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A large and highly accessible human organ, the skin protects the body against external insults to maintain internal homeostasis. Its biological sophistication allows it to perform a myriad of functions above and beyond that of a suit of armor. Physiologically, the skin participates directly in thermal, electrolyte, hormonal, metabolic, antimicrobial and immune regulation, without which a human would perish. Rather than merely repelling noxious physical agents, the skin may react to them with a variety of defensive mechanisms that prevent internal or widespread cutaneous damage. If an insult is severe or sufficiently intense to overwhelm the protective function of the skin, acute or chronic injury becomes readily manifest in various ways. The specific presentation depends on a variety of intrinsic and extrinsic factors including body site, duration of exposure, and other environmental conditions (Table 19-1).
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The skin consists of two major components—the outer epidermis and the underlying dermis, which are separated by a basement membrane (Fig. 19-1). The junction ordinarily is not flat but has an undulating appearance (rete ridges). In addition, epidermal appendages (hair follicles, sebaceous glands, and eccrine glands) span the epidermis and are embedded in the dermis. In thickness, the dermis comprises approximately 90% of the skin and has largely a supportive function. It has a high content of collagen and elastin secreted by scattered fibroblasts, thus providing the skin with elastic properties. Separating the dermis from underlying tissues is a layer of adipocytes, whose accumulation of fat has a cushioning action. The blood supply to the epidermis originates in the capillaries located in the rete ridges at the dermal–epidermal junction. Capillaries also supply the bulbs of the hair follicles and the secretory cells of the eccrine (sweat) glands. The ducts from these glands carry a dilute salt solution to the surface of the skin, where its evaporation provides cooling.
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The interfollicular epidermis is a stratified squamous epithelium consisting primarily of keratinocytes. These cells are tightly attached to each other by desmosomes and to the basement membrane by hemidesmosomes. Melanocytes are interspersed among the basal cells and distributed in the papilla of hair follicles. In the epidermis, these cells are stimulated by ultraviolet light to produce melanin granules. The granules are extruded and taken up by the surrounding keratinocytes, which thereby become pigmented. Migrating through the epidermis are numerous Langerhans cells, which are important participants in the immune response of skin to foreign agents.
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Keratinocytes of the basal layer comprise the germinative compartment. When a basal cell divides, one of the progeny detaches from the basal lamina and migrates outward. As cells move toward the skin surface, they undergo a remarkable program of terminal differentiation. They gradually express new protein markers and accumulate keratin proteins, from which the name of this cell type is derived. The keratins form insoluble intermediate filaments accounting for nearly 40% of the total cell protein in the spinous layer. At the granular layer, the cells undergo a striking morphological transformation, becoming flattened and increasing in volume nearly 40-fold. Lipid granules fuse with the plasma membrane at the granular layer/stratum corneum interface, filling the intercellular spaces of the stratum corneum with lipid, as opposed to the aqueous intercellular solution in the viable epidermis. This lipid performs a variety of defensive functions, both in preventing diffusion of water and ions out of the body, and preventing access of toxins and bacteria into the body. It thus constitutes the major barrier to toxin entry into the skin (Elias, 2005). Meanwhile, the plasma membranes of these cells become permeable, resulting in the loss of their reducing environment and consequently in extensive disulfide bonding among keratin proteins. Cell organelles are degraded, while a protein envelope is synthesized immediately beneath the plasma membrane. The membrane is altered characteristically by the loss of phospholipid and the addition of sphingolipid.
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This program of terminal differentiation, beginning as keratinocytes leave the basal layer, produces the outermost layer of the skin, the stratum corneum. No longer viable, the mature cells (called corneocytes) are approximately 80% keratin in content. They are gradually shed from the surface and replaced from beneath. The process typically takes two weeks for basal cells to reach the stratum corneum and another two weeks to be shed from the surface. In the skin disease psoriasis, the migration of cells to the surface is nearly 10-fold faster than normal, resulting in a stratum corneum populated by cells that are not completely mature. In instances in which the outer layer is deficient due to disease or physical or chemical trauma, the barrier to the environment that the skin provides is inferior to that provided by normal, healthy skin.
