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Classic Reducing-Type Air Pollution
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The acute air pollution episodes of the 20th century showed that high concentrations of the reducing-type air pollution, characterized by SO2 and smoke, are capable of producing dramatic human health effects. Empirical studies in human subjects and animals have long stressed the irritancy of SO2 and its role in these incidents, while the full potential for interactions among the copollutants in the smoky, sulfurous mix has a mixed record of replication in the human exposure laboratory. Because of its history, SO2 is often thought of as the prototypic air pollutant of the industrial age, and as such it has a detailed historical literature base. It is an irritant gas that has a toxicology of its own and, through atmospheric reactions, can transform photochemically into sulfites or sulfates within a secondarily irritant particle. The impact of SO2 emissions on irritancy, the formation of secondary particles that affect visibility, and its damaging acidification of the environment have been the focus of much regulation for coal-fired power plants since 1970. Levels are greatly reduced (>60%) in the United States and continue to fall under new cross-state regulations.
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Sulfur dioxide is a water-soluble irritant gas. As such, it is absorbed predominantly in the upper airways. It is a sensory irritant and can stimulate bronchoconstriction and mucus secretion in a number of species, including humans. Early studies with relatively high exposure concentrations of SO2 showed airway cellular injury and subsequent proliferation of mucus-secreting goblet cells. This attribute of SO2 has led to its use (>250 ppm) in the production of laboratory animal models of bronchitis and airway injury (Kodavanti et al., 2000). At much lower concentrations (<1 ppm), such as might be encountered in the polluted ambient air of industrialized areas, long-term residents experience a higher incidence of bronchitis. In fact, prior to the breakup of the Soviet block, many eastern European cities were renowned for widespread public affliction with bronchitis; 20 years later, the prevalence of bronchitis was greatly reduced (von Mutius et al., 1994). While other factors (diet, access to health care, other pollutants) may well have been involved in this reversal, reductions in ambient smoke and SO2 are generally thought to be the most important.
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The concentrations of SO2 likely to be encountered in the United States are lower still—on average, considerably less than 0.01 ppm. Rats exposed for 70 to 170 hours to 0.1, 1.0, and 20 ppm exhibited reduced clearance of inert particles, while dogs exposed to 1 ppm for a year had slowed tracheal mucociliary transport. Analogously, mouse models show that mice exposed to 0.1 ppm were more susceptible to bacterial infections. The fact that the low-concentration exposures showed marked effects when extended over longer periods is consistent with the epidemiological associations between SO2 exposure and bronchitis. The evidence is not clear, however, as some studies show no overt long-term pulmonary pathology. Guinea pigs and monkeys, for example, showed no effect on lung function or pathology after a year of continuous exposure to concentrations of 0.1 to 5 ppm SO2 (Alarie et al., 1970, 1972).
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The penetration of SO2 into the lungs is greater during mouth as opposed to nose breathing. An increase in the airflow during deep rapid breathing augments penetration of the gas into the deeper lung. As a result, persons exercising would inhale more SO2 and, as noted with asthmatics, are likely to experience greater irritation. Once deposited along the airway, SO2 dissolves into surface lining fluid as sulfite or bisulfite and is readily distributed throughout the body. It is thought that the sulfite interacts with sensory receptors in the airways to initiate local and centrally mediated bronchoconstriction.
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Pulmonary Function Effects
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The basic pulmonary response to inhaled SO2 is mild bronchoconstriction, which is reflected as a measurable increase in airflow resistance due to narrowing of the airways. Concentration-related increases in resistance have been observed in guinea pigs, dogs, and cats as well as humans. Exposure of isolated segments of the nose or airways of dogs and guinea pigs appeared to alter resistance in a manner consistent with receptor-mediated sensory stimulation. Airflow resistance increased more when the gas was introduced through a tracheal cannula than via the nose, since nasal scrubbing of the water-soluble gas was bypassed. Isolated nasal exposures increased airflow resistance through the nose largely as a result of mucosal swelling, but the irritant effect appeared to signal the more distal airways as well. Direct exposure of the trachea had a more dramatic effect on airflow resistance. Exposure of the intact nose also induced some response, consistent with the existence of a nasal neural network being involved in bronchoconstriction (Frank and Speizer, 1965; Nadel et al., 1965). Intravenous injection of atropine (a parasympathetic receptor blocker) or cooling of the cervical vagal nerves abolishes bronchoconstriction in the cat model; rewarming of the nerve reestablishes the response. The rapidity of the response and its reversal emphasize the parasympathetic tonal change in airway smooth muscle. Studies in human subjects have confirmed the predominance of parasympathetic mediation, but histamine from inflammatory cells may play a secondary role in the bronchoconstrictive responses of asthmatics.
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Human subjects exposed to 1, 5, or 13 ppm SO2 for just 10 minutes exhibit a rapid bronchoconstrictive response, with 1 to 3 ppm being a threshold for most if exercise is involved. Exposures to 0.25 to 1 ppm for a few hours can induce bronchoconstriction in adult and adolescent subjects with clinically defined mild asthma (Sheppard et al., 1981; Koenig et al., 1981). Findings such as these (responses <0.5 ppm) have raised concerns about potential adverse effects in this sensitive subpopulation when it is exposed to peaks of SO2 that are known to occur near point sources.
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Only a few long-term studies have been conducted with SO2 at levels approaching those found in ambient air. Alarie et al. (1970) exposed guinea pigs to 0.1, 1.0, or 5.7 ppm SO2 continuously for a year without adverse impact on lung mechanics. Similarly, monkeys exhibited no alteration in pulmonary function when exposed continuously for 78 weeks to 0.1, 0.6, and 1.3 ppm SO2 (Alarie et al., 1972). Even in the presence of 0.1 mg/m3 sulfuric acid, dogs exposed 16 hours a day for 18 months to 0.5 ppm SO2 showed no impairment in pulmonary function (Vaughan et al., 1969). Higher levels of SO2 for protracted periods of time (dogs to 5 ppm for 225 days [Lewis et al., 1969]; rats to 350 ppm for 30 days [Reid, 1963]) have been shown to alter airway mucus secretion, goblet cell topography, or lung function, but these results are of little relevance to typical SO2 levels in ambient air.
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Sulfuric Acid and Related Sulfates
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The conversion of SO2 to sulfate is favored in the environment with subsequent ammonia neutralization to ammonium sulfate [(NH4)2SO4] or ammonium bisulfate [NH4HSO4]. During oil and coal combustion or the smelting of metal ores, sulfuric acid condenses downstream of the combustion processes with available metal ions and water vapor to form submicrometer sulfuric acid fume and sulfated fly ash. Sulfur dioxide continues to oxidize to sulfate within dispersing smokestack plumes, which can be augmented by the presence of free soluble or partially coordinated transition metals such as iron, manganese, and vanadium within the effluent ash. When coal is burned, the acid may adsorb to the surface or solubilize in ultrafine (<0.1 μm) metal oxide particles during emission. Photochemical reactions also promote acid sulfate formation via both metal-dependent and independent mechanisms, but studies have shown that most of the oxidation of SO2 occurs within plumes as they disperse in the atmosphere. Stack emissions may undergo long-range transport to areas distant from the emission source, allowing considerable time for sunlight-driven chemistry. Although the fine particle sulfates may exist as fine sulfuric acid (the primary source of free H+), partially or fully neutralized forms of sulfate predominate due to the abundance of natural atmospheric ammonia. As fine PM sulfates are transported long distances, they may contribute to regional summer haze and pose a health hazard to certain groups such as asthmatics (Koenig et al., 1989), and may also stress the general environment as acid rain (Calvert et al., 1985) (Fig. 29-13). The CAA and subsequent regulations for the reduction of sulfur emissions (eg, the 1990 Acid Rain Program and the 2012 Cross State Air Pollution Rule) continue to reduce the environmental acidification and visibility problems associated with transport and deposition of acidic products formed from stack emissions in North America.
