to tag the aberrant proteins for proteasomal degradation (Bedford et al., 2011).

Induction of Chaperones Repairing Misfolded Proteins—The Endoplasmic Reticulum Stress and the Unfolded Protein Response

All proteins that are destined for export or insertion into cellular membranes pass through the EPR. In this Ca²⁺-rich oxidative environment, they may be subjected to N-glycosylation at asparagine residues by oligosaccharyltransferase, formation of disulfide bonds by protein disulfide isomerases, and folding assisted by EPR-resident chaperones, such as the glucose-regulated proteins Grp78 (also called binding immunoglobulin protein [BiP]) and Grp94 as well as the Ca²⁺-binding proteins calreticulin and calnexin. Damage of proteins being processed in the EPR by reactive metabolites produced in situ by CYP enzymes (eg, the quinoneimine metabolite of acetaminophen, Cl₃C^• and Cl₃COO^• radicals formed from CCl₄, and ROS generated by CYP2E1 during oxidation of ethanol) and/or depletion of Ca²⁺ in the EPR lumen (eg, by inactivation of the EPR Ca²⁺-ATPase by reactive metabolites) causes accumulation of unfolded or misfolded proteins, a condition known as EPR stress (Cribb et al., 2005; Nagy et al., 2007; Malhi and Kaufman, 2011). Protein folding disorder can also be experimentally induced by some natural compounds that disturb the homeostasis of EPR at specific steps, such as thapsigargin (which blocks SERCA-mediated Ca²⁺ uptake into EPR; see Table 3-7), tunicamycin (which inhibits N-glycosylation of proteins), castanospermine (which interferes with maturation of nascent glycoproteins by deglucosylation), and brefeldin A (which inhibits transport of proteins from EPR to Golgi).

When the load of unfolded or misfolded proteins in the EPR exceeds the capacity of EPR-resident chaperones, these proteins trigger a complex adaptive response, called unfolded protein response (UPR). This may constrain exacerbation of the disorder (1) by attenuation of mRNA translation to decrease the functional load on the EPR, (2) by increased transcription of EPR chaperones to boost the folding capacity, (3) by initiating the translocation of aberrant proteins via translocon peptide channels from the EPR into the cytosol for proteasomal degradation, and (4) by eliminating the affected cell via apoptosis, if the EPR stress is sustained or massive. The eukaryotic translation initiation factor 2α (eIF2α) and TFs X-box binding protein 1 (XBP1), ATF6, ATF4, and C/EBP homologous protein (CHOP) (or Gadd153) are the major executioners of this adaptive response.

Three transmembrane EPR proteins sense the overload of the EPR lumen with misfolded proteins and transduce this signal to initiate the UPR. These are (1) PERK, (2) inositol-requiring enzyme 1α (IRE1α), a protein with kinase and endoribonuclease (RNase) activities, and (3) the precursor form ATF6 (activating TF 6). Normally, these sensor proteins are turned off by being associated at their luminal domain with the EPR-resident soluble chaperone BiP (or Grp78). However, when EPR stress occurs, the unfolded proteins that accumulate in the lumen of the EPR sequester BiP away from these sensors, thereby turning them on to signal for the UPR. PERK stripped off BiP is activated by dimerization and trans-autophosphorylation. With its kinase activity thus raised, PERK catalyzes the phosphorylation of the eIF2α, with the phosphorylated eIF2α causing a global translation attenuation and decreased synthesis of most (but not all) proteins. The second EPR stress sensor IRE1α also undergoes dimerization and trans-autophosphorylation on dissociation from BiP. These changes boost the RNase activity of IRE1α, enabling it to cleave and process a mRNA into one that translates into XBP1, an active TF, another executioner of UPR. The third sensor ATF6 resides as a 90 kDa precursor protein in the EPR with its Golgi localization sequences masked by BiP. On dissociation from BiP, ATF6 translocates to the Golgi, undergoes a so-called regulated intramembrane proteolysis (by site-1 and site-2 proteases), yielding a 50 kDa fragment, the active TF ATF6. XBP1 and ATF6 bind alone or as heterodimers to cognate DNA sequences, such as EPR response element (ERSE) and unfolded protein response element (UPRE), and activate the transcription of genes coding for chaperone proteins that promote protein folding (eg, Grp78, Grp94, Mdg1/Erdj4, Herp) and for proteins that assist in degradation of misfolded proteins (eg, EPR degradation-enhancing α-mannosidase-like protein [EDEM]).

On excessive EPR stress, the three activated sensors, that is, PERK, ATF6, and IRE1α, may act in concert to initiate apoptosis (Malhi and Kaufman, 2011). While PERK-catalyzed phosphorylation of eIF2α decreases the synthesis of most proteins (as stated above), it increases the synthesis of some, including that of ATF4. This TF (as well as ATF6) promotes the expression of CHOP, yet another TF. CHOP in turn can transcriptionally induce the expression of the proapoptotic Bim protein and the cell surface death receptor TRAIL-2, while inhibiting the expression of the antiapoptotic Bcl-2 protein (see Fig. 3-19). The activated IRE1α may initiate cell death via recruiting the adaptor protein TNF receptor-associated factor-2 (TRAF2), with subsequent activation of apoptosis signal-regulating kinase 1 (ASK1) and JNK. By phosphorylation, JNK can activate the proapoptotic Bim and inactivate the antiapoptotic Bcl-2 proteins (Malhi and Kaufman, 2011). The IRE1α–TRAF2 complex may also recruit and activate procaspase 12. Alternatively, Ca²⁺ released from the EPR can activate calpains, which cleave the EPR-associated procaspase 12 into active caspase 12. The latter then engages the caspase cascade (see Fig. 3-19) by activating effector caspases therein to sacrifice the cell by apoptosis. Several toxicants have been shown to induce EPR stress in experimental animals and in isolated cells by demonstrating expression of some elements of the UPR on toxicant exposure (Cribb et al., 2005; Nagy et al., 2007; Malhi and Kaufman, 2011).

Induction of Enzymes Repairing DNA—The DNA Damage Response (DDR)

DDR is initiated by detection of the DNA damage. Double-stranded DNA breaks are recognized by the MRN complex (the trimer of Mre11, Rad50, and Nbs1 proteins) or the Ku protein (the dimer of Ku70 and Ku80; see the section “Repair of DNA”) that bind and activate protein kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs, respectively. A third kinase, ataxia telangiectasia and Rad3-related (ATR), is activated in response to persistent single-stranded DNA coated with replication protein A (RPA), an intermediate during nucleotide excision or recombination DNA repair processes. Directly or through checkpoint kinases (Chk1, Chk2), these kinases phosphorylate p53 (Christmann et al., 2003; McGowan and Russell, 2004), a protein that can play the role of a TF regulating gene expression and the role of an associate protein affecting the function of its interacting protein partner. Normally, p53 is kept inactive and at low levels by its binding protein mdm2 (see Fig. 3-33), which ubiquitinates p53, facilitating its proteasomal degradation. On phosphorylation, p53 escapes from mdm2, allowing its activation and stabilization. Indeed, the levels of p53 protein in cells increase dramatically in response to DNA damage caused by UV or gamma irradiation or genotoxic chemicals. p53 then facilitates DNA repair by a number of mechanisms. For example, mainly by transcriptionally upregulating the cyclin-dependent kinase inhibitor protein p21, p53 arrests cells in G1 phase of the cell cycle (see Fig. 3-33), allowing more time for DNA repair. As a TF, p53 also increases expression of proteins directly involved in DNA repair (Harms et al., 2004). Such proteins include (a) growth arrest and DNA damage inducible (gadd45), which interacts with histones and facilitates access of proteins (eg, topoisomerase) to DNA, (b) XPE and XPC, members of the xeroderma pigmentosum group of proteins important in UV-induced DNA damage recognition before nucleotide excision repair, (c) MSH2 operating in mismatch repair, (d) PCNA that holds DNA polymerase-δ to DNA during DNA replication as well as reparative DNA synthesis in the excision and the recombination repair processes, and (e) a form of ribonucleotide reductase that provides deoxyribonucleotides for sealing DNA gaps. As a partner protein, p53 supports the function of several proteins of the nucleotide excision machinery (eg, TFIH, XPB, XPD). (Other roles p53 plays in apoptosis and in carcinogenesis as a tumor suppressor protein are illustrated in Figs. 3-19 and 3-33 and discussed elsewhere in this chapter.) The DNA-damage-activated protein kinases (ATM, ATR, and DNA-PK) also phosphorylate histone H2AX adjacent to the damage, which then becomes ubiquitinated. These markings in turn recruit repair proteins (including BRCA1) and induce chromatin relaxation for better access of repair enzymes to the lesion. The DDR-initiating kinases have numerous other protein substrates, including other kinases; therefore, DDR may extend to diverse cellular functions. It is important to realize that while both the complex DDR and distinct DNA repair mechanisms (eg, direct repair by MGMT) protect normal cells that suffered DNA damage from transformation into cancer cells (a process to be discussed below), these mechanisms, if remain operative in cancer cells, can protect them from mortal DNA damage inflicted by radiotherapy or chemotherapy, and thereby DDR and DNA repair can contribute to resistance of tumor cells to anticancer treatments.

Adaptive Increase in Tissue Repair—A Proliferative Response

Many toxicants potentially injurious to cells, for example, electrophiles, oxidants, and those inducing oxidative stress, can initiate mitogenic signaling as a prelude to tissue repair via cell replacement. It appears that the need for mitogenesis is sensed by PTP (eg, PTP1B) and the lipid phosphatase PTEN, which contains reactive cysteine thiols at their active site (Rhee et al., 2005). These phosphatases serve as brakes on the growth factor receptor–initiated mitogenic signaling, as PTPs dephosphorylate (and inactivate) the receptors themselves (eg, EGFR, PDGFR, IGFR) as well as some protein kinases (eg, Src and JAK), whereas PTEN dephosphorylates PIP₃, an important second messenger in the PI3K–Akt–IKK–NF-κB pathway (Fig. 3-12). Electrophiles covalently bind to essential cysteine-SH groups in these phosphatases. HOOH can oxidize the critical -SH group in PTEN to an intramolecular disulfide, whereas it oxidizes the -SH group of PTP1B, through sulfenic acid (-S-OH), to a 5-membered cyclic sulfenyl amide species in which the sulfur atom is covalently linked to the nitrogen of the neighboring serine (Rhee et al., 2005). Inactivation of PTPs and PTEN, which decrease the proliferative signal transduction, amplifies intracellular signaling for mitosis and survival.