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Percutaneous Absorption
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Until the turn of the century, the skin was believed to provide an impervious barrier to exogenous substances. Gradually, the ability of substances to penetrate the skin, though this process generally is very slow, became appreciated. During the past 50 years, the stratum corneum has been recognized as the primary barrier (Scheuplein and Blank, 1971). Diseases (eg, psoriasis) or other conditions (eg, abrasion, wounding) in which this barrier is compromised can permit greatly increased uptake of poorly permeable substances, as does removal of the stratum corneum by tape stripping or organic solvents such as acetone. The viable layer of epidermis provides a much less effective barrier, because hydrophilic chemicals readily diffuse into the intercellular water, while hydrophobic agents can partition into cell membranes, and each can diffuse readily to the blood supply in the rete ridges of the dermis.
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Probably the best-known biological membrane barrier for this purpose, the stratum corneum prevents water loss from underlying tissues by evaporation. Its hydrophobic character reflects the lipid content of the intercellular space, approximately 15% of the total volume (Elias, 1992). The lipids, a major component being sphingolipids, have a high content of long-chain ceramides, removal of which seriously compromises barrier function as measured by transepidermal water loss. The stratum corneum ordinarily is hydrated (typically 20% water), the moisture residing in corneocyte protein, but it can take up a great deal more water upon prolonged immersion, thereby reducing the effectiveness of the barrier to chemicals with a hydrophilic character. Indeed, occlusion of the skin with plastic wrap, permitting the retention of perspiration underneath, is a commonly employed technique to enhance uptake of chemicals applied to the skin surface. Although penetration from the air is generally too low to be of concern, protection from skin uptake may be advisable for some compounds (eg, nitrobenzene) at concentrations high enough to require respirator use.
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Finding the rate at which the uptake of agents through the skin occurs is important for estimating the consequences of exposure to many agents we encounter in the environment. Indeed, a regulatory strategy permitting bathing in water considered barely unfit for drinking was revised when it was realized that exposure from dermal/inhalation uptake during bathing could be comparable to that from drinking 2 L of the water (Brown et al., 1984). Uptake through the skin is now incorporated in pharmacokinetic modeling to estimate potential risks from exposures. The degree of uptake depends upon the details of exposure conditions, being proportional to solute concentration (assuming it is dilute), time, and the amount of skin surface exposed. In addition, two intrinsic factors contribute to the absorption rate of a given compound: its hydrophobicity, which affects its ability to partition into epidermal lipid, and its rate of diffusion through this barrier. A measure of the first property is the commonly used octanol/water partitioning ratio (Kow). This is particularly relevant for exposure to contaminated water, as occurs during bathing or swimming. However, partitioning of a chemical into the skin is greatly affected by its solubility in or adhesion to the medium in which it is applied (including soil). Similarly, very hydrophobic compounds, once in the stratum corneum, may diffuse only very slowly into less hydrophobic regions below. The second property is an inverse function of molecular weight (MW) or molecular volume. Thus, hydrophobic agents of low MW permeate the skin better than those of high MW or those that are hydrophilic. For small molecules, hydrophobicity is a dominant factor in penetration.
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Although only small amounts of chemicals may penetrate the stratum corneum, those of high potency may still be very dangerous. For example, hydrophobic organophosphorus and carbamate pesticides can be neurotoxic to humans and domestic animals by skin contact. Children and adolescents harvesting tobacco are especially susceptible to poisoning by contact with nicotine, a natural pesticide present in moisture on the leaves (McKnight and Spiller, 2005). Conditions of topical treatment of livestock for pest control must take into consideration not only the tolerance of the animals but also residues in meat and milk resulting from skin penetration. High level skin exposure to chemicals considered safe at low levels can be dangerous, evident from the nervous system toxicity and deaths of babies exposed to hexachlorophene mistakenly added to talcum powder for their diapers (Martin-Bouyer et al., 1982). Previous findings of N-nitrosamines that penetrate skin well (such as N-nitrosodiethanolamine) in cutting oils and cosmetics raised concern and led to their monitoring and to reduction of exposure.