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Sulfuric acid irritates by virtue of its ability to protonate (H+) receptor ligands and other biomolecules. This action can either directly damage membranes or activate sensory reflexes that initiate inflammation. Ammonia, which exists in free air at about 25 ppb and in much higher concentrations within the mammalian naso-oropharynx (in the human up to 350 ppm), is capable of neutralizing most of the irritant acidic sulfates (Utell et al., 1989). Neutralization can also be quite efficient in standard whole-body animal exposure studies, in which excreta and bacteria in the chambers interact, giving rise to in-chamber ammonia concentrations up to 1100 ppb—more than enough to fully neutralize neat sulfuric acid up to several mg/m3 (Higuchi and Davies, 1993).
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Interestingly, there is considerable species variability in sensitivity to sulfuric acid, with guinea pigs being quite responsive to acid sulfates, in contrast to rats, which seem generally resistant. The reasons for this difference relate to sensory fiber network density in the airways, and probably not on differences in neutralization by ammonia in the airway. The sensitivity of healthy humans appears to fall somewhere in between, with asthmatic humans being perhaps best modeled by the guinea pig. Overall, however, the collective data involving animals and humans are remarkably coherent (Amdur, 1989).
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Unlike other irritants, such as O3 (see below), inhaled sulfuric acid does not appear to stimulate a classic neutrophilic lung inflammation. Rather, eicosanoid homeostasis appears to be disturbed resulting in macrophage dysfunction and altered host defense.
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Pulmonary Function Effects
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Sulfuric acid produces an increase in flow resistance in guinea pigs due to reflex airway narrowing, or bronchoconstriction, which impedes the flow of air into and out of the lungs. This response might be thought of as a defensive measure to limit the inhalation of air containing noxious gases, but this explanation may be more teleological than fact. The magnitude of the response is related to both acid concentration and particle size (Amdur et al., 1978). Early studies indicated that as particle size was reduced from 7 μm to the submicrometer range, the concentration of sulfuric acid necessary to induce a response and the time to the onset of the response fell significantly. With large particles, even the sensitive guinea pig was able to withstand an exceedingly high (30 mg/m3) challenge with little change in pulmonary resistance, in contrast to the <1 mg/m3 challenge needed with the 0.3-μm particles (Amdur et al., 1978). Human asthmatics exposed to 2 mg/m3 of acid fog (10 μm) for one hour, a very high concentration for an asthmatic, experienced variable respiratory symptoms suggesting irritation, but no changes in spirometry were elicited (Hackney et al., 1989). The apparent reason for this PM size–based differential is probably the scrubbing of large particles in the nose, while small particles are able to penetrate deep into the lung. The thicker mucus blanket of the nose may also blunt (by dilution or neutralization by mucus buffers) much of the irritancy of the deposited acid. In contrast, the less shielded distal airway tissues, with higher receptor density, would be expected to be more sensitive to the acid particles reaching that area (Costa and Schlegele, 1998). Regional sensitivity and the longer residence times of a deposited particle relative to SO2 gas are reflected in the relatively protracted recovery times observed in acid-exposed guinea pigs compared with those in animals exposed to SO2 alone.
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Asthmatics appear to be somewhat more sensitive to the bronchoconstrictive effects of sulfuric acid than are healthy individuals, but published studies have been inconsistent in this finding (Koenig et al., 1989; Utell et al., 1984). Asthma generally is characterized by hyperresponsive airways, so their tendency to constrict at low acid concentrations would be expected, just as asthmatic airways are sensitive to nonspecific airway smooth muscle agonists (eg, carbachol, histamine, exercise). The variability may well relate to differences in the degree of impairment or underlying inflammation in the subjects, but this hypothesis remains to be confirmed. Airway hyperreactivity has been observed as an acute response in guinea pigs two hours after a one-hour exposure to 200 μg/m3 sulfuric acid and appears to be associated with pulmonary inflammation. Likewise in rabbits, increased airway reactivity was associated with arachidonate metabolites, products of both epithelial and inflammatory cells. The general correlation between airway responsiveness and inflammation that appears to be important in grading asthma severity and risk of negative clinical outcomes may also be predictive of responses to environmental stimuli.
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Effects on Mucociliary Clearance and Macrophage Function
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Sulfuric acid alters the clearance of particles from the lung. Using insoluble, radioactively labeled ferric oxide particles as a probe, as little as a single one-hour exposure in donkeys, rabbits, and human subjects can slow clearance. Mucus clearance appears to vary directly with the acidity ([H+]) of the acid sulfate, with sulfuric acid having the greatest effect and ammonium sulfate the smallest (Schlesinger, 1984). Curiously, there appears to be a biphasic response to acid. In general, brief, single exposures of <250 μg/m3 accelerate clearance, while high concentrations of >1000 μg/m3 clearly depress clearance. With repeated daily exposures to low levels, there appears to be a cumulative (concentration times duration) dose-related depression of clearance. Longer-term exposure of rabbits to low-level acid also results in hyperplasia of airway mucosecretory cells (Gearhart and Schlesinger, 1989).
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Collectively, there seems to be coherence in the data to rank sulfate irritancy: sulfuric acid > ammonium bisulfate > ammonium sulfate. Acidity [H+] appears to be the primary driver on most respiratory effects attributable to the acid sulfates even at the level of pulmonary macrophages. Lavaged rabbit macrophage phagocytosis was affected more after a single exposure to 500 μg/m3 sulfuric acid than after exposure to 2000 μg/m3 ammonium bisulfate (Schlesinger et al., 1990). Nevertheless, in the complexity of summer haze, it remains unclear whether the bioactive form of [H+] is more appropriately assayed as free ion concentration (as pH) or as total available ion concentration (titratable H+).
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Not surprisingly, sulfuric acid induces qualitatively similar effects in the airways as found at high concentrations of SO2. As a fine aerosol, sulfuric acid deposits deeper along the respiratory tract, and its high specific acidity imparts greater effect on various cells (eg, phagocytes and epithelial cells). Thus, a primary concern with regard to chronic inhalation of acidic aerosols is the potential for bronchitis, since this has been a problem in occupational settings in which employees are exposed to sulfuric acid mists (eg, battery plants). Early studies in the donkey (later confirmed in a rabbit model) have provided fundamental data on this issue. The depression of clearance observed in donkeys exposed repeatedly (100 μg/m3 one hour per day for six months) raises concerns that a similar response (potentially contributing to chronic bronchitis) can occur in humans. Studies with cigarette smoke showed parallel responses.
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Studies conducted with sulfuric acid in the rabbit are in general agreement with the early findings in the donkey (Schlesinger, 1984). The initial early stimulation of clearance with subsequent depression has been shown to occur over 12 months with as little as two hours per day at 125 μg/m3 sulfuric acid (Schlesinger et al., 1992). Related studies also have demonstrated that the airways of exposed animals become progressively more sensitive to challenge with acetylcholine, show a progressive decrease in diameter, and experience an increase in the number of secretory cells, especially in the smaller airways. These studies have expanded our knowledge of the biological response and its exposure-based relationship to sulfuric acid. It seems reasonable to postulate that chronic daily exposure of humans to ~100 μg/m3 sulfuric acid may lead to impaired clearance and mild chronic bronchitis. The possibility that chronic irritancy may elicit bronchitis-like disease in susceptible individuals (perhaps over a lifetime or in children because of dose differences) appears to be reasonable.