It has been known for some time that oxidative stress, if not severe, activates the TF NF-κB (Dalton et al., 1999). For example, silica, which can produce ROS on its surface, activates NF-κB as well as PI3K when added to various cells (Castranova, 2004). In light of new information discussed above, NF-κB activation is now attributed to the fact that this TF is situated downstream of growth factor receptors (which are negatively controlled by the ROS-sensitive PTP) and PIP₃ (which is eliminated by ROS-sensitive PTEN) (Fig. 3-12). Furthermore, NF-κB is at the focal point of proliferative and prolife signaling, as it transactivates genes producing cell cycle accelerators (eg, cyclin D1 and c-Myc) and apoptosis inhibitors (eg, antiapoptotic Bcl proteins and the caspase IAPs) (Karin, 2006). In addition, NF-κB also transactivates the genes of ferritin, GST, SOD1, HO-1, a proteasome subunit, and gadd45, facilitating detoxication and molecular repair. All these roles of NF-κB explain its involvement in tolerance to chemically induced tissue injury, resistance against cholestatic liver injury caused by bile acids, adaptation to ionizing radiation, as well as the phenomenon termed preconditioning. This is a tolerance to ischemic tissue injury (eg, myocardial infarction), a tolerance induced by temporarily enhanced ROS formation evoked by hyperoxia or brief periods of ischemia–reperfusion.

In addition to signaling for cell replacement in damaged tissue—in which NF-κB plays a leading role—the growing cells need to boost protein synthesis. This is done under the control of the protein kinase mTOR. As shown in Fig. 3-29, mTOR activation results from signaling through both pathways coupled to growth factor receptors, that is, the MAPK pathway leading to phosphorylation of the MAPK isoform Erk and the PI3K pathway leading to phosphorylation of Akt (see Fig. 3-12). Importantly, these pathways are subject to activation in response to oxidant or electrophile exposure as they are controlled by PTPs and PTEN (Fig. 3-12). Erk and Akt protein kinases activate mTOR through a complex mechanism (Fig. 3-29), and mTOR in turn phosphorylates and regulates effectors of protein synthesis, such as the translation repressor protein 4EBP1 and the protein kinase S6K, which modifies ribosomes increasing their translational efficiency (Shaw and Cantley, 2006). As described in the sections “Adaptation to Hypoxia—The Hypoxia Response” and “Adaptation to Energy Depletion—The Energy Stress Response,” mTOR signaling is switched off in the cell to save energy as a measure to adapt to the energy shortage caused by hypoxia or toxic impairment of ATP synthesis.

Adaptation by Compensating Dysfunction

Dysfunctions caused by toxicants or drug overdose manifested at the level of organism (eg, hypoxia), organ system (eg, hypotension and hypertension), or organ (eg, renal tubular dysfunction) may evoke compensatory mechanisms.

Adaptation to Hypoxia—The Hypoxia Response

When O₂ delivery is impaired and hypoxia persists for more than a few minutes, a response involving gene expression alteration is initiated. This reaction is mainly orchestrated by hypoxia-inducible factor-1α (HIF-1α), a ubiquitous TF whose activity and cellular abundance is greatly increased in response to hypoxia (Maxwell and Salnikow, 2004; Pouyssegur et al., 2006). HIF-1α is maintained at very low intracellular levels because of continuous hydroxylation of its 2 proline residues by HIF-prolyl hydroxylases. This permits a ubiquitin ligase subunit (called von Hippel Lindau protein [VHL]) to capture HIF-1α and initiate its destruction by proteasomal degradation. Indeed, HIF-1α is one of the shortest lived proteins with a half-life of less than 5 minutes. In addition, HIF-1α is kept transcriptionally inactive by hydroxylation at one of its asparagine residues by HIF-asparagine hydroxylases, which prevents interaction of HIF-1α with transcriptional coactivators, such as p300 and CREB-binding protein (CBP). These two types of HIF hydroxylases are O₂ sensors: they use O₂ as a substrate to carry out proline/asparagine hydroxylations with concomitant oxidative decarboxylation of 2-oxoglutarate to succinate, with the K_M of O₂ being close to the ambient O₂ concentration. As the O₂ concentration falls, decreases in the hydroxylation rate of HIF-1α as well as its VHL-mediated ubiquitination and proteasomal degradation occur, and this increases its abundance and transcriptional activity. HIF hydroxylases belong to the Fe²⁺ and ascorbate-dependent dioxygenases (the largest group of nonheme oxidases); therefore, not only hypoxia but also Fe²⁺ deficiency impairs their activity. The latter feature explains why iron chelators (eg, deferoxamine) or Fe²⁺-mimicking metal ions (eg, Co²⁺ and Ni²⁺) also induce and activate HIF-1α (Maxwell and Salnikow, 2004). When induced after hypoxic conditions, HIF-1α dimerizes with HIF-1β (also called Arnt, which, coincidentally, is the dimerization partner for the Ah receptor as well). The HIF complex transactivates a vast array of genes with hypoxia response element (HRE) in their promoter. Many of the gene products assist in acclimatization to hypoxia (Pouyssegur et al., 2006). These include (1) erythropoietin (EPO) that is produced largely in kidney and activates erythropoiesis in bone marrow, (2) proteins involved in iron homeostasis (eg, transferrin, TFR, ceruloplasmin, and heme oxygenase) that may increase availability of iron for erythropoiesis, (3) vascular endothelial growth factor (VEGF) and angiopoietin-2 that stimulate blood vessel growth (ie, angiogenesis), (4) proteins facilitating anaerobic ATP synthesis from glucose (ie, glycolysis), such as the glucose transporter GLUT1 and some glycolytic enzymes, (5) proteins that correct acidosis caused by glycolytic overproduction of lactate (eg, a monocarboxylate transporter and a Na⁺/H⁺ exchanger for export of lactate and H⁺, respectively), (6) the REDD1 signal transducer protein that initiates a complex signaling pathway that leads to suspension of the ATP-consuming protein synthesis via inactivation of the protein kinase mTOR (Fig. 3-29), and (7) many other proteins, such as those that promote ECM remodeling (eg, matrix metalloproteinase-2) and cell migration (perhaps to facilitate access of the cells to the blood vessel), as well as BNIP3, a proapoptotic MOM protein that is also involved in autophagy of mitochondria in reticulocytes (perhaps to induce apoptosis of cells subjected to extreme hypoxia and to facilitate the terminal differentiation of red blood cells). Experiments on mice kept in low O₂ environment (hypoxic preconditioning) demonstrated that HIF-1α became stabilized in the retina of these animals, hypoxia-responsive genes (EPO, VEGF) were induced, and the retina became resistant to light toxicity (Grimm et al., 2005). Adaptation to hypoxia also occurs, for example, in response to high-altitude hypoxia, chronic cardiorespiratory dysfunction, and ischemic preconditioning, along with other adaptive responses discussed above. The hypoxia response is also expected to develop as a result of toxicities causing hypoxia acutely or subacutely (eg, respiratory muscle weakness after organophosphate intoxication, diquat-induced pulmonary injury) or as a delayed sequel (eg, respiratory surface restriction in hard metal disease).

Adaptation to Energy Depletion—The Energy Stress Response

Cells try to maintain their adenosine nucleotide pool in triphosphorylated, energized state, which is in the form of ATP. When the rephosphorylation rate of AMP and ADP to ATP does not keep up with the rate of ATP use, because, for example, oxidative phosphorylation is impaired or ATP use for muscle contraction or ion pumping is excessive, the ratio of AMP to ATP increases. A cellular mechanism has evolved to sense this menacing energy deficit and, in order to compensate, boosts ATP production and curtails ATP consumption (Hardie et al., 2006). The sensor is a ubiquitous heterotrimeric intracellular protein complex called AMP-activated protein kinase (AMPK). AMP strongly activates AMPK allosterically, and also by making it susceptible for phosphorylation by protein kinase LKB1 (or by the calmodulin-dependent protein kinase kinase [CaMKK] in neurons). The phosphorylated, and thus activated AMPK, targets 2 sets of proteins. One set includes those whose activation facilitates ATP production from catabolism of glucose and fatty acids as well as by promoting the biogenesis of mitochondria. For example, AMPK activation increases (a) glucose uptake (via recruiting to the cell membrane or activating glucose transporters GLUT4 and GLUT1), (b) glycolysis (via phosphorylation and activation of 6-phosphofructo-2-kinase [PFK-2] whose product, fructose-2,6-bisphosphate, is a glycolytic activator), and (c) fatty acid oxidation in mitochondria (via phosphorylation and inactivation of acetyl-CoA-carboxylase, whose product, malonyl-CoA, is an allosteric inhibitor of carnitine palmitoyltransferase-1 [CPT-1], which mediates uptake of long-chain fatty acid CoA esters into mitochondria). Another set of proteins, which are inactivated by AMPK (directly or indirectly), includes those that are involved in biosynthetic ATP-consuming reactions. Thus, AMPK inhibits (a) glycogen synthesis via phosphorylation and inactivation of glycogen synthase, (b) lipid synthesis by phosphorylating and inactivating acetyl-CoA-carboxylase, whose product, malonyl-CoA, is an essential substrate for fatty acid synthesis, (c) cholesterol synthesis by phosphorylating and inactivating HMG-CoA reductase, (d) glucose synthesis via inactivating phosphorylation of a transcriptional coactivator, TORC2, which then decreases expression of key gluconeogenetic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, and (e) protein synthesis, and thus cell growth, by inhibiting the protein kinase mTOR (Fig. 3-29). AMPK-mediated modulation of cellular energy supply and consumption involves mainly kinase reactions rather than new protein synthesis. Therefore, this adaptation is a rapid process. It can be a response to any harmful condition that compromises oxidative phosphorylation, such as hypoxia, hypoglycemia (especially in neurons), and chemically induced mitochondrial toxicity. For example, cells exposed to arsenite exhibit rapid increases in the AMP/ATP ratio and AMPK activity, with concomitant declines in HMG-CoA reductase activity as well as fatty acid and cholesterol synthesis (Corton et al., 1994).