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Considerable empirical information has been collected on some compounds of special interest (including pharmaceuticals, pesticides, and pollutants) for use in quantifying structure–penetration relationships. From such information, relations can be obtained for skin penetration (Pcw) using empirically derived constants (C1, C2, C3) that have the form shown below (Potts and Guy, 1992).
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log Pcw = C1 − C2(MW) + C3 log Kow
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Such relations describe steady-state conditions, in which a chemical leaves the stratum corneum at the same rate it enters. Because rates of transfer of very hydrophobic agents into the aqueous phase of the spinous layer are slow, saturation of the stratum corneum provides a depot, leading to continued penetration into the body for relatively long time periods after external exposure to a chemical stops. Recognition that such estimates overpredict uptake from short exposures has led to modeling of limited doses (Guy, 2010a).
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Diffusion through the epidermis is considerably faster at some anatomical sites than others. A list in order of decreasing permeability gives the following hierarchy: foot sole > palm > forehead > abdomen (Scheuplein and Blank, 1971). Scrotal skin reportedly has the highest permeability for some topical chemicals (Fisher, 1989). Under ordinary conditions, absorption through the epidermal appendages is generally neglected, despite the ability of chemicals to bypass the stratum corneum by this route, because the combined appendageal surface area is such a small fraction of the total available for uptake. However, because loading of the stratum corneum is slow, penetration through the appendages can constitute an appreciable fraction of the total for short exposures. In some cases, the effects of appendages can even be dominant. For instance, benzo[a] pyrene penetrates the skin of haired mice several fold faster than that of hairless strains (Kao and Hall, 1987). Recent studies indicate the hair follicles serve as important routes of entry for hydrophilic agents and pharmaceuticals as well, a property that could be exploited for therapeutic purposes (Otberg et al., 2008).
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Transdermal Drug Delivery
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The ability of the stratum corneum to serve as a reservoir for exogenously applied agents is well illustrated by the recent development of methods for the delivery of pharmaceuticals. Application of drugs to the skin can produce systemic effects, a phenomenon observed unintentionally before the ability of the skin to serve as a delivery system was appreciated. For example, topical exposure of young girls to estrogens has led to reports of pseudoprecocious puberty, while in young or adult males, such exposure has produced gynecomastia (Amin et al., 1998). Transfer of topically applied hormonal medication from a treated to an untreated individual with consequent effects on the latter is also observed (Busse and Maibach, 2011).
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Specially designed patches are currently in use to deliver at least 17 different drugs (including estradiol, testosterone, nitroglycerin, scopolamine, clonidine, fentanyl, and nicotine) for therapeutic purposes (Guy, 2010b), and others are under development. The advantages of this approach over oral dosing include providing a steady infusion for extended periods (typically one-seven days), thereby avoiding large variations in plasma concentration, preventing exposure to the acidic pH of the stomach, and avoiding biotransformation in the gastrointestinal tract or from first-pass removal by the liver. The contrast in plasma concentration kinetics between different methods of delivery is particularly evident for chemicals that are rapidly metabolized, such as nitroglycerin, which has a half-life of minutes. A variety of chemicals, chosen carefully to minimize irritation or allergenicity, have been incorporated into pharmaceutical preparations to enhance absorption and penetration. In addition, encapsulating a drug in small vesicles of phospholipid or nonionic surfactant or coating nanoparticles can improve delivery through the epidermis and hair follicles (Desai et al., 2010). Finally, various microneedle systems have been studied to deliver drugs, vaccines, and gene therapy across the skin. These systems may prove especially important in delivering vaccines in areas where refrigeration for conventional vaccines is not available (Prausnitz et al., 2009).