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PM was referred to as “soot” in the “reducing-type” air pollution of the classic episodes. The major constituents of this soot consisted of incompletely burned carbonaceous materials, acid sulfates, various metals, and silicates associated with the solid nature of the fuel. Metals were abundant, with a considerable amount of zinc in the form of zinc sulfate—as reported from postepisode analyses of the Donora PM. Soot is indicative of poorly (inefficiently) combusted fuel. Over time, combustion technology and improved fuels provided gains by increasing efficiency and minimizing gross soot emissions. Improvements in combustion methods simultaneously reduced the size of emitted particles and overall less mass. A side benefit of the smaller particles was the reduction in light diffraction through the emissions and hence a less visible plume. As such, much of the early cleanup was largely achieved through technological improvements. Carbon in fuel that is not fully oxidized to CO2 persists in elemental form or as products of incomplete combustion—organic carbon. Oxidized S becomes sulfate and many of the metals appear as oxides. As noted above, sulfate was long suspected as the culprit of most health impacts associated with stationary sources, but this relationship is less discernable in contemporary particle epidemiology and toxicology.
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In the last 20 years PM has reemerged as the dominant issue in the air pollution community, overtaking O3 as the pressing air pollution health issue. The reason for this shift was the emergence of epidemiology data consistently showing increased mortality, an adverse effect of greatest import with the major impact on the cost/benefit analyses. As already noted, the collective studies showed health impacts (both morbidity and mortality) at levels of PM thought to be “safe” with no apparent threshold (reviewed in Pope and Dockery, 2006). Over time, science advances and reanalyses have confirmed the initial mortality findings. Several hypotheses that lend “biological plausibility” to the findings have emerged along with several “ancillary” impacts of PM on health not previously realized. The impacts of PM on health appear to be ever expanding, beyond lung effects, perhaps with even greater cardiovascular effects and an ever growing body of data suggesting impacts on neurologic and reproductive health, as well as growth and development.
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PM in the atmosphere can be solid, liquid, or a combination of both with a mélange of organic, inorganic, and biological compounds. The compositional matrix of PM can vary significantly depending on the emission source and secondary transformations, many of which involve gas to particle conversions. Long-range transport of emissions or transformation products can contribute significantly to the regional matrix of PM, particularly in the case of sulfate of East Coast PM2.5. Particles of larger size tend to have more local sources, the reason being that they are formed from dispersed dust and attrition of materials. Being of larger size, they tend to “fall out” or settle from the air due to gravity (although winds can in fact carry these particles great distances—eg, Sahara desert particles have been found on the US East Coast). Particles in the range of 10 to 2.5 μm (PM10-2.5—coarse PM) are highly inhalable by humans. In the urban setting there is considerable spatial and temporal heterogeneity of coarse PM while PM2.5 appears more homogenous throughout a regional environment. The size designation of fine and coarse PM is based on their relative respirability—those in the range of PM10 are inhalable into the larger thoracic airways while the PM2.5 is inhalable into the deeper reaches (gas exchange areas) of the lung (see Chap. 15).
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The large epidemiological database contending that PM elicits both short- and long-term health effects is largely founded on data from monitoring networks used for PM regulation. As such, the PM-associated effects appear to be dependent on the gravimetric measures that these networks yield. From this, it might then be argued that the effects are not influenced by particle composition (eg, inorganic and organic components). Further, because the findings strengthen with decreasing particle size (eg, total suspended particulate mass [pre-1978 index], PM10, PM2.5), it is the mass concentration of PM, despite compositional complexity and variation, that is the index of choice. However, toxicologists argue that a mass-based relationship contradicts the basic tenets of conventional air pollution toxicology, which is rooted in the concept of chemical-specific toxicities. A number of hypotheses that draw on various physical and chemical attributes of PM have also been offered in search of a “biologically plausible” explanation for the reported epidemiological observations. However, no one constituent has been identified as singularly determinant of the spectrum of health impacts. The smallest particles, which derive from anthropogenic combustion activities, appear to drive many of the health effects of particles. Prominently included among the causative hypotheses that tie toxicity to particle characteristics are metals, organics, acidity, size distribution (focusing on the unique bioactivity of ultrafine PM—PM <0.1 μm), PM oxidant activity or reactivity, and the presence of potentially toxic or allergenic biologicals. However, at present, there remains insufficient understanding of the relative importance of these theories to choose one over another, especially if the spectrum of heath effects is included. As a result, there seems to be insufficient reason to unseat the PM mass-based correlation with health outcomes as the driver of regulatory policies. Although the laboratory animal and human toxicological database is growing rapidly with regard to the issue of causation, much remains to be learned before new regulatory indices can be considered.
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From research directed initially toward potential occupational hazards, it is known that several metals and silicates that make up much of the inorganic phase of PM can be cytotoxic to lung cells. Organic constituents, as well, can induce toxicity either directly or via metabolism product—some of which are genotoxic. Other PM attributes may also come into play. Studies focusing on very small, ultrafine particles suggest that although these particles are low in mass, they are high in number and thus provide substantial reactive particle surface to interact with biological substances. Less is known about the role of biologically derived materials, such as endotoxin, glucans, plant glycoproteins, and bioallergen fragments, which may elicit rudimentary inflammatory responses in the lung. The involvement of biologicals may be greatest in agricultural and indoor exposure environments. There has been considerable interest in PM–copollutant interactions, but our knowledge in this area is somewhat limited and has spurred toxicologists to better access the experience and knowledge of atmospheric scientists as to the atmospheric interactions that are most relevant to biological outcomes.
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There have been many standard acute and subchronic rodent inhalation studies with specific metal compounds, often as oxides, chlorides, or sulfates. These exposure studies relate most appropriately to occupational exposures. The varied systemic toxicities of metal compounds are presented in detail elsewhere in this book; however, it should be appreciated that the effects of metals delivered by inhalation may differ from their impacts when administered by other routes. Metals may arise from natural as well as anthropogenic activities, and as a result metals are a common constituent in ambient PM. The metal profiles among regions differ appreciably in concentration and type and they also differ by the size mode of PM. Coarse PM (2.5–10 μm) arises largely from natural sources and thus has prominent earthen metals such as iron, sodium, silica, and magnesium—usually in oxide forms. Combustion-derived metals reflect the fuel source. For example, oil may have vanadium, nickel, and perhaps zinc and iron, while coal may have zinc and selenium. Their chemical forms vary from water-soluble salts to oxide and phosphate forms. Other metals are emitted from vehicles burning fuels to which metal compounds were added to alter functionality (eg, lead, manganese, platinum) or as engine wear and catalyst by-products. Similarly, metals may also derive from brake (copper, iron), tire (zinc), and dispersed road (earthen silicates) wear. Metals have many biological properties, some essential to life while others being directly toxic to cells or act indirectly in a pro-oxidant toxic fashion. Thus, metals have garnered considerable interest regarding their role in PM toxicity (Costa and Dreher, 1997).