Apart from the above-described rapid AMPK-dependent reprogramming of the cell’s intermediary metabolism from energy-consuming operation to the energy-producing mode, there is an ultimate means for the cell to generate fuel for ATP production in case of nutrient shortage. This slower process involves consumption of the cell’s own constituents (lipid droplets, glycogen particles, proteins, and organelles) by nonselective or bulk autophagy (Rabinowitz and White, 2010) involving their lysosomal hydrolysis in order to gain fuel (amino acids, fatty acids, nucleosides, and carbohydrates). An essential actor in this form of autophagy is a protein complex of unc-51-like kinase-1 (ULK-1), autophagy-related protein 13 (Atg13), and focal adhesion kinase family-interacting protein of 200 kDa (FIP200), which is an initiator of autophagosome formation (Fig. 3-29). When nutrients are abundant, the ULK-1–Atg13–FIP200 complex associates with mTOR complex 1 (mTORC1), with both ULK-1 and mTOR kinase being bound by Raptor, a scaffolding protein in mTORC1. Then mTOR phosphorylates and inactivates ULK-1 and Atg13, thus inhibiting autophagy. In nutrient shortage, signaling pathways, such as those activated by AMP and hypoxia (see Fig. 3-29), inactivate mTOR kinase, thereby forcing mTORC1 to release the complex of ULK-1–Atg13–FIP200, which in turn initiates autophagosome nucleation (Hosokawa et al., 2009).

In summary, mTOR kinase is a key regulator of the cell’s energy homeostasis, permitting or limiting cell growth according to availability of resources, the use of which can also be controlled by mTOR through switching autophagy on and off. In the presence of abundant nutrients and growth factors, mTOR is activated, thereby promoting cell growth and metabolic activity while suppressing nonselective autophagy. In nutrient deprivation or stress (eg, energetic failure, hypoxia), signaling pathways inactivate mTOR kinase activity. This both suppresses cell growth to reduce energy demand and induces autophagy to enable stress adaptation and survival (Fig. 3-29).

After surveying the major cellular adaptation mechanisms to toxicants, it is easy to recognize that one noxious effect may initiate several adaptive responses. For example, cells exposed to a hypoxic environment can rapidly respond with both AMPK-mediated program of energy stabilization and HIF-1α-directed adaptation to oxygen shortage. Theoretically, an electrophile toxicant that can bind covalently to cellular macromolecules and can also generate oxidative stress, such as a redox cycling quinone, would be expected to induce a number of adaptive processes, including the electrophile response, the heat-shock response, the EPR stress response, the DDR, and the proliferative response, and if it compromises ATP synthesis as well, it even induces the energy stress response.

Adaptation by Neurohumoral Mechanisms

There are numerous adaptive responses to dysfunctions of organs or organ systems that are mediated by humoral or neuronal signals between cells located in the same or different organs. For example, the rapid hyperventilation evoked by acute hypoxia or HCN inhalation is mediated by a neural reflex initiated by glomus cells in the carotid body. These chemosensitive cells generate a Ca²⁺ signal via the above-described AMP sensor, AMPK, which becomes activated by hypoxia or CN⁻ through impairment of oxidative phosphorylation in these cells, causing rise in the AMP/ATP ratio (Evans et al., 2005). Besides CN⁻, mitochondrial electron transport inhibitors (eg, rotenone, antimycin A, myxothiazole), uncouplers (eg, 2,4-dinitrophenol), or ATP-synthase inhibitors (eg, oligomycin) (see Table 3-6 and Fig. 3-16) as well as AMPK activators mimic the response to hypoxia in these cells. There are numerous other neurohumoral adaptive mechanisms in the body, such as the sympathetic reflex as well as activation of the renin–angiotensin–aldosterone system in response to hypotension, and the feedback systems between endocrine glands and the hypothalamus–hypophysis, which correct abnormal hormone levels. For information on these and other mechanisms, the reader is referred to textbooks of physiology.

When Repair and Adaptation Fail

When Repair Fails

Although repair mechanisms operate at molecular, cellular, and tissue levels, for various reasons they often fail to provide protection against injury. First, the fidelity of the repair mechanisms is not absolute (eg, the nonhomologous end joining that restores DNA DSB), making it possible for some lesions to be overlooked or erroneously fixed. However, repair fails most typically when the damage overwhelms the repair mechanisms, as when protein thiols are oxidized faster than they can be reduced. In other instances, the capacity of repair may become exhausted when necessary enzymes or cofactors are consumed. For example, alkylation of DNA may lead to consumption of MGMT (a self-sacrificing enzyme), lipid peroxidation can deplete α-tocopherol, and overproduction of oxidized or otherwise damaged proteins can exhaust the pool of ubiquitin. Sometimes the toxicant-induced injury adversely affects the repair process itself. For example, ethanol generates ROS via CYP2E1 that impairs the proteasomal removal of damaged proteins. Autophagic removal of cell constituents may be compromised by lysosomotropic drugs (lipophilic amines) that increase the pH in these organelles, thus decreasing the activity of lysosomal hydrolases. This may underlie the mechanism of chloroquine-induced myopathy in rats. Thiol-reactive chemicals may inactivate ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and the HECT domain-containing ubiquitin ligases (E3), each of which contains a catalytically active cysteine, as well as the lysosomal cysteine proteases (eg, cathepsins B, H, and L), thereby compromising the clearance of damaged proteins by both the UPS and autophagy. Indeed, diminished proteolysis occurs in hepatocytes exposed to toxic concentrations of acetaminophen. These repair mechanisms are especially important in neurons, as genetic deletion of proteins mediating UPS and autophagy causes accumulation of intraneuronal inclusions, neuronal loss, or neurodegeneration. In fact, human neurodegenerative diseases can be directly attributed to some dysfunction of the UPS and/or autophagy (Bedford et al., 2011). The finding that individual overexpression of some E2 and E3 enzymes confers resistance to methylmercury toxicity in yeast (Hwang et al., 2006) lends some support to the speculation that impairment of the UPS and/or autophagy might contribute to the neurotoxicity of methylmercury. After exposure to necrogenic chemicals, mitosis of surviving cells may be blocked and restoration of the tissue becomes impossible (Mehendale, 2005). Finally, some types of toxic injuries cannot be repaired effectively, as occurs when xenobiotics are covalently bound to proteins or when protein carbonyls are formed. Thus, toxicity is manifested when repair of the initial injury fails because the repair mechanisms become overwhelmed, exhausted, or impaired or are genuinely inefficient.

It is also possible that repair contributes to toxicity. This may occur in a passive manner, for example, if excessive amounts of NAD⁺ are cleaved by PARP when this enzyme assists in repairing broken DNA strands, or when too much NAD(P)H is consumed for the repair of oxidized proteins and endogenous reductants. Either event can compromise oxidative phosphorylation, which is also dependent on the supply of reduced cofactors (see Fig. 3-16), thus causing or aggravating ATP depletion that contributes to cell injury. Excision repair of DNA and reacylation of lipids also contribute to cellular deenergization and injury by consuming significant amounts of ATP. However, repair also may play an active role in toxicity. This is observed after chronic tissue injury, when the repair process goes astray and leads to uncontrolled proliferation instead of tissue remodeling. Such proliferation of cells may yield neoplasia, whereas overproduction of ECM results in fibrosis. A specific case is when the cellular repair processes that normally degrade damaged proteins, such as the UPS and the autophagy pathway, become inappropriately stimulated by adverse signaling, causing degradation of ordinary intracellular proteins. This occurs in muscle wasting induced by glucocorticoids, such as cortisol and dexamethasone. This condition appears to be secondary to suppression of the growth-promoting IGF-1–PI3K–Akt signaling that leaves FoxO TFs unchecked, which in turn increase the expression of muscle-specific ubiquitin ligases (MAFbx, also called atrogin-1, and MuRF1) as well as several components of the autophagy–lysosome pathway (Fig. 3-30).

When Adaptation Fails

Although adaptation mechanisms, such as the Nrf2-mediated electrophile response and the NF-κB-induced proliferative reaction, boost the capacity of the organism to withstand toxicant exposure and damage, excessive exposure can overwhelm these protective responses. Moreover, toxicants may impair the adaptive process. For example, moderate oxidative stress activates NF-κB, AP-1, and Nrf2 to initiate adaptive protection. However, extensive oxidant exposure aborts this program because it leads to oxidation of thiol groups in the DNA-binding domain of these TFs (Hansen et al., 2006). Similarly, Hg²⁺ can incapacitate NF-κB, thus inhibiting the prolife program activated by this TF. This promotes Hg²⁺-induced renal tubular cell injury (Dieguez-Acuna et al., 2004).

Some adaptive mechanisms may be harmful under extreme conditions. For example, acute tubular injury, which impairs tubular reabsorption and causes polyuria, triggers a tubuloglomerular feedback mechanism that reduces glomerular blood flow and filtration. Ultimately, this may precipitate anuric renal failure. It is possible that an adaptive mechanism that is beneficial in the short term may become harmful when forced to operate for a prolonged period of time. Bulk autophagy likely contributes to lethal wasting syndrome induced by TCDD in experimental animals. Chronic inflammation, tissue injury, or cancer may lead to iron deficiency and anemia because IL-6 (the acute-phase-triggering cytokine overproduced in these conditions) upregulates hepcidin secretion from the liver, which in turn reduces intestinal iron absorption. As discussed earlier, NF-κB activation is indispensable for repair via proliferation of the acutely injured tissue. However, NF-κB also targets cytokine genes, and the cytokines (eg, TNF, IL-1β) in turn activate NF-κB through their receptors (see Fig. 3-12). This vicious cycle may lead to chronic inflammation and cancer when repetitive tissue injury maintains NF-κB signaling (Karin, 2006). This occurs after occupational exposure to silica (Castranova, 2004). Sustained activation of HIF-1α in tumors facilitates invasiveness, in part by increasing VEGF expression and angiogenesis. In the kidney, HIF-1α may be involved in fibrogenesis, as it targets critical genes, such as tissue inhibitor of metalloproteinase-1 (TIMP-1).