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Measurements of Penetration
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A pharmacokinetic approach with intact subjects has been commonly employed with experimental animals. To simplify determination of penetration kinetics, skin flaps may be employed and the capillary blood flow monitored to measure penetration. For this purpose, pig skin has particular utility (Riviere and Brooks, 2005). Because penetration through rodent skin is usually faster than through human skin, the former can provide an overestimate for behavior of the latter. Without verification using human skin, such measurements are subject to large uncertainties due to species differences in density of epidermal appendages, stratum corneum properties (eg, thickness, lipid composition), and biotransformation rates. A promising variation minimizing species differences is to use skin grafts on experimental animals for these measurements. Human skin persists well on athymic mice and retains its normal barrier properties.
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For risk assessment and pharmaceutical design, the most useful subject for experimentation is human skin. Volunteers are dosed, plasma and/or urine concentrations are quantified at suitable intervals, and amounts excreted from the body are estimated. Past measurements of penetration often used 14C-labeled agents. This approach is not preferred, but use of isotopic labels now is readily feasible when coupled to ultrasensitive detection by accelerator mass spectrometry (Buchholz et al., 1999). For in vitro work, excised split-thickness skin can be employed in special diffusion chambers, though care is needed to preserve the viability of the living layer of epidermis. The chemical is removed for measurement from the underside by a fluid into which it partitions, thereby permitting continued penetration. Commonly employed is a simpler setup using cadaver skin with the lower dermis removed. This lacks biotransformation capability, but retains the barrier function of the stratum corneum. Accurate testing of percutaneous absorption of poorly soluble chemicals from environmental and pharmaceutical substrates requires attention to details of particle size, component complexes, vehicle, application rate, and skin contact. A long time goal has been to predict chemical permeability from chemical structure. Comparisons of predictive models with results from transdermal patches show good correlations, despite large discrepancies in fluxes of some compounds, and emphasize the importance of experimental details (Farahmand and Maibach, 2009).
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The ability of the skin to metabolize chemicals that diffuse through it contributes to its barrier function. In specific activity, phase I metabolism in the skin usually is only a small fraction (≈3%) of that in the liver (Rolsted et al., 2008), but it is capable of affecting the outcome of exposure. Biotransformation influences the biological activity of xenobiotics and topically applied drugs, leading to their degradation or their activation as skin sensitizers or carcinogens (Svensson, 2009). While overall activities are normally low, they can be induced (or suppressed). In fact, a large fraction of the pharmaceuticals used in clinical dermatology are cytochrome P450 inducers, inhibitors, or substrates (Ahmad and Mukhtar, 2004). For example, cytochromes P450 1A1 and 1B1 are inducible in the epidermis to high levels by crude coal tar (Smith et al., 2006), which is used in dermatological therapy. Well-known xenobiotic inducers of these isozymes include tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons, and coplanar polychlorinated biphenyls. Exposure to such inducers could influence skin biotransformation and even sensitize epidermal cells to other chemicals that are not good inducers themselves, a phenomenon observable in cell culture (Walsh et al., 1995).
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Biotransformation of a variety of compounds in the skin has been detected, including arachidonic acid derivatives, steroids, retinoids, amines, and polycyclic aromatic hydrocarbons, suggesting that multiple P450 activities are expressed. Using sensitive technologies, it is now evident that numerous distinct isozymes are expressed at widely varying levels. Evidence for expression of >30 CYP genes has been obtained by DNA microarray and real-time polymerase chain reaction (Hu et al., 2010; Luu-The et al., 2009). A recent survey of those in the CYP1-4 families indicated that half were expressed at substantially higher levels in differentiating keratinocytes (similar to spinous cells) than in basal-like cells (Du et al., 2006). Species differences are apparent in the amounts of P450 activities detectable. For example, measured ethoxycoumarin-O-deethylase activity is 20-fold higher in mouse than human (or rat) skin. Differences of such magnitude help rationalize the observation that the rate of penetration of ethoxycoumarin is sufficient to saturate its metabolism in some species such as human but not in others such as mouse or guinea pig (Storm et al., 1990). Beyond the cytochromes P450, other phase I enzymes expressed in the skin include flavin-dependent monooxygenases, aldehyde dehydrogenases, carboxylesterases, and glutathione peroxidases (Hu et al., 2010). To the extent that phase I and II metabolism influences sensitization to exogenous chemicals (Svensson, 2009), they may also help rationalize species differences in allergic response.