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Metal compounds can be separated nominally by physicochemical characteristics: those that are essentially water-insoluble (eg, metal oxides and hydroxides such as those that might be released from high-temperature combustion sources or derived from the geocrustal matrix) and those that are soluble or somewhat soluble in water (often chlorides or sulfates such as those that might form under acidic conditions in a smoke plume or leach from acid-hydrated silicate particles in the atmosphere). Solubility appears to play a role in the toxicity of many inhaled metals by enhancing metal bioavailability (eg, nickel from nickel chloride vs nickel oxide), but insolubility can also be a critical factor in determining toxicity by increasing pulmonary residence time within the lung (eg, insoluble cadmium oxide vs soluble cadmium chloride). Moreover, some metals, either in their soluble forms or when partially coordinated on the surface of silicate or bioorganic materials, can promote electron transfer to form reactive oxidants. Complexes with particulate organic material in a partially hydrated form (as might be promoted by the presence of sea salt) have been shown to interact with poorly soluble metals to free coordination sites that again are pro-oxidant (Kieber et al., 2005). Thus, caution is warranted in assessing inhaled PM-associated metals, as both their chemical and physical attributes and their interaction with cocontaminants in PM may influence their apparent toxicity. Simply measuring total metal mass to estimate effects in the lung can be misleading.
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Perhaps the most compelling evidence for a role of soluble metals in PM toxicity derives from a series of studies on PM sampled in Utah Valley where a large open hearth steel mill went from full production to a one-year hiatus due to a strike and then resumed its open hearth operations. These studies related the metal content of PM sampled in the area to plant operation showing a reduction in total PM as well as a reduction of >90% PM metal content during the strike. Studies of hospital admissions for a variety of lung ailments and death rates showed reductions during the strike and toxicological studies where PM extractions from the operational and shutdown periods were tested in human and animal lungs, as well as on human lung epithelial cells, all correlated with plant operations, PM metal content, and the findings in parallel population studies (Pope, 1996; Dye et al., 2001; Ghio, 2004).
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Gas–Particle Interactions
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As already noted, these gas–particle interactions can be extremely complex involving multiple components of the particles, gases/vapors, and sunlight. However, more than 40 years ago, generic binary interactions between particles and gases in the absence of light were shown to alter the toxicity of either the particle or the gas acting alone. The guinea pig bronchoconstriction model used for many years by Amdur and associates showed early on that SO2 can interact with hydrated metal salts to potentiate particle irritancy. The mechanism(s) behind this interaction has yet to be fully discerned, but it appears to involve solubility of SO2 in a hydrated aerosol and the ability of the metal to catalyze the oxidation of the dissolved SO2 to sulfate. In the case of sodium chloride aerosol, potentiation appeared to be governed primarily by the solubility of SO2 in the salt droplet and enhanced respiratory penetration. The metal salts of manganese, iron, and vanadium, on the other hand, catalyzed the formation of sulfate. Studies in humans have been less revealing about such interactions, but this database affirms the need to consider the complexity of the atmospheric challenge in estimating biological outcomes.
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Complex chemistry also occurs within the effluent of the combustion source. Using a laboratory-scale furnace, the emission mix of sulfuric acid and metal oxide particles common to metal smelting and coal combustion was used to explore potential plume interactions that might impact respiratory irritancy (Amdur et al., 1986). These emitted metal oxide particles, once aged and cooled, were a mixture of singlet and agglomerated ultrafine particles that would be expected to distribute throughout the lung on inhalation. Exposures in guinea pigs of 30 to 60 μg/m3 sulfuric acid combined with ultrafine zinc oxide produced progressive decreases in DLCO, total lung capacity, and vital capacity and increases in cells, protein, and a variety of enzymes in lavage fluid that were not completely resolved 96 hours after exposure (Amdur, 1989). It is unclear whether the acid was on the surface of the particles or made the metal more soluble, but the combination was clearly more toxic than acid alone. These effects greatly exceeded the changes in airway resistance found with relatively simple, binary mixture of SO2 and water-soluble metal salts.
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Combustion studies using different coals again emphasized the significance of surface-associated acidic S-compounds. Ultrafine combustion particles from Illinois No. 6 coal had a layer of sulfuric acid adsorbed on the surfaces resulting in greater effects in guinea pigs than the more alkaline Montana lignite. Despite the greater sulfur content of the Montana coal, this emission ultrafine particle had neutralized the irritating sulfate (Chen et al., 1990). Similar studies using inert carbon black appear consistent with its role as carrier for reactive gases such as O3 and various aldehydes to enhance delivery of toxic materials to the deep lung (Jakab, 1992). The result of the latter study was enhanced infectivity when the carbon-gas preexposed test animals were subsequently exposed to pathologic bacteria.
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Similar interactions may result from gaseous pollutants that impair the clearance of particles from the lung or otherwise alter their metabolism. Studies by Laskin at New York University in the 1960s showed an intriguing interaction of SO2 and benzo(a)pyrene. It was thought that impaired clearance and greater residence time in the lung led to the enhanced probability of carcinogenic expression of the particle. Similarly, rats exposed to an urban eight-hour daily profile of O3 for six weeks, followed by a five-hour exposure to asbestos, were found to retain three times as many fibers as did the controls 30 days postexposure. The fibers were deposited in the distal airways and penetrated more deeply into airway tissues making them less accessible to phagocytic removal (Pinkerton et al., 1989). These studies, together with those focusing on irritancy and infectivity, raise the prospect that realistic exposure scenarios of gaseous and particulate pollutants can interact through either chemical or physiologic mechanisms to enhance health risks of complex polluted atmospheres.
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Ultrafine Carbonaceous Matter
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Ultrafine carbon particles (often called black carbon) typically result from high-temperature pyrolysis or as the product of atmospheric transformation involving organic vapors and sunlight. The size of these particles allows them to slip between gas molecules moving primarily by diffusion and principles of Brownian motion. Agglomeration on surfaces or other particles in the air is their primary mode of dissipation. When concentrations exceed ~1 million/cm3, they rapidly agglomerate with each other to form larger clumps or chains of ultrafine particles. As an air pollutant, therefore, elemental carbon particles generally do not exist as singlets except near their emission points—for example, traffic or other high-temperature sources. Fine PM consists in part of agglomerates of carbonaceous organic material that if partially oxidized may be somewhat soluble in water. Some organic materials, which exist in the vapor form, condense on the ultrafine carbon (eg diesel PM). Estimates of the carbonaceous (including organic) content of ambient fine PM vary considerably but are nominally considered to be about 10% to 60% of the total mass depending on the urban or regional area. The sources of organic carbon are varied and include the combustion products of natural smoke (eg, forest fires), engine exhaust, and stationary sources as well as transformed condensates from VOCs in the air. Elemental carbon in diesel PM frequently combines into long ultrafine chains, with variably complex organics associated with its surface depending on the combustion conditions. It has been estimated that diesel contributes about 7% of the fine urban PM emissions, which, when expressed as an annual US average, is about 2 μg/m3 (USEPA, 1993)—but focus estimates (urban canyons) vary widely. The higher use of diesel fuel in Europe and areas of concentrated trucking in the United States has led to estimates as high as 30% of ambient fine PM mass. Elemental carbon has traditionally been associated with traffic, but it is a better marker of older vehicles as new engine technology for diesels, designed to meet new emission standards in 2007 and 2010, emits substantially (>98%) less black carbon. Automobiles release even less. The expected result over the next 20 years, with the turnover of truck fleets in particular, is a great reduction in ambient levels of elemental carbon.
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Recent epidemiological and field studies with PM have been focusing on black carbon as an index of traffic and have reported strong associations between traffic (through the carbon indicator) and health effects. Most notably the observed adverse effects have been cardiovascular, thus giving some credence to hypotheses that ultrafine particles somehow find their way into the systemic circulation (see below) or trigger systemic inflammation that links to the cardiovascular effects (Peters, 2006; Tong et al., 2010). The penetration of ultrafine particles into the circulation under experimental conditions appears to be composition dependent (Kreyling et al., 2009).
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Diesel particles vary widely in the ratio of organic and elemental carbonaceous materials, which in empirical studies has been shown to influence toxic outcomes, such as to their inflammatory and carcinogenic potential (Singh et al., 2004). Some diesel particles also appear to have adjuvant activity when tested with bioallergens in both animals and humans (Diaz-Sanchez et al., 1999). When reacted in vitro with O3, there appears to be an enhancement of lung inflammation relative to the diesel or O3 alone (Madden et al., 2000). However, it is important to realize that diesel particles should not be equated with whole diesel exhaust that also contains significant amounts of gaseous pollutants: NOx, CO, and SOx as well as various VOCs and carbonyl irritants. Exposure to diluted diesel exhaust in humans reveals that the exhaust mix is inflammogenic and to a degree cytotoxic to airway cells (Ghio et al., 2012). The use of diesel particles alone in toxicology studies does not seem to display similar toxicity, thus underscoring the potential importance of interactions among air pollutants as a critical consideration in air pollution toxicity (Mauderly and Samet, 2009).
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Elemental carbon itself is generally considered to be of low toxicity, although long-term, high-concentration exposure conditions in rats can lead to lung “overload” where there is evidence of lung damage and carcinogenicity (addressed below). In the environment, carbon has the potential to act as a carrier of certain irritant gases as was noted earlier. However, carbon in the ultrafine mode (<0.1 μm) has been suggested to be more toxic than the fine mode (2.5 μm) form, perhaps due to enhanced surface reactivity or tissue penetration (Oberdorster et al., 2002; Donaldson et al., 1998). Size is not the only factor as it appears that composition of the ultrafine particle also contributes to its effects and behavior (Kreyling et al., 2009). Ultrafine particles in the environment exist in extremely high numbers but contribute negligibly to mass. Recent commercial introduction of “engineered” nanoparticles brings many of the same concerns as ultrafines by virtue of their similar sizes. Additionally, being “engineered” particles, they may possess design features that “natural” combustion ultrafine (or nano) particles do not.
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Chronic Effects and Cancer
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The role of air pollution in human lung cancer is difficult to assess because the vast majority of respiratory cancers result from cigarette smoking. The ACS study noted above (Pope et al., 2011) showed a significant linkage between PM and lung cancer, but in general it has been difficult to show these effects for the many HAP compounds that occur as urban air pollutants and are also thought to be carcinogenic. However, most of the HAPs and even fewer (about 10%) of the more than 2800 compounds that have been identified in the air have been assayed for carcinogenic potency. Fig. 29-14 gives estimates of the relative contributions of various chemicals to the lung cancer rate that are not associated with cigarette smoking, which, for outdoor air, is estimated to be about 2000 cases per year (Lewtas, 1993). This compares with about 2000 cases per year for passive environmental tobacco smoke and >100,000 cases per year for smokers. VOCs and nitrogen-containing and halogenated organics account for most of the compounds that have been studied with animal and genotoxicity bioassays. Most of these compounds are derived from combustion sources ranging from tobacco to power plants to incinerators to motor vehicles. Other potential carcinogens arise from mobile sources (including diesel) as products of incomplete combustion as well as their atmospheric transformation products. Fugitive or accidental chemical releases also figure into the many chemicals in ambient air. The National Scale Air Toxics Assessment (NATA) is an assessment of the cancer and noncancer risks associated with ambient HAPs (www.epa.gov/nata2005/). The profile of outdoor carcinogens contrasts with that of indoor air, where the sources are thought to derive largely from environmental tobacco smoke and radon, with some contribution from off-gassed organics (eg, adhesives, carpet polymers, cleaning agents). Human exposure to airborne toxicants is highly complex compositionally as well as in its temporal and spatial heterogeneity.
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The lung cancer risk of any individual is some function of the carcinogenic nature of the substance, the amount of material deposited in the lungs, which is itself a function of the concentration in the ambient air, the physical and chemical properties of the inhalant that may determine deposition efficiency, and the cumulative volume of air inhaled. Of course, the innate susceptibility of the individual (including genotype and environmental factors such as diet, etc) is also likely to be important. A significant body of data suggests that the majority of lung cancer risk from ambient air pollution lies within the PM fraction. Among the many potent chemicals are the polycyclic organic chemicals, along with a group of less-volatile organics sometimes referred to as “semivolatiles” (including nitroaromatics). These persistent organics associate with the PM matrix and thus could have a prolonged residence time at deposition sites within the respiratory tract. Genetic bioassays have revealed the potent mutagenicity, and presumably carcinogenicity, of various chemical fractions of ambient aerosols (Lewtas, 2007). Some of these compounds require metabolic transformation to activate their potency while others may be detoxified by their metabolism. Not to be forgotten, although not a feature of this chapter, are carcinogenic vapors such as benzene that are inhaled but have target tissues away from the lung—in the case of benzene, bone marrow leukemia.
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The cells lining the respiratory tract turn over relatively quickly, since they interface with the ambient environment with every breath. Conceptually, their DNA would thus be vulnerable to carcinogenic or oxidant-induced replication errors that, when fixed as mutations, could give rise to tumors. Copollutants, such as irritant gases, that initiate inflammation may promote carcinogenic activity by damaging cells and further enhancing their turnover. For example, there is experimental evidence that benzo(a)pyrene inhaled by rats whose respiratory tracts have been chronically exposed to SO2 are prone to bronchogenic carcinomas. Likewise, epidermoid carcinomas were produced in mice that inhaled ozonized gasoline vapors, containing many reactive organic products, but only if these mice had been previously infected with influenza virus and presumably had inflamed lungs. Many believe that the so-called rural–urban gradient of lung cancer, apparent even when corrected for cigarette smoking, is a product of such complex interactions. Thus, while the phenomenon of environmental lung cancer remains poorly understood, there is general sentiment for the early opinion expressed by Kotin and Falk in 1963: “Chemical, physical and biological data unite to form a constellation that strongly implicates the atmosphere as one dominant factor in the pathogenesis of lung cancer.” At the time of this statement, however, the role of tobacco smoke was not widely appreciated.
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To understand the role of inhaled particles to non-cigarette-smoke-related lung cancer, chronic exposure studies have been conducted with a number of particles ranging from titanium dioxide and carbon to diesel exhaust and coal fly ash aerosol. Of these substances, diesel exhaust has been the most extensively studied (reviewed by Cohen and Nikula, 1999; Ghio et al., 2012). The primary concern with diesel has been the suspicion that it can induce lung cancer (thus its IARC classification as a Group 1 human carcinogen). However, the evidence from over 40 occupational studies (primarily railway yard, truck, and bus workers) implicating diesel exhaust as a mild carcinogen continues to be debated because of a variety of confounding issues (Gamble, 2010). Taking the empirical data alone, however, carcinogenicity is suggested by several chronic exposure studies in animals and in vitro data indicating mutagenicity in Salmonella bacteria and enhanced sister chromatid exchange rates in Chinese hamster ovary cells. (The latter genotoxic effects have been linked to the nitroarenes associated with the diesel PM.) Rodent studies, unfortunately, have not fully resolved the question of human carcinogenic risk because of the overload needed to yield positive results, and the tumors develop only in the rat. At high concentrations of diesel PM (3.5 and 7 mg/m3), normal mucociliary clearance in the rodent models becomes overwhelmed, resulting in a progressive buildup of particles in the lungs. By 12 months in the rat, clearance irreversibly decreases to cessation with concomitant inflammation, oxidant generation, epithelial hyperplasia, and fibrosis. Rats seem to react more to this circumstance. Particle agglomerates within the alveolar lumen become the focus of inflammation, injury, and the eventual development of adenosarcomas and squamous cell carcinomas. At lower concentrations, where the particle buildup does not occur, tumors do not develop.
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“Overload” is a common finding at the highest exposure concentrations of chronic particle inhalation bioassays. While this phenomenon is not likely in humans exposed to ambient PM, it is a hypothesis worthy of consideration in a discussion of particle health effects, as it relates to the interpretation of toxicological data relevant to product use and industrial exposures. Several poorly soluble particles (PSPs) have induced lung tumors in chronic rat bioassays under conditions of overload; tumors have not developed under similar conditions in mice and hamsters. Among these particles are titanium dioxide, carbon black, toner dust, talc, and diesel emission; the potential for tumors is especially marked when the particles are in the ultrafine mode. In the rat, the timecourse and pattern of accumulation, chronic inflammation, epithelial hyperplasia, and tumorigenesis are essentially the same for all of the particles. In contrast, the degree of active inflammation in the mouse and hamster under similar overload conditions appears less intense, and thus is an important distinction among the species that relates to their relative sensitivities. On one hand, such exposures do not typically demonstrate classic in vivo genotoxicity, although bulky adducts and other modifications to DNA have been described after in vivo exposure to carbon black and diesel exhaust (reviewed by Schins and Knaapen, 2007). Also, mutation of the hprt (hypoxanthine guanine phosphorybosyl transferase) gene was found in rat epithelial cells cultured with bronchoalveolar lavage from chronic carbon black– and titanium dioxide–treated rats (Driscoll et al., 1997). Even though these few studies have demonstrated inert particle-induced modifications to DNA, generalizing this finding to overload cancers, not surprisingly, remains controversial. Typically, the pathways leading to lung cancer by relatively inert and PSPs (eg, carbon black and diesel exhaust particles) are thought to be secondary and involve pathways such as oxidative stress rather than a primary carcinogenesis mechanism such as DNA mutations (Greim et al., 2001). The closest analogy in humans would be coal miners who do not appear to have an enhanced risk of lung cancer except when smoking is not involved.
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The issue, then, is whether rat bioassay cancer data under conditions of overload are relevant to risk assessment. A review by an expert panel (ILSI, 2000) concluded that rats, while apparently unique in this response, may represent a sensitive subgroup, and that tumorigenesis data from the rat bioassay under conditions of overload cannot be summarily dismissed as not relevant to the consideration of cancer risk in humans. However, the data should be interpreted and weighed in the context of lower concentrations and the tumor incidence and pathology found therein.
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Photochemical Air Pollution
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Photochemical air pollution (notably O3) remains the most elusive of the criteria pollutants to bring under control. It arises secondarily from a series of complex reactions in the troposphere activated by the ultraviolet (UV) spectrum of sunlight. In addition to O3, it comprises a mixture of nitric oxides (NOx), aldehydes, peroxyacetyl nitrates (PAN), and a myriad of aromatics and alkenes along with analog reactive radicals. If SO2 is present, sulfates may also be formed and, collectively, they yield “summer haze.” Likewise, the complex chemistry can generate organic PM, nitric acid vapor, and various condensates. Attempts to mitigate photochemical smog (especially O3) by controlling hydrocarbon and/or NOx emissions have proven difficult due to the complex stoichiometry of atmospheric photochemistry. Some progress has been made in controlling peak values in the United States (regulated by the pre-1997 one-hour NAAQS), but the longer-time-frame eight-hour NAAQS has seen generally less progress. Because of a particularly strong regulatory effort in Southern California, however, the average number of high ozone days per year dropped by 33% between 2000 and 2010 (State of the Air, 2012) in the Los Angeles–Long Beach–Riverside metropolitan area, the most polluted part of the United States, in terms of ambient ozone levels.
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From the point of view of the toxicology of photochemical air pollutant gases, O3 is by far the toxicant of greatest concern. It is highly reactive and more toxic than NOx, and because its generation is fueled through cyclic hydrocarbon radicals, it reaches greater concentrations than the hydrocarbon radical intermediates. The gaseous hydrocarbons, so integral to the chemistry, are no longer listed collectively as a criteria pollutant in the United States since they do not have a strong health-based driver. In general, the concentrations of the hydrocarbon precursors in ambient air do not reach levels high enough to produce acute toxicity, although some individual compounds fall under the HAPs rule. Rather, the importance of these hydrocarbons stems from their role in the chain of photochemical reactions.
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Although O3 is of toxicological importance in the troposphere, in the stratosphere it plays a critical protective role. About 10 to 50 km above the earth’s surface, UV light directly splits molecular O2 into atomic O•, which then combines with O2 to form O3. The O3 also dissociates back but much more slowly. The result is an accumulation of O3 to several ppm within a relatively thin strip of the stratosphere forming an effective “permanent” barrier by absorbing the short-wavelength UV in the chemical process. This barrier had in recent years been threatened by various anthropogenic emissions (Cl2 gas and certain chlorofluorocarbons) that enhance O3 degradation (creation of an “O3 hole”), but recent restrictions on the use of these degrading chemicals seem to have been effective in reversing this process. The benefits are believed to be a reduction of excess UV light infiltration to the earth’s surface and reduced skin cancer risk.
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This protective issue is quite different in the troposphere, where accumulation of O3 serves no known purpose and poses a threat to the respiratory tract. Near the earth’s surface, NO2 arising from combustion processes efficiently absorbs longer-wavelength UV light, from which a free O atom is cleaved, initiating the following simplified series of reactions:
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(29-1)
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This process is inherently cyclic, with NO2 regenerated by the reaction of the NO• and O3. In the absence of unsaturated hydrocarbons (olefins and substituted aromatics) arising from fuel vaporization or combustion, as well as biogenic terpenes, this series of reactions would approach a steady state with little buildup of O3. The free electrons of the double bonds of unsaturated hydrocarbons are attacked by free atomic O•, resulting in oxidized compounds and radicals that react further with NO• to produce more NO2. Thus, the balance of the reactions sequence shown in Eqs. (29-1) to (29-3) is tipped to the right, leading to buildup of O3. This reaction is particularly favored when the sun’s intensity is greatest at midday, utilizing the NO2 provided by morning rush-hour traffic. Carbonyl compounds (especially short-chained aldehydes) are also by-products of these reactions. Formaldehyde and acrolein account for about 50% and 5%, respectively, of the total aldehyde content in urban atmospheres. Peroxyacetyl nitrate (CH3COONO2), often referred to as PAN, and its homologs also arise in urban air, most likely from the reaction of the peroxyacyl radicals with NO2. In the evening when sun intensity wanes, the second rush-hour peak of NO2 shifts the balance back by reacting with O3 bringing the daytime peak concentration slowly down. In assessing the excess adverse effects of O3, it is important to determine the background level of ambient O3. Interestingly, background levels of ozone in North America are known to peak as high as 0.14 ppm (one hour), particularly in high elevation regions such as Wyoming, although the contribution of these transient one-hour levels of ozone to public health is unclear. This high altitude peak concentration is thought to be due to a combination of high VOC levels, NOx, and the additional UV light reflected off snow cover. However, other factors such as season, thickness of boundary layers, vertical intrusions from the stratosphere, and long-range transport also play roles in determining background levels of O3 in North America.
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Short-Term Exposures to Smog
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In the 1950s to 1960s, early air pollution toxicologists were challenged by the complexity of photochemical air pollution both to ascertain its potential to adversely affect human health and to determine what about these atmospheres was responsible for the effects. The focus was immediately placed on O3 because its toxicity was found to be very high even at low ppm concentrations. Concerns that the complex atmosphere was even more hazardous led to a number of studies with actual (outdoor-derived) or synthetic (photolyzed laboratory-prepared atmospheres) smog in an attempt to assess the potency of the actual pollution mix. When human subjects were exposed to real-world photochemical air pollution (Los Angeles ambient air pumped into a laboratory exposure chamber), they experienced changes in lung function similar to those described in controlled clinical studies of O3 alone (ie, reduction in spirometric lung volumes; see below), thus supporting the view that this oxidant was the pollutant of primary concern. Acute animal studies using synthetic atmospheres (usually irradiated auto exhaust) provided supportive evidence indicating deep lung damage, primarily within the small airway and proximal alveolar epithelium.
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Thus, O3 appeared to be the prime toxicant in many of these early studies; there was some evidence that other copollutants were involved in the effects observed with smog. When guinea pigs were exposed to irradiated auto exhaust, airway resistance increased quickly, in contrast to the pattern of O3 alone, where less effect is seen on resistance than on breathing rate. This indicated that a more soluble irritant(s) probably was active, presumably reactive aldehydes. Thus, the array of effects of a complex atmosphere may be more diverse than would be predicted if it were assumed that O3 alone was responsible. Interestingly, the focus over time has been almost exclusively on O3, perhaps with the emphasis coming from the regulatory perspective tied to this single pollutant.
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Chronic Exposures to Smog
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Epidemiological studies in human populations as well as empirical studies in laboratory animals have attempted to link degenerative lung disease with chronic exposure to photochemical air pollution. Cross-sectional and prospective field studies have suggested an accelerated loss of lung function in people living in areas of high pollution. However, as with many studies of this type, there were problems with confounding factors (meteorology, imprecise exposure assessment, and population variables). Studies have been conducted in children living in modern-day Mexico City, which has oxidant and PM levels far in excess of any city in the United States. These studies have focused on the nasal epithelium as an exposure surrogate for pulmonary tissues, using biopsy and lavage methodologies to assess damage. Dramatic effects were found in exposed children, consisting of severe epithelial damage and metaplasia as well as permanent remodeling of the nasal epithelium. When children migrated into Mexico City from cleaner, nonurban regions, even more severe damage was observed, suggesting that the tissue remodeling in the permanent residents imparted some degree of incomplete adaptation. Because the children were of middle-class origin, these observations were less likely confounded by socioeconomic variables (Calderon-Garcidueñas et al., 1992). Changes in lung hyperinflation as estimated by x-ray tomography and impaired lung function have been reported among similarly exposed children living in Mexico City (Calderon-Garcidueñas et al., 2003) raising further concerns of long-term effects. Recently, a more mechanistic panel study demonstrated that the epithelial cell damage in the nasal cavity of Mexico City children was inversely correlated with glutathione peroxidase, a marker of oxidative stress (Hernández-Escobar et al., 2009).
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A now classic synthetic smog study in animals was undertaken at the Cincinnati EPA laboratory in the mid-1960s in an attempt to address the potential for long-term lung disease. Beagle dogs were exposed on a daily basis (16 hours) for nearly six years, followed by a clean air recovery period of about three years (Lewis et al., 1974). A series of physiologic measurements were made on the dogs after the exposure, and after their three-year recovery. They were then moved to the College of Veterinary Medicine at the University of California at Davis. The lungs of the dogs then underwent extensive morphologic examination to correlate with the physiology. The dogs had been exposed to nonirradiated auto exhaust (group 1), irradiated auto exhaust (group 2), SO2 plus sulfuric acid (group 3), the two types of exhaust plus the sulfur mixture (groups 4 and 5), and a high and a low level of NOx (groups 6 and 7). The irradiated exhaust contained oxidant (measured as O3) at about 0.2 ppm and NO2 at about 0.9 ppm. The raw exhaust contained minimal concentrations of these materials and about 1.5 ppm NO. Both forms of exhaust also contained about 100 ppm CO. The control group did not show time-related lung function changes, but all the exposure groups had functional abnormalities, most of which persisted or worsened over the three-year recovery period in clean air. Enlargement of airspaces and loss of interalveolar septa in proximal acinar regions were most severe in dogs that were exposed to NOx and SOx with irradiated exhaust (Hyde et al., 1978). These studies described a morphologic lesion that was degenerative and progressive in nature, not unlike that of chronic obstructive pulmonary disease (COPD)—a condition most often associated with lifelong tobacco smoking.
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Long-term “sentinel animal” studies in polluted cities have been attempted whereby the animals live in the same highly polluted air to which people are exposed. Such approaches have had a troubled past, but newer studies appear to be better controlled for the problems of infection, inappropriate animal care (eg, heat), and variable exposure atmospheres. One such study, conducted in rats exposed for six months to the air of São Paulo, Brazil, found considerable airway damage, lung function alterations, and altered mucus rheology (Saldiva et al., 1992). The concentrations of O3 and PM in São Paulo frequently exceed daily maximum values (in the summer months of February and March) of 0.3 ppm and 75 μg/m3, respectively. This collage of effects is not unlike a composite of injury one might suspect from a mixed atmosphere of oxidants and acid PM in controlled laboratory animal studies. Strangely enough, however, a seven-week study in rats exposed to the polluted Mexico City air, which had induced significant lesions in children, did not reveal any nasal or lung histopathology in F-344 rats (Moss et al., 2001). While there is no clear reason for the apparent differences in the findings, it is important to appreciate that all sentinel studies have elements of exposure that may be uncontrolled and hence at times can yield conflicting findings.
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Ozone is the primary oxidant of concern in photochemical smog because of its inherent bioreactivity and its concentration relative to other reactive species. Although it depends on the meteorological conditions of a given year, approximately 129 million Americans lived in areas not in absolute compliance with the 8-hour NAAQS in 2010. Progress has been made in reducing ambient O3 levels and Los Angeles, for example, has not exceeded the Stage 1 smog alert (one-hour peak of 0.20 ppm or more) since 1998 and dropped the number of exceedances of the 0.075 ppm NAAQS in half from 1990 to 2011. Unlike SO2 and the reducing-type pollution profile discussed above, current mitigation strategies for O3 have been only marginally successful despite significant reductions in individual automobile emissions. These reductions have been offset by population growth, which brings with it additional vehicles and vehicle miles driven. With suburban sprawl and the downwind transport of air masses from populated areas to more rural environments, the geographic distribution of those exposed has also expanded, as has the temporal profile of individual exposure. In other words, ambient O3 exposures are no longer stereotyped as brief one- to two-hour peaks. Instead, there is more typically a prolonged period of exposure of six hours or more at or near the NAAQS level. This important change in the exposure profile to O3 has given rise to concerns that cumulative damage over an exposure of several hours may be more significant than brief pulse-like exposures, and that, as a result, many more people are at risk than was previously thought. With the 1997 revision of the US O3 NAAQS to include an eight-hour daily average of 0.08 ppm, more cities and suburban areas find themselves in violation of the standard. If indeed the damage to the lung is cumulative over an eight-hour time period, then people in areas of compliance but with exposure levels near the standard may be affected. The American Lung Association estimates that nearly 50% of US inhabitants live in counties that are not in O3 compliance (State of the Air, 2012). Perhaps of greater significance is that of those who might be considered susceptible due to age and/or preexistent cardiopulmonary impairments, 80% to 90% live in those areas that fail to comply with the present O3 NAAQS.
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Ozone induces a variety of effects in humans and experimental animals at concentrations that occur in many urban areas (reviewed by Lippmann, 1989; U.S. EPA, 2004). These effects include morphologic, functional, immunologic, and biochemical alterations. Because of its low water solubility, a substantial portion of inhaled O3 penetrates deep into the lung, but its reactivity is such that about 17% and 40% is scrubbed by the nasopharynx of resting rats and humans, respectively (Hatch et al., 1994; Gerrity et al., 1988). The reason for the higher degree of scrubbing in humans is unclear, but the finding is reproducible. Moreover, the mouth as well as the nose appears to scrub. Nevertheless, regardless of species, the region of the lung that is predicted to have the greatest O3 deposition (dose per surface area) is the centriacinar region, from the terminal bronchioles to the alveolar ducts, also referred to as the proximal alveolar ductal region (Overton and Miller, 1987). Because O3 penetration increases with increased tidal volume and flow rate, exercise increases the dose to the target area. Using 18O3 (a nonradioactive isotope of oxygen), Hatch and coworkers have shown that the dose to the distal lung and the degree of damage to the lung as determined by leakage of plasma protein into the alveolar space (as collected by bronchoalveolar lavage) in exercising human subjects exposed to 0.4 ppm for two hours with intermittent periods of 15 minutes of exercise (threefold normal ventilation on average) are similar to those in resting rats exposed for the same length of time to 2.0 ppm. Thus, it is important to consider the role of exercise-associated dosimetry in a study of O3 or any inhalant before making cross-study comparisons, especially if that comparison is across species.
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Animal studies indicate that the acute morphologic responses to O3 involve epithelial lining cells along the entire respiratory tract. The pattern of injury parallels the dosimetry profile, with the majority of damage occurring in the distal lung. Along the conducting airways, ciliated cells appear to be most sensitive to O3, while Clara cells and mucus-secreting cells are the least sensitive. Studies in the rat nose indicate that O3 also is an effective mucus secretagogue. In the alveolar region, type 1 epithelia are very sensitive to O3 relative to type 2 cells, which have an active metabolic machinery. The more resistant type 2 cells serve as the stem cells for the replacement of type 1 cells. Ultrastructural damage to alveolar epithelia can be observed in rats after a few hours at 0.2 ppm, but sloughing of cells in the distal airway generally requires concentrations above 0.8 ppm. Recovery occurs within a few days, and there appears to be no residual pathology. Hence, from animal studies, it would appear that a single exposure to O3 at a relatively low concentration is not likely to cause permanent damage. On a gross level, when a bronchoscope is used to peer into the human bronchus after O3 exposure, the airways appear “sunburned” and, as with mild skin sunburn, recovery is typical. What is uncertain is the impact of repeated “sunburning” of the airways and lung.
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Studies have shown particular sensitivity to O3-induced inflammation in neonatal rats that decreased with age into adulthood. However, enhanced sensitivity to O3 again appears in older rats as evidenced by interstitial edema that is not seen in younger adult animals. Older rats have lower ascorbate in the lung and this diminishment of antioxidant capacity may be in part responsible. Elderly humans, however, seem less responsive to the lung function impacts of high ambient O3. The reasons for this are unclear, but caution in linking lung function and inflammation in the O3 response is warranted.
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The mechanisms by which O3 causes injury have been studied using cellular as well as cell-free systems. As a powerful oxidant, O3 seeks to extract electrons from other molecules. The surface fluid lining the respiratory tract and the cell membranes that underlie the lining fluid contain a significant quantity of polyunsaturated fatty acids (PUFA), either free or as part of the lipoprotein structures of the cell. The double bonds within these fatty acids have a labile, unpaired electron that is easily attacked by O3 to form ozonides that progress through a less stable zwitterion or trioxolane (depending on the presence of water); these ultimately recombine or decompose to lipohydroperoxides, aldehydes, and hydrogen peroxide. These pathways are thought to initiate propagation of lipid radicals and auto-oxidation of cell membranes and macromolecules (Fig. 29-15).
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Evidence of free radical–related damage in vivo includes detection of exhaled breath pentane and ethane and tissue measurements of diene conjugates. Damage to the air–blood interface disrupts its barrier function and promotes inflammation. Inflammatory cytokines (eg, interleukins 6, 8, and others, TNF) are released from epithelial cells and macrophages that mediate early responses and initiate repair. This inflammatory process is generally transient, but it may also interact with neural irritant responses to affect lung function acutely. The latter response may have implications for those with preexistent inflammation or disease.
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Pulmonary Function Effects
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Exposure to O3 produces a variety of pulmonary function changes and human subjects appear to be more sensitive than animal models. Early studies showed that exercising human subjects exposed for two to three hours to 0.12 to 0.4 ppm O3 experience reversible concentration-related decrements in forced exhaled volumes (FVC and forced expiratory volume in one second [FEV1]) (McDonnell et al., 1983). Because of the concern that prolonged periods of exposure (six–eight hours) may lead to cumulative effects, similar protocols with lower exposure and exercise levels were extended up to 6.6 hours. In these studies, exposures to 0.12, 0.10, and 0.08 ppm induced progressive lung function impairment during the course of the exposure (Horstman et al., 1989). The pattern of response was a function of exposure time such that functional changes were not detectable at 1 or 2 hours but reached significance by four to six hours. Decrements in FEV1 after 6.6 hours at 0.12 ppm averaged 13.6% and were comparable to that observed after a two-hour exposure to 0.22 ppm with much heavier exercise. These studies were then followed up with exposures to even lower O3 concentrations. The latter controlled human exposure studies demonstrated that multihour exposures to 0.06 and 0.08 ppm O3 (with exercise) produced small but statistically significant mean decreases in FEV1 (in the range of 3%–4%) as well as increases in respiratory symptoms and inflammation. It is noteworthy that in these and other controlled human exposure studies with O3, considerable interindividual differences in response were observed, thus confirming the many animal studies showing that the response to ozone is dependent on many innate factors such as age, diet, genetics, preexisting disease, and gender.
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It is not clear what mechanisms underlie the altered lung function (in terms of changes in FEV1) produced by O3. Although chest pain/discomfort is thought to contribute to O3-induced decreases in an effort-dependent lung function maneuver such as FEV1, there is also evidence that the decrements in lung function are vagally mediated, and that the response can be abrogated by analgesics, such as ibuprofen and opiates, which also reduce pain and inflammation (see below). Thus, pain reflexes involving C-fiber networks are thought to be important in the reduction in forced expiratory volumes. On the other hand, animal studies show a prominent role for vagal reflexes in altered airway reactivity and bronchoconstriction. There is also evidence to implicate vagal reflexes in cardiac as well as thermal regulation, at least in rodents.
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Airway responsiveness to specific (eg, allergen) and nonspecific (eg, cold air, inhaled methacholine) bronchoconstriction is another commonly used test of the pulmonary response to inhaled pollutants such as O3. These types of tests are very important because airway hyperresponsiveness is a central feature of asthma and asthmatics are a sizeable subpopulation (7%–9% of the total population in the United States) that may be particularly sensitive to the adverse respiratory effects of inhaled pollutants. A number of animal models have demonstrated that O3 induces airway hyperresponsiveness and that innate factors such as genetics, diet, and obesity can modify this effect. For the most part, many of these studies have utilized O3 exposure concentrations that are far above ambient levels. Importantly, controlled human exposure studies have not only verified the findings of the high-dose animal studies but also demonstrated adverse effects at concentrations relevant to ambient O3 levels. Airway hyperreactivity to nonspecific agonists in humans has been reported after acute exposure to as little as 0.08 ppm O3. It is widely thought that hyperreactive airways may be a marker of predisposition to other pollutants such as sulfuric acid or aeroallergens, but such evidence is limited.
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Inflammation of the Lung and Host Defense
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The mechanism by which O3 produces decrements in pulmonary function is not fully understood and its link to airway inflammation is unclear. For example, O3-induced lung dysfunction does not appear to be enhanced in asthmatics