Toxicity Resulting from Inappropriate Repair and Adaptation

Like repair, dysrepair occurs at the molecular, cellular, and tissue levels. Some toxicities involve dysrepair at an isolated level. For example, hypoxemia develops after exposure to methemoglobin-forming chemicals if the amount of methemoglobin produced overwhelms the capacity of methemoglobin reductase. Because this repair enzyme is deficient at early ages, neonates are especially sensitive to chemicals that cause methemoglobinemia. Formation of cataracts purportedly involves inefficiency or impairment of lenticular repair enzymes, such as the endopeptidases and exopeptidases, which normally reduce oxidized crystalline and hydrolyze damaged proteins to their constituent amino acids. Dysrepair also is thought to contribute to the formation of Heinz bodies, which are protein aggregates formed in oxidatively stressed and aged red blood cells. Defective proteolytic degradation of the immunogenic trifluoroacetylated proteins may make halothane-anesthetized patients victims of halothane hepatitis.

Several types of toxicity involve failed and/or derailed repairs at different levels before they become apparent. This is true for the most severe toxic injuries, such as tissue necrosis, fibrosis, and chemical carcinogenesis.

Tissue Necrosis

As discussed above, several mechanisms may lead to cell death. Most or all involve molecular damage that is potentially reversible by repair mechanisms. If repair mechanisms operate effectively, they may prevent cell injury or at least retard its progression. For example, prooxidant toxicants cause no lipid fragmentation in microsomal membranes until α-tocopherol is depleted in those membranes. Membrane damage ensues when this endogenous antioxidant, which can repair lipids containing peroxyl radical groups (Fig. 3-24), becomes unavailable (Scheschonka et al., 1990). This suggests that cell injury progresses toward cell necrosis if molecular repair mechanisms are inefficient or the molecular damage is not readily reversible.

Progression of cell injury to tissue necrosis can be intercepted by 2 repair mechanisms working in concert: apoptosis and cell proliferation. As discussed above, injured cells can initiate apoptosis, which counteracts the progression of the toxic injury. Apoptosis does this by preventing necrosis of injured cells and the consequent inflammatory response, which may cause injury by releasing cytotoxic mediators. Indeed, the activation of Kupffer cells, the source of such mediators in the liver, by the administration of bacterial LPS (endotoxin) greatly aggravates the hepatotoxicity of galactosamine. In contrast, when the Kupffer cells are selectively eliminated by pretreatment of rats with gadolinium chloride, the necrotic effect of carbon tetrachloride is markedly alleviated (Edwards et al., 1993). Blockade of Kupffer cell function with glycine (via the inhibitory glycine receptor; see item 4 in Fig. 3-15) also protects the liver from alcohol-induced injury (Yin et al., 1998).

Another important repair process that can halt the propagation of toxic injury is proliferation of cells adjacent to the injured cells. This response is initiated soon after cellular injury. A surge in mitosis in the liver of rats administered a low (non-necrogenic) dose of carbon tetrachloride is detectable within a few hours. This early cell division is thought to be instrumental in the rapid and complete restoration of the injured tissue and the prevention of necrosis. This hypothesis is corroborated by the finding that in rats pretreated with chlordecone, which blocks the early cell proliferation in response to carbon tetrachloride, a normally non-necrogenic dose of carbon tetrachloride causes hepatic necrosis (Mehendale, 2005). The sensitivity of a tissue to injury and the capacity of the tissue for repair are apparently two independent variables, both influencing the final outcome of the effect of injurious chemical—that is, whether tissue restitution ensues with survival or tissue necrosis occurs with death. For example, variations in tissue repair capacity among species and strains of animals appear to be responsible for certain variations in the lethality of hepatotoxicants (Soni and Mehendale, 1998).

It appears that the efficiency of repair is an important determinant of the dose–response relationship for toxicants that cause tissue necrosis. Following chemically induced liver or kidney injury, the intensity of tissue repair increases up to a threshold dose, restraining injury, whereupon it is inhibited, allowing unrestrained progression of injury (Mehendale, 2005). Impaired signaling to mitosis (see Fig. 3-12), caused by high tissue concentrations of toxicants (eg, acetaminophen in the liver or S-(1,2-dichlorovinyl)-l-cysteine in the kidney) and their reactive metabolites may account for lagging tissue repair (Boulares et al., 1999; Vaidya et al., 2003), but maintenance of DNA and protein synthesis, mitotic machinery, and energy supply may also be impaired at high-dose chemical exposures. That is, tissue necrosis is caused by a certain dose of a toxicant not only because that dose ensures sufficient concentration of the ultimate toxicant at the target site to initiate injury but also because that quantity of toxicant causes a degree of damage sufficient to compromise repair, allowing for progression of the injury. Experimental observations with hepatotoxicants indicate that apoptosis and cell proliferation are operative with latent tissue injury caused by low (non-necrogenic) doses of toxicants, but are inhibited with severe injury induced by high (necrogenic) doses. For example, 1,1-dichloroethylene, carbon tetrachloride, and thioacetamide all induce apoptosis in the liver at low doses, but cause hepatic necrosis after high-dose exposure (Corcoran et al., 1994). Similarly, there is an early mitotic response in the liver to low-dose carbon tetrachloride, but this response is absent after administration of the solvent at necrogenic doses (Mehendale, 2005). This suggests that tissue necrosis occurs because the injury overwhelms and disables the repair mechanisms, including (1) repair of damaged molecules, (2) elimination of damaged cells by apoptosis, and (3) replacement of lost cells by cell division.

As in tissues and organs several types of cells are integrated and support the function of each other, toxic injury to different cell types may exacerbate the tissue damage and promote its progression to tissue necrosis. This principle is exemplified by the acetaminophen-induced hemorrhagic hepatic necrosis. Even before causing manifest injury to the parenchymal liver cells, acetaminophen overdose in mice has a deleterious effect on the sinusoidal endothelial cells (McCuskey, 2008). These cells swell and lose normal function (eg, endocytosis); their fenestrae coalesce into gaps that permit red blood cells to penetrate into the space of Disse. The subsequent collapse of sinusoids reduces blood flow, thus impairing oxygen and nutrient supply of hepatocytes that also endure direct damage by the reactive metabolite of acetaminophen.

Fibrosis

Fibrosis is a pathologic condition characterized by excessive deposition of an ECM of abnormal composition. Hepatic fibrosis, or cirrhosis, results from chronic consumption of ethanol or high-dose retinol (vitamin A), treatment with methotrexate, and intoxication with hepatic necrogens such as carbon tetrachloride and iron. Pulmonary fibrosis is induced by drugs such as bleomycin and amiodarone and prolonged inhalation of oxygen or mineral particles. Doxorubicin may cause cardiac fibrosis, whereas drugs acting as agonists on 5-HT_2B receptors of human valvular interstitial cells, such as bromocriptine, ergotamine, methysergide, and fenfluramine after long-term use as well as 5-HT itself when overproduced in carcinoid syndrome, induce proliferative valve disease with fibrosis. Exposure to high doses of ionizing radiation induces fibrosis in many organs. Most of these agents generate free radicals and cause chronic cell injury.

Fibrosis is a specific manifestation of dysrepair of the chronically injured tissue. As discussed above, cellular injury initiates a surge in cellular proliferation and ECM production, which normally ceases when the injured tissue is remodeled. If increased production of ECM is not halted, fibrosis develops.

The cells that manufacture the ECM during tissue repair (eg, stellate cells and myofibroblasts in liver, mesangial cells in the kidney, fibroblast-like cells in lungs and skin) are the ones that overproduce the matrix in fibrosis. These cells are controlled and phenotypically altered (“activated”) by cytokines and growth factors secreted by nonparenchymal cells, including themselves (see Fig. 3-26). TGF-β appears to be the major mediator of fibrogenesis, although other factors are also involved. These include growth factors, such as connective tissue growth factor (CTGF, a TGF-β-induced growth factor) and PDGF, vasoactive peptides, such as endothelin-1 and angiotensin-II, and the adipocyte-derived hormone leptin (Lotersztajn et al., 2005). The evidence is compelling to indicate that TGF-β, acting through its receptor (item 8 in Fig. 3-12), and receptor-activated TFs (Smad2 and 3), is a highly relevant causative factor of fibrosis. For example, subcutaneous injection of TGF-β induces local fibrosis, whereas overexpression of TGF-β in transgenic mice produces hepatic fibrosis. Smad3-null mice are relatively resistant to radiation-induced cutaneous fibrosis, bleomycin-induced pulmonary fibrosis, and CCl₄-induced hepatic fibrosis. TGF-β antagonists, such as anti-TGF-β immunoglobulin and decorin, as well as Smad3 antagonists, such as halofuginone and overexpressed Smad7 protein (which is antagonistic to Smad2 and 3), ameliorate chemically induced fibrogenesis (Flanders, 2004). In several types of experimental fibrosis and in patients with active liver cirrhosis, overexpression of TGF-β in affected tissues has been demonstrated. Specific factors may also be involved in the pathomechanism of chemically induced fibrosis. For example, in alcoholic liver cirrhosis stellate cells may be activated directly by acetaldehyde (formed by alcohol dehydrogenase), by ROS (generated by the ethanol-induced CYP2E1), and by bacterial endotoxin (LPS), which is increasingly absorbed from the gut, the permeability of which is enhanced by chronic alcohol exposure. LPS can stimulate stellate cells both directly and indirectly through Kupffer cells (see Fig. 3-26) because both cells express TLR through which LPS acts.

The increased expression of TGF-β is a common response mediating regeneration of the ECM after an acute injury. However, whereas TGF-β production ceases when repair is complete, this does not occur when tissue injury leads to fibrosis. Failure to halt TGF-β overproduction could be caused by continuous injury or a defect in the regulation of TGF-β. Indeed, after acute CCl₄-induced liver injury, hepatic stellate cells exhibit a TGF-β-mediated induction of Smad7 (which purportedly terminates the fibrotic signal by inhibiting activation of Smad2 and Smad3 by TGF-β receptor); however, after chronic injury, Smad7 induction fails to occur (Flanders, 2004).

The fibrotic action of TGF-β is due to increased production and decreased degradation of ECM components. TGF-β stimulates the synthesis of individual ECM components (eg, collagens) by specific target cells via the Smad pathway (see Fig. 3-12) and also by downregulation of the transcription of miR-29 miRNA family members, which inhibit the translation of collagen. Downregulation of miR-29 family members occurs in murine hepatic stellate cells exposed to TGF-β, in the stellate cells of mice with hepatic fibrosis induced by CC1₄ or bile duct ligation, and in the liver of patients with advanced liver cirrhosis (Roderburg et al., 2011). TGF-β inhibits ECM degradation by disproportionately increasing the expression of inhibitor proteins that antagonize ECM-degrading enzymes, such as TIMP-1 and plasminogen activator inhibitor-1 (PAI-1), compared with the expression of ECM-degrading metalloproteinases (Arthur et al., 1999; Flanders, 2004). Interestingly, TGF-β induces transcription of its own gene in target cells (Flanders, 2004), suggesting that the TGF-β produced by these cells can amplify in an autocrine manner the production of the ECM. This positive feedback (autoinduction) may facilitate fibrogenesis.

Fibrosis involves not only excessive accumulation of the ECM but also changes in its composition. The basement membrane components, such as collagen IV and laminin, as well as the fibrillar-type collagens (collagen I and III), which confer rigidity to tissues, increase disproportionately during fibrogenesis (Gressner, 1992).

Fibrosis is detrimental in a number of ways:

1. The scar compresses and may ultimately obliterate the parenchymal cells and blood vessels.

2. Deposition of basement membrane components between the capillary endothelial cells and the parenchymal cells presents a diffusional barrier that contributes to malnutrition of the tissue cells.

3. An increased amount of ECM and its rigidity unfavorably affect the elasticity and flexibility of the whole tissue, compromising the mechanical function of organs such as the heart and lungs.

4. Furthermore, the altered extracellular environment is sensed by integrins. Through these transmembrane proteins and the coupled intracellular signal transducing networks (see Fig. 3-12), fibrosis may modulate several aspects of cell behavior, including polarity, motility, and gene expression (Raghow, 1994).

Carcinogenesis

Chemical carcinogenesis involves malfunctions in various repair and adaptive mechanisms. At the molecular level, a crucial feature of carcinogenesis is altered expression of critical proteins, that is, proto-oncogenic proteins and tumor suppressor proteins. This may result (1) from mutation of critical genes due to insufficient adaptive response to DNA damage and missed DNA repair or (2) from inappropriate transcriptional control at the regulatory regions of critical genes that gives rise to overexpression or underexpression of their products. (Note that the genes that we designate here as “critical” include those that are transcribed into mRNAs that in turn translate into proto-oncogenic or tumor suppressor proteins, as well as those that are transcribed into miRNAs that in turn regulate the translation of proto-oncogenic or tumor suppressor proteins. These proteins will be defined below.) At the cellular level, the fundamental feature of tumorigenesis is proliferation of cells, which may result (1) from failure to execute apoptosis and/or (2) from failure to restrain cell division.

As to be described in more detail later, carcinogenesis entails gene expression alterations initiated by two fundamentally distinct types of mechanisms that often work simultaneously and in concert, that is, genetic and epigenetic mechanisms. Genetic mechanisms bring about a qualitative change in gene expression, that is, expression of an altered gene product, a mutant protein, or miRNA, with gain or loss in activity. In contrast, epigenetic mechanisms cause quantitative change in gene expression resulting in more or less gene product. Whereas genetic mechanisms alter the coding sequences of critical genes, epigenetic mechanisms eventually influence the regulatory (promoter) region of genes. Thus, chemical and physical insults may induce neoplastic transformation of cells by affecting critical genes through genotoxic and nongenotoxic (ie, epigenetic) mechanisms. However, either mechanism ultimately induces cancer by causing cellular failures in executing apoptosis and/or restraining cell division, thereby giving rise to an uncontrollably proliferating cell population.

Genotoxic Mechanisms of Carcinogenesis: Chemical Damage and Disrepair in the Coding Region of Critical Genes Leading to Mutation

Chemicals that react with DNA may cause damage such as adduct formation, oxidative alteration, and strand breakage. In most cases, these lesions are repaired or the injured cells are eliminated. If neither event occurs, a lesion in the parental DNA strand may induce a heritable alteration, or mutation, in the daughter strand during replication. The mutation may remain silent if it does not alter the protein encoded by the mutant gene or if the mutation causes an amino acid substitution that does not affect the function of the protein. Alternatively, the genetic alteration may be incompatible with cell survival. The most unfortunate scenario for the organism occurs when the altered genes express mutant proteins that reprogram cells for multiplication and escaping apoptosis (ie, immortalization). When such cells undergo mitosis, their descendants also have a similar propensity for proliferation. Moreover, because enhanced DNA replication and cell division increases the likelihood of mutations (for reasons discussed below), these cells eventually acquire additional mutations that may further augment their growth advantage over their normal counterparts. The final outcome of this process is a nodule, followed by a tumor consisting of rapidly proliferating transformed cells (Fig. 3-31).

The critical role of DNA repair in preventing carcinogenesis is attested by the human heritable disease xeroderma pigmentosum. Affected individuals lack excision repair proteins of the XP series and exhibit a greatly increased incidence of sunlight-induced skin cancers. Cells from these patients are also hypersensitive to DNA-reactive chemicals, including aflatoxin B₁, aromatic amines, polycyclic hydrocarbons, and 4-nitroquinoline-1-oxide (Lehmann and Dean, 1990). Also, mice with ablated PARP gene are extremely sensitive to γ-rays and N-methyl-N-nitrosourea and show genomic instability, as indicated by sister chromatid exchanges and chromatid breaks following genotoxic insult (D’Amours et al., 1999).

A small set of cellular genes is the target for genetic alterations that initiate neoplastic transformations. Included are proto-oncogenes and tumor suppressor genes (Barrett, 1992).

Mutation of Proto-Oncogenes

Proto-oncogenes are highly conserved genes encoding proteins that stimulate the progression of cells through the cell cycle, or oppose apoptosis (Smith et al., 1993). The products of proto-oncogenes that accelerate the cell division cycle include (1) growth factors; (2) growth factor receptors; (3) intracellular signal transducers such as G proteins, protein kinases, cyclins, and cyclin-dependent protein kinases; and (4) nuclear TFs (see Figs. 3-12 and 3-32). A notable proto-oncogene product that inhibits apoptosis is Bcl-2 (see Fig. 3-19).

Fig. 3-32 depicts several proto-oncogene products that are closely involved in initiating the cell division cycle. The legend of that figure outlines some important details on the function of these proteins and their interaction with tumor suppressor proteins (to be discussed below). Transient increases in the production or activity of proto-oncogene proteins are required for regulated growth, as during embryogenesis, tissue regeneration, and stimulation of cells by growth factors or hormones. In contrast, permanent activation and/or overexpression of these proteins favor neoplastic transformation. One mechanism whereby genotoxic carcinogens induce neoplastic cell transformation is by producing an activating mutation of a proto-oncogene. Such a mutation is so named because the altered gene (then called an oncogene) encodes a permanently active protein that forces the cell into the division cycle.

An example of mutational activation of an oncogene protein is that of the Ras proteins. Ras proteins are G-proteins with GTP/GDP-binding capacity as well as GTPase activity (Anderson et al., 1992). They are localized on the inner surface of the plasma membrane and function as crucial mediators in the signaling pathways initiated by growth factors (see Figs. 3-12 and 3-32). Ras is located downstream from growth factor receptors and nonreceptor protein tyrosine kinases and upstream from mitogen-activated protein kinase (MAPK) cascade whose activation finally upregulates the expression of cyclin D and initiates the mitotic cycle (Fig. 3-32). In this pathway, Ras serves as a molecular switch, being active in the GTP-bound form and inactive in the GDP-bound form. Some mutations of the Ras gene (eg, a point mutation in codon 12) dramatically lower the GTPase activity of the protein. This in turn locks Ras in the permanently active GTP-bound form. Continual rather than signal-dependent activation of Ras can lead eventually to uncontrolled cell division and transformation. Indeed, microinjection of Ras-neutralizing monoclonal antibodies into cells blocks the mitogenic action of growth factors as well as cell transformation by several oncogenes. Ionizing radiation and carcinogenic chemicals (eg, N-methyl-N-nitrosourea, polycyclic aromatic hydrocarbons, benzidine, aflatoxin B₁) induce mutations of Ras proto-oncogenes that lead to constitutive activation of Ras proteins (Anderson et al., 1992). Most of these chemicals induce point mutations by transversion of G₃₅ to T in codon 12.

Another example for activating mutation of a proto-oncogene is B-Raf mutation, although Ras and Raf mutations are mutually exclusive (Shaw and Cantley, 2006). Raf proteins are protein kinases, lying just downstream from Ras and being the first signal transducers in the MAP kinase pathway (see Fig. 3-12). After recruitment by Ras to the cell membrane, Raf is activated by the growth factor receptor (see item 4 in Fig. 3-12) through phosphorylation in its activating segment. B-Raf mutations occur in mouse liver tumors induced by diethylnitrosamine (Jaworski et al., 2005) in 66% of malignant melanomas and a wide range of human cancers. All mutations are within the activation segment of B-Raf, with a single amino acid substitution (V599E) accounting for the majority. The mutant B-Raf protein has elevated kinase activity probably because substitution of the nonpolar valine with the negatively charged glutamate mimics an activating phosphorylation. Thus, the constitutively active B-Raf continually sends Ras-independent proliferative signal down the MAPK pathway. Indeed, transfection of the mutant B-Raf gene into cells induced neoplastic transformation even in the absence of Ras proteins (Davies et al., 2002). Another proto-oncogene product that often undergoes activating mutation in breast and colon tumors is p110-α, the catalytic subunit of PI3K (Shaw and Cantley, 2006). This can cause permanent proliferative signaling via the GF receptor– PI3K–Akt pathway (see Fig. 3-12).

Whereas constitutive activation of oncogene proteins, as a result of point mutation, is a common initiator of chemical carcinogenesis, permanent overexpression of such proteins also can contribute to neoplastic cell transformation. Overexpression of proto-oncogene proteins may result from amplification of the proto-oncogene, that is, the formation of more than one copy (Anderson et al., 1992). Such an event may be initiated by DNA strand breaks, and therefore often observed after exposure to ionizing radiation; however, proto-oncogene amplification also occurs in spontaneous human cancer. An example for a proto-oncogene protein that is overexpressed in response to gene damage is the antiapoptotic Bcl-2 protein (see Fig. 3-19). The aberrantly increased expression of Bcl-2 is caused by a chromosomal translocation and is responsible for B-cell lymphoma, a spontaneous human malignancy (see later). Overexpression of proto-oncogene proteins as a result of nongenotoxic, epigenetic mechanisms will be discussed later.

Mutation of Tumor Suppressor Genes

Tumor suppressor genes encode proteins that inhibit the progression of cells in the division cycle, or promote DNA repair or apoptosis on DNA damage. Fig. 3-32 depicts such proteins, which include, for example, cyclin-dependent protein kinase inhibitors (eg, p15, p16, and p21), TFs (eg, p53 and Smad) that activate genes encoding cyclin-dependent protein kinase inhibitors, proteins (eg, pRb) that block TFs involved in DNA synthesis and cell division, and proteins (eg, ARF) that block inhibitors of tumor suppressor proteins. Other notable tumor suppressor gene products include, for example, the protein kinases (eg, ATM, ATR) that sense the DNA damage and signal for the p53-controlled response shown in Fig. 3-33, proteins involved in DNA repair, such as O⁶-methyguanine-DNA methyltransferase (which removes alkyl groups adducted to guanine) as well as BRCA1 and BRCA2 proteins (which contribute to recombinational DNA repair), proapoptotic proteins (eg, Bax, Puma, Noxa, and Bim) induced after DNA damage (Fig. 3-33), the suppressor of cytokine signaling (Socs) protein (Fig. 3-12), the phosphatase PTEN (which dephosphorylates the membrane lipid PIP₃, an essential intermediate in the PI3K–Akt pathway) that turns off the PI3K–Akt pathway-mediated proliferative signaling (Fig. 3-12), and the tuberous sclerosis complex-2 (TSC2) that prevents activation of mTOR (Fig. 3-29). Uncontrolled proliferation can occur when the mutant tumor suppressor gene encodes a protein that cannot suppress cell division. Inactivating mutations of specific tumor suppressor genes in germ cells are responsible for the inherited predisposition to cancer, as in familial retinoblastoma (pRb; see Fig. 3-32), Wilms tumor (WT1, WTX, and β-catenin; see Fig. 3-13), familial polyposis (Smad4; see Figs. 3-12 and 3-32), and Li–Fraumeni syndrome (p53; see Figs. 3-19, 3-32, and 3-33). Mutations of tumor suppressor genes in somatic cells contribute to nonhereditary cancers. The genes of p16, PTEN, and pRb are frequently mutated in human cancer. The best known tumor suppressor gene involved in both spontaneous and chemically induced carcinogenesis is p53.

The p53 tumor suppressor gene encodes a 53 kDa protein with multiple functions (Fig. 3-33). Acting as a transcriptional modulator, the p53 protein (1) activates protein-coding genes whose products arrest the cell cycle (eg, p21 and gadd45), repair damaged DNA (eg, XPE, MSH2), or promote apoptosis (eg, Bax, Puma, and Fas receptor); (2) activates miRNA-coding genes whose products (eg, miR-34a) repress the translation of mitogenic TFs and cell cycle accelerator proteins (eg, Myc, E2F3, cyclin D, and CDK4/6); and (3) represses protein-coding genes that encode cell cycle accelerators (eg, cyclin B1, Cdk1), or antiapoptotic proteins (eg, Bcl-2 and IGF-1 receptor) (Bennett et al., 1999; Liu and Chen, 2006). DNA damage activates protein kinases (ATM, ATR, Chk1, Chk2) to phosphorylate and stabilize the p53 protein, causing its accumulation (Fig. 3-33). The accumulated p53 may induce cell cycle arrest and apoptosis of the affected cells. Thus, p53 eliminates cancer-prone cells from the replicative pool, counteracting neoplastic transformation; therefore, it is commonly designated as guardian of the genome.

Indeed, cells that have no p53 are a million times more likely to permit DNA amplification than are cells with a normal level of this suppressor gene. Furthermore, mice with the p53 gene deleted develop cancer by 6 to 9 months of age, attesting to the crucial role of the p53 tumor suppressor gene in preventing carcinogenesis.

Mutations in the p53 gene are found in 50% of human tumors and in a variety of induced cancers. The majority are “missense mutations” that change an amino acid and result in a faulty or altered protein (Bennett et al., 1999). The faulty p53 protein forms a complex with endogenous wild-type p53 protein and inactivates it. Thus, the mutant p53 not only is unable to function as a tumor suppressor protein but also prevents tumor suppression by the wild-type p53.

Carcinogens may cause characteristic mutations in the p53 tumor suppressor gene. An example is the point mutation in codon 249 from AGG to AGT, which changes amino acid 249 in the p53 protein from arginine to serine. This mutation predominates in hepatocellular carcinomas in individuals living where food is contaminated with aflatoxin B₁ (Bennett et al., 1999). Because in human hepatocytes the CYP-activated metabolites of aflatoxin B₁ induce the transversion of G to T in codon 249 of the p53 tumor suppressor gene (Aguilar et al., 1993), it appears likely that this mutation in primary human liver cancer is indeed caused by this mycotoxin. Although the incriminated mutation probably contributes to the hepatocarcinogenicity of aflatoxin B₁ in humans, it is not involved in aflatoxin B₁–induced hepatocarcinogenesis in rats, as the transformed liver cells from the toxin-exposed rats do not show this mutation.

Another example for inactivating mutation of a tumor suppressor gene of environmental origin is that of Patched, which often occurs in basal cell carcinoma of the skin that may be induced by UV and ionizing radiation, as well as arsenic exposure (Tang et al., 2007). Normal Patched, the membrane receptor of the Hh ligand, suppresses the mitogenic Hh pathway in absence of Hh ligand, which would inhibit Patched (Fig. 3-14). Mutant Patched loses its power to restrain this signaling network, which thus promotes cell division even in absence of its inhibitor ligand.

In addition to aberrations in critical protein-coding genes, damage in genes coding for miRNA (which repress the translation of critical proteins; see Fig. 3-11) may also contribute to carcinogenesis. For example, amplification of miR-21 (which is oncogenic by repressing the translation of PTEN; see Fig. 3-12) and deletion of miR-15 and miR-16 (which is tumor suppressive by repressing the translation of cdc25A; see Fig. 3-25) occur in spontaneous human malignancies. Therefore, miRNA gene damage may also underlie the mechanism of chemical carcinogenesis. The role of altered miRNA expression as an epigenetic mechanism of chemical carcinogenesis will be discussed below.

Epigenetic Mechanisms in Carcinogenesis: Inappropriate Activation or Responsiveness of the Regulatory Region of Critical Genes

Whereas some chemicals cause cancer by reacting with DNA and inducing a mutation, others do not damage DNA, yet induce cancer after prolonged exposure (Barrett, 1992). These chemicals are designated nongenotoxic (or epigenetic) carcinogens and include (1) xenobiotic mitogens, that is, chemicals that promote proliferative signaling, such as the PKC activator phorbol esters and fumonisin B₁, as well as the protein phosphatase inhibitor okadaic acid (see Fig. 3-12); (2) endogenous mitogens, such as growth factors (eg, TGF-α) and hormones with mitogenic action on specific cells, for example, estrogens on mammary gland or liver cells, TSH on the follicular cells of the thyroid gland, and luteinizing hormone on Leydig cells in testes; (3) toxicants that, when given chronically, cause sustained cell injury (such as chloroform and d-limonene); (4) xenobiotics that are nongenotoxic carcinogens in rodents but not in humans, such as phenobarbital, DDT, TCDD, and peroxisome proliferators (eg, fibrates, WI-14643, dihalogenated and trihalogenated acetic acids); and (5) ethionine and diethanolamine, which interfere with formation of the endogenous methyl donor S-adenosyl methionine (Poirier, 1994). Because several of the listed chemicals promote the development of tumors after neoplastic transformation has been initiated by a genotoxic carcinogen, they are referred to as tumor promoters. Despite the initial belief that promoters are unable to induce tumors by themselves, studies suggest that they can do so after prolonged exposure.

It appears that nongenotoxic chemicals, like the genotoxic ones, eventually also influence the expression of proto-oncogenes and/or tumor suppressor genes, but in a different manner. When continuously present, nongenotoxic carcinogens can permanently induce the synthesis of normal proto-oncogene proteins and/or repress the synthesis of normal tumor suppressor proteins, rather than inducing the synthesis of permanently active mutant proto-oncogene proteins or permanently inactive mutant tumor suppressor proteins, as the mutagenic genotoxic carcinogens do.

Carcinogens may alter the synthesis of proto-oncogene proteins and tumor suppressor proteins at transcriptional and/or translational levels. Chemicals may modify transcription of the genes into mRNAs for proto-oncogene and tumor suppressor proteins by perturbing the signal transduction to the promoter region of these genes and/or by altering the signal receptivity of this gene region by DNA methylation and histone modifications. Translation of proto-oncogene proteins and tumor suppressor proteins from their mRNA may be controlled by specific miRNAs, which repress this process (Fig. 3-11). Carcinogen-induced perturbations in signaling and promoter alterations may not only influence transcription of protein-coding genes but also affect the transcription of miRNA-coding genes. The resultant changes in the abundance in miRNAs in turn alter, in a reciprocal (inverse) manner, the synthesis of proto-oncogene proteins and tumor suppressor proteins, provided the miRNA in question targets the mRNA of such a protein. The forthcoming discussion deals with the importance of signaling, DNA as well as histone modifications, and miRNA expression in carcinogenesis.

Role of Signaling in Carcinogenesis

An important target of nongenotoxic carcinogens is the promoter region of critical genes where they can act via two distinct modes. First, they may alter the abundance and/or activity of TFs, typically by influencing upstream signaling elements ranging from extracellular signaling molecules (eg, TSH) to intracellular transducer proteins (eg, PKC). Obviously both xenobiotic and endogenous mitogens mentioned above can thus activate proliferative pathways (see Fig. 3-12) that descend to TFs (eg, Myc) that act on the promoter of proto-oncogenes coding for mRNAs of proto-oncogene proteins (eg, cyclins), and, as to be discussed below, on the promoter of genes coding for oncogenic miRNAs (eg, miR-17-92). It is easy to recognize that even nongenotoxic carcinogens of the cytotoxic type act in this manner. As described under tissue repair, cell injury evokes the release of mitogenic growth factors such as HGF and TGF-α from tissue macrophages and endothelial cells. Thus, cells in chronically injured tissues are exposed continuously to endogenous mitogens. Although these growth factors are instrumental in tissue repair after acute cell injury, their continuous presence is potentially harmful because they may ultimately transform the affected cells into neoplastic cells. Indeed, transgenic mice that overexpress TGF-α develop hepatomegaly at a young age and tumors by 12 months (Fausto et al., 2006). Mitogenic cytokines secreted by Kupffer cells are apparently involved in hepatocyte proliferation and, possibly, tumor formation induced by peroxisome proliferators in rats (Rose et al., 1999) and in the formation of the endothelial cell–derived hepatic hemangiosarcoma in mice exposed to 2-butoxyethanol (Corthals et al., 2006).

Role of DNA Methylation and Histone Modification in Carcinogenesis

As a second mode of action, nongenotoxic carcinogens may alter expression of critical genes by modifying the responsiveness of the promoter region of these genes to TFs. Promoter responsiveness is typically controlled by DNA methylation. This takes place at C₅ of specific cytosine residues located in CpG islands (ie, clusters of CpG dinucleotides) in the promoter and is catalyzed by DNA cytosine methyltransferases (eg, DNMT1, DNMT3a, and DNMT3b) using S-adenosyl methionine as the methyl donor. It is well known that promoter methylation decreases the transcriptional activity of genes. For example, tissue-specific genes (eg, the genes of GI TFFs) are hypermethylated in tissues where they are not expressed, but are typically hypomethylated in tissues where they are expressed.

Promoter methylation can silence genes because it weakens binding of TFs to the promoter and because it triggers secondary alterations in histone proteins that reduce the accessibility of the promoter for TFs (Esteller, 2005). The latter mechanism involves modification of the protruding amino-terminal tails of core histone proteins by deacetylation and methylation of lysine residues. This makes the histone more compact, thereby diminishing access of TFs and other proteins involved in transcription initiation to the gene promoter. Histone deacetylases that remove the acetyl group from histone tails may be recruited by DNMTs directly, or through proteins that recognize and bind to the methylated CpG dinucleotides (eg, MeCP1, MeCP2, MBD2, MBD3). Some of these methyl-CpG-binding domain-containing proteins (eg, MBD2, MBD3) also have histone deacetylase activity. Finally, both DNMTs and MBDs can recruit histone methyltransferases to methylate histone tail lysines.

While methylation of CpG dinucleotides codes for gene silencing via histone deacetylation and methylation (“histone code”), hypomethylated genes are alert. In fact, CpG islands in the 5′-end region (promoter, untranslated region, exon 1) of genes are relatively hypomethylated in normal tissues, except for tissue- or germ-line-specific genes, and genomically imprinted genes (see earlier). Thus, if the appropriate TFs are available for a particular gene, and if the CpG island remains hypomethylated, the histones acetylated and hypomethylated, then the gene will be transcribed (Esteller, 2005). Histones are acetylated by histone acetyltransferases, such as p300 and CBP, which also acetylate other proteins, including p53 (see Fig. 3-33), and are also transcriptional coactivators.

It is well documented that the normal methylation pattern of DNA is disrupted in cancer cells. Such cells are characterized by global (average) hypomethylation, that is, decreased content of genomic 5′-methylcytosine. Paradoxically, DNA hypomethylation occurs in the face of hypermethylation of the CpG islands in the promoter region tumor suppressor genes, which are normally demethylated (Esteller, 2005). The frequently hypermethylated tumor suppressor genes in human cancers are, for example, the Cdk inhibitor p15 and p16, the mdm2-binding protein ARF, pRb that tethers E2F (see Fig. 3-30), the DNA repair enzyme MGMT, and the lipid phosphatase PTEN (see Fig. 3-12). Importantly, both global hypomethylation and tumor suppressor gene hypermethylation intensify with increased malignancy of the tumor. Relevance of hypermethylation-induced silencing of tumor suppressor genes in carcinogenesis is supported by the finding that inhibitors of DNMTs (such as 5-aza-2′-deoxycytidine) can stop the growth of cancer cells and induce their differentiation by demethylating the dormant tumor suppressor genes, thereby restoring their expression (Esteller, 2005). The consequence of global hypomethylation of DNA is less clear. Nevertheless, hypomethylation of proto-oncogenes and increased expression of their products is a plausible mechanism (Goodman and Watson, 2002), although others have also been proposed (eg, chromosomal instability, reactivation of transposable DNA elements, and loss of genomic imprinting). On comparing mouse strains, it has been suggested that their sensitivity to chemical carcinogens may be related inversely to their capacity to maintain normal patterns of DNA methylation. It is worth noting that DNA methylation is more stable in human cells than in rodent cells (Goodman and Watson, 2002).

It appears that some of the nongenotoxic carcinogens alter DNA methylation. Inhibition of DNA methylation is a plausible mechanism that underlies tumorigenesis induced by ethionine, which depletes S-adenosyl-methionine (the methyl donor for DNMTs), and diethanolamine, which inhibits cellular uptake of choline, a dietary methyl group source for methylation of homocysteine to methionine. On prolonged administration, virtually all nongenotoxic rodent liver carcinogens listed above decrease DNA methylation. In addition to global DNA hypomethylation, promoter hypomethylation of the following proto-oncogenes have been observed in mouse or rat liver: Ras or Raf, following phenobarbital treatment, c-Jun, c-Myc, and IGF-2, after treatment with dichloroacetic acid, dibromoacetic acid, or trichloroacetic acid, and c-Myc after WY-14643 dosing. Long-term arsenic exposure of mice also induces hypomethylation of hepatic DNA globally and in the promoter of ERα, a hormone-activated TF. This is associated with an increase of cyclin D, a potentially ERα-linked gene (Chen et al., 2004). In contrast, in human keratinocytes TCDD induces promoter hypermethylation in the tumor suppressor genes p16 and p53 as well as immortalization of these cells (Ray and Swanson, 2004). Although the mechanisms that initiate altered promoter methylation are currently unknown, it is possible that that altered promoter methylation plays a role in tumor promotion by these nongenotoxic carcinogens.

Role of MicroRNAs in Carcinogenesis

As miRNAs almost always repress the translation of proteins from their mRNAs (Fig. 3-11), a miRNA plays an oncogenic role if it represses the translation of tumor suppressor proteins, that is, proteins that counter cell division, repair DNA, or promote apoptosis. For example, the miR-17-92 cluster is considered oncogenic, because its members promote mitosis by repressing the translation of PTEN (an inhibitor of the mitogenic PI3K–Akt signaling; see Fig. 3-12) and p21 (a cyclin-dependent kinase inhibitor; see Fig. 3-32) and because they inhibit apoptosis by repressing the translation of the proapoptotic protein Bim. Other examples for oncogenic miRNAs include miR-29b (which also targets the mRNAs of p21 and Bim), as well as miR-221/222, the targets of which include mRNAs translating into tumor suppressor proteins, such as PTEN, p27, p57, FOXO3 (see Fig. 3-30), TIMP3 (metalloproteinase inhibitor 3), and Bim (Chen et al., 2010; Garofalo and Croce, 2011). Overexpression of such oncogenic miRNAs promotes neoplastic cell transformation, whereas their underexpression opposes it.

Conversely, a miRNA has tumor suppressive role if it represses the translation of proto-oncogene proteins, that is, proteins that facilitate cell division or oppose apoptosis. For example, the let-7 family of miRNAs is regarded tumor suppressive as they silence the translation of Ras and Myc proteins, components of proliferative signaling (see Fig. 3-12). Furthermore, miR-15a and miR-16-1 repress the translation of protein phosphatase cdc25, which dephosphorylates and thus activates CDK1 and 2 that drives the cell cycle (see Fig. 3-25), and of Bcl-2, which inhibits apoptosis (see Fig. 3-19). Another tumor suppressor miRNA is miR-34, whose targets include the mRNAs that translates into the mitogenic TFs Myc and E2F3, as well as the cell cycle accelerator proteins cyclin D and CDK4/6 (see Figs. 3-25 and 3-32) (Chen, 2010; Garofalo and Croce, 2011). Upregulation of such tumor suppressive miRNAs counters neoplastic cell transformation, whereas their downregulation stimulates it.

Expression of miRNAs, like that of mRNAs coding for proteins, can be regulated at the promoter region of their genes by TFs as well as DNA methylation and histone modification. For example, Myc activates the transcription of the oncogenic miR-17-92, whereas p53 activates the transcription of the tumor suppressive miR-34. Therefore, the latter event is part of the adaptive DNA damage stress response discussed earlier and depicted in Fig. 3-33. Indeed, genotoxic carcinogens such as N-ethyl-N-nitrosourea and tamoxifen (which forms a DNA-reactive metabolite in rats, but not in humans) as well as ionizing radiation typically induce overexpression of mi-34.

The role of miRNAs in tumorigenesis induced by nongenotoxic carcinogens is well exemplified by the rodent hepatocarcinogenesis induced by the PPARα-activator WY-14643. This process involves transcriptional downregulation of the gene coding for let-7c and consequential derepression of the translation of c-Myc mRNA into c-Myc protein. Due to its increased abundance, this TF (see Figs. 3-12 and 3-32) increasingly activates the gene coding for cyclin D, a mitogenic protein, as well as the gene coding for miR-17-92, an oncogenic miRNA. miR-17-92 in turn represses the translation of proteins that counter mitosis (eg, PTEN and p21) and promote apoptosis (eg, Bim), thus forcing hepatocytes to proliferate (Gonzalez and Shah, 2008).

Cooperation of Genotoxic and Epigenetic Mechanisms in Carcinogenesis

Genotoxic and epigenetic mechanisms most likely complement and amplify each other in chemical carcinogenesis. One chemical may exert both genotoxic and epigenetic effects. For example, estrogens produce mutagenic free radicals via redox cycling of their quinone and hydroquinone metabolites and induce receptor-mediated proliferative effect (Newbold, 2004). Well-known genotoxic carcinogens can also cause epigenetic alterations. For example, 2-acetylaminofluorene does not only form DNA adducts but also induces DNA methylation in the promoter of Rassf1a and p16 genes as well as histone methylation around the promoter of Rassf1a, p16, Socs1, Cdh1, and Cx26 genes, thereby silencing all these genes that encode tumor suppressor proteins (Pogribny et al., 2011). The p16, ARF, and MGMT tumor suppressor genes are also hypermethylated in aflatoxin B₁–induced mouse lung tumor and in 7,12-dimethylbenzanthracene-induced skin tumor. The antiestrogen tamoxifen, a hepatocarcinogen in rats (but not in humans), exerts genotoxic effect and induces global hypomethylation of DNA and histones in rat liver (Tryndyak et al., 2006). Thus, epigenetic alterations evoked by genotoxic carcinogens may drive the process of carcinogenesis after the initiating genotoxic event (Pogribny et al., 2011). Conversely, global DNA hypomethylation and tumor suppressor gene hypermethylation may increase the occurrence of mutations in cells exposed to nongenotoxic carcinogens. In this sense, epigenetic mechanisms may also initiate carcinogenesis (Goodman and Watson, 2002).

Failure to Execute Apoptosis Promotes Mutation and Clonal Growth

As discussed earlier, in cells suffering DNA damage, the stabilized p53 protein may induce cell death by apoptosis (Fig. 3-33). Apoptosis thus eliminates cells with DNA damage, preventing mutations to initiate carcinogenesis. Initiated preneoplastic cells have much higher apoptotic activity than do normal cells (Bursch et al., 1992) and this can counteract their clonal expansion. In fact, facilitation of apoptosis can induce tumor regression. This occurs when hormone-dependent tumors are deprived of the hormone that promotes growth and suppresses apoptosis. This is the rationale for the use of tamoxifen, an antiestrogen, and gonadotropin-releasing hormone analogues to combat hormone-dependent tumors of the mammary gland and the prostate gland, respectively (Bursch et al., 1992).

Thus, inhibition of apoptosis is detrimental because it facilitates both mutations and clonal expansion of preneoplastic cells. Indeed, apoptosis inhibition plays a role in the pathogenesis of human B-cell lymphomas, in which a chromosomal translocation results in aberrantly increased expression of Bcl-2 protein, which overrides programmed cell death after binding to and inactivating the proapoptotic Bax protein (see Fig. 3-19). Increased levels of Bcl-2 are also detected in other types of cancer, and a high Bcl-2/Bax ratio in a tumor is a marker for poor prognosis (Jäättelä, 1999). Other antiapoptotic proteins, such as Hsp27, Hsp70, and Hsp90 (Sreedhar and Csermely, 2004) and IAP family members, may also contribute to progression of neoplasia. Survivin, a member of the IAP family, is expressed in all cancer cells but not in adult differentiated cells (Jäättelä, 1999).

Inhibition of apoptosis is one mechanism by which the rodent tumor promoter phenobarbital promotes clonal expansion of preneoplastic cells. This has been demonstrated in rats given a single dose of N-nitrosomorpholine followed by daily treatments with phenobarbital for 12 months to initiate and promote, respectively, neoplastic transformation in liver (Schulte-Hermann et al., 1990). From 6 months onward, phenobarbital did not increase DNA synthesis and cell division in the preneoplastic foci, yet it accelerated foci enlargement. The foci grow because phenobarbital lowers apoptotic activity, allowing the high cell replicative activity to manifest itself. The peroxisome proliferator nafenopin, a nongenotoxic hepatocarcinogen, also suppresses apoptosis in primary rat hepatocyte cultures (Bayly et al., 1994). Based on a recent study with WY-14643, another peroxisome proliferator (Gonzalez and Shah, 2008), it may be suggested that miR-17-92 upregulation and consequential suppression of Bim translation accounts for apoptosis inhibition.

Failure to Restrain Cell Division Promotes Mutation, Proto-Oncogene Expression, and Clonal Growth

Enhanced mitotic activity, whether it is induced by oncogenes inside the cell or by external factors such as xenobiotic or endogenous mitogens, promotes carcinogenesis for a number of reasons:

1. First, the enhanced mitotic activity increases the probability of mutations. This is due in part to activation of the cell division cycle, which invokes a substantial shortening of the G_l phase, allowing less time for the repair of injured DNA before replication, thus increasing the chance that the damage will yield a mutation. Although repair still may be feasible after replication, postreplication repair is error-prone. In addition, activation of the cell division cycle increases the proportion of cells that replicate their DNA at any given time. During replication, DNA becomes unpacked and its amount doubles, greatly increasing the effective target size for DNA-reactive mutagenic chemicals, including ROS.

2. Enhanced mitotic activity may compromise DNA methylation, which occurs in the early postreplication period and is carried out by DNMTs that copy the methylation pattern of the parental DNA strand to the daughter strand (maintenance methylation). Limitation of DNMTs by shortened G₂ phase or by presence of other transacting factors might impair methylation and may contribute to overexpression of proto-oncogens, starting a vicious cycle.

3. During cell division, the cell-to-cell communication through gap junctions (constructed from connexins) and AJ (constructed from cadherins) is temporarily disrupted. Several tumor promoters, such as phenobarbital, phorbol esters, and peroxisome proliferators, decrease gap junctional intercellular communication. It has been hypothesized that this contributes to neoplastic transformation. Increased susceptibility of connexin-knockout mice to spontaneous and chemically induced liver tumors supports this hypothesis (Chipman et al., 2003). Lack of the AJ contributes to the invasiveness of tumor cells (see below).

4. Another mechanism by which proliferation promotes the carcinogenic process is through clonal expansion of the initiated cells to form nodules (foci) and tumors.

In summary, transformation of normal cells with controlled proliferative activity to malignant cells with uncontrolled proliferative activity is driven by 3 major forces: (1) accumulation of genetic damage in the form of mutant proto-oncogenes (which encode permanently activated cell cycle accelerator and/or antiapoptotic proteins) and mutant tumor suppressor genes (which encode permanently inactivated cell cycle decelerator and/or proapoptotic proteins); (2) increased transcription and/or translation of normal proto-oncogenes (causing expression of more cell cycle accelerator and/or antiapoptotic proteins); and (3) silencing of normal tumor suppressor genes at transcriptional and/or translational level (causing expression of less cell cycle decelerator and/or proapoptotic proteins). Uncontrolled proliferation results from offset of the balance between mitosis and apoptosis (Fig. 3-34).

Genotoxic carcinogens appear to induce cancer primarily by inducing activating mutations in proto-oncogenes or inactivating mutations in tumor suppressor genes, and secondarily by causing inappropriate expression rate of these critical genes. In contrast, nongenotoxic carcinogens cause cancer primarily by transcriptional or translational upregulation of the synthesis of proto-oncogene proteins or downregulation of the synthesis of tumor suppressor proteins. Secondarily, however, nongenotoxic carcinogens may also increase mutation of critical genes, which is initiated by genotoxic agents or spontaneous DNA damage. In human cells, spontaneous DNA damage may give rise to mutation at a frequency of 1 out of 10⁸ to 10¹⁰ base pairs (Barrett, 1992). Genotoxic carcinogens increase this frequency 10- to 1000-fold. Nongenotoxic carcinogens also increase the rate of spontaneous mutations through a mitogenic effect (see above) and increase the number of cells with DNA damage and mutations by facilitating their division and inhibiting their apoptosis. Both enhanced mitotic activity and decreased apoptotic activity expand the population of transformed cells.

According to an emerging theory, cancers may form by genotoxic and/or epigenetic mechanisms in pluripotent stem cell populations. Such cells are characterized by quiescence, self-renewal, and conditional immortality, thus would potentially supply a lifelong, latent neoplastic population after carcinogen attack. Stem cells may be an especially likely target for transplacental carcinogens, such as DES or arsenic (Tokar et al., 2011), because they are abundant during fetal development and have the longevity to carry the molecular lesion to the adult period of life.

Finally, further changes in gene expression may occur in these proliferating cells making them capable of invading the tissue and forming metastasis. These alterations confer the cells capacity for increased mobility by (1) their transdifferentiation into a mobile fibroblastoid phenotype (called epithelial-to-mesenchymal transition [EMT]), (2) disruption of the AJ that anchor cells to their neighbor (by downregulation of E-cadherin expression), and (3) degradation of the surrounding ECM (by upregulation of matrix metalloproteinases). Signaling systems that orchestrate EMT include those triggered by TGF-β (Fig. 3-12), the Wnt ligand (see Fig. 3-13), and the Hh ligand (see Fig. 3-14) (Katoh and Katoh, 2008).

This overview systematically surveys the mechanisms of the potential events that follow toxicant exposure and contribute to toxicity.

This approach is also useful in the search for mechanisms responsible for (1) selective toxicity, that is, differences in the sensitivity to toxicants of various organisms, such as different species and strains of animals, organs, and cells, and (2) alteration of toxicity by exogenous factors such as chemicals and food and physiologic or pathologic conditions such as aging and disease. To identify the mechanisms that underlie selective toxicity or alterations in toxicity, all steps where variations might occur must be considered systematically. Selective or altered toxicity may be due to different or altered (1) exposure; (2) delivery, thus resulting in a different concentration of the ultimate toxicant at the target site; (3) target molecules; (4) biochemical processes triggered by the reaction of the chemical with the target molecules; (5) repair at the molecular, cellular, or tissue level; or (6) altered gene expression–based stress responses as well as circulatory and thermoregulatory reflexes by which the affected organism can adapt to some of the toxic effects.

In this chapter, a simplified scheme has been used to give an overview of the development of toxicity (Fig. 3-1). In reality, the route to toxicity can be considerably more diverse and complicated. For example, one chemical may yield several ultimate toxicants, one ultimate toxicant may react with several types of target molecules, and reaction with one type of target molecule may have a number of consequences. Thus, the toxicity of one chemical may involve several mechanisms that can interact with and influence each other in an intricate manner.

This chapter has emphasized the significance of the chemistry of a toxicant in governing its delivery to and reaction with the target molecule as well as the importance of the biochemistry, molecular and cell biology, immunology, and physiology of the affected organism in its response to the action of the toxicant. An organism has mechanisms that (1) counteract the delivery of toxicants, such as detoxication; (2) reverse the toxic injury, such as repair mechanisms; and (3) offset some dysfunctions, such as adaptive responses. Thus, toxicity is not an inevitable consequence of toxicant exposure because it may be prevented, reversed, or compensated for by such mechanisms. Toxicity develops if the toxicant exhausts or impairs the protective mechanisms and/or overrides the adaptability of biological systems.