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Enzymes participating in phase II metabolism are expressed in skin. Several isozymes each of UDP glucuronosyltransferase, sulfotransferase, and glutathione S-transferase have been detected in human (Hu et al., 2010; Luu-The et al., 2009) and rodent skin (Oesch et al., 2007). In general, this activity occurs at a lower rate than observed in the liver, but exceptions are evident, as in the case of quinone reductase (Khan et al., 1987). Different species express different relative amounts of the various isozymes, which could alter resulting target specificities or degrees of responsiveness. Glutathione S-transferase, for instance, catalyzes the reaction of glutathione with exogenous electrophiles or provides intracellular transport of bound compounds in the absence of a reaction. It also facilitates the reaction of glutathione with endogenous products of arachidonate lipoxygenation (leukotrienes) to yield mediators of anaphylaxis and chemotaxis, which are elements of the inflammatory response in skin. Of the first three major transferase forms characterized in liver, the dominant form in skin of humans and rodents is the P isozyme. A comparison of human with rodent skin indicates the former has higher glutathione S-transferase activity but lower glutathione content, suggesting that it may be less susceptible to low doses of electrophilic substrates, whereas rodent skin could be less susceptible at higher doses when glutathione is depleted in human but not mouse skin (Jewell et al., 2000).
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A variety of other metabolic enzyme activities have also been detected in human epidermal cells, including sulfatases, β-glucuronidase, N-acetyl transferases, esterases, and reductases (Hu et al., 2010; Luu-The et al., 2009). In addition, the intercellular region of the stratum corneum has catabolic activities (eg, protease, lipase, glycosidase, and phosphatase) supplied by the lamellar bodies along with their characteristic lipid (Elias, 1992). The epidermis and pilosebaceous units are the most important drug targets and sites of toxic effects sites and, indeed, are the major sources of biotransformation activity in the skin. However, other cell types such as fibroblasts are known to participate in biotransformation, helping to rationalize observations that organotypic cell cultures containing underlying fibroblasts resemble natural skin in biotransformation better than those without fibroblasts (Gibbs et al., 2007; Hu et al., 2010; Luu-The et al., 2009). Such cultures, where keratinocytes float on collagen gels at the air–liquid interface, are better skin mimics than conventional ones using established keratinocyte lines. Their development has stimulated aspirations that these in vitro models can replace much animal testing of cosmetics and potential skin toxicants.
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The report that cholesterol sulfotransferase is regulated by ligands of LXR and PPAR illustrates the potential for exogenous chemicals, including pharmaceuticals, to influence such activities and thereby the barrier function of the skin (Jiang et al., 2005), where penetration can be influenced by cell differentiation and biotransformation. The influence of hydroxysteroid dehydrogenases and microsomal reductase activities during percutaneous absorption is evident in studies on skin in organ culture. Biotransformation can activate prodrugs, an example being minoxidil, applied to prevent hair loss, which undergoes sulfation locally to its active form. To permit increased penetration of pharmaceuticals through the lipid barrier of the stratum corneum, prodrug design strategies have been developed that depend on epidermal esterase activity. Thus, hydrophobic esters of pharmaceuticals targeting the skin (eg, acne) could be applied to the skin surface, de-esterified during transit, and yield slow release of the active forms (Fang and Leu, 2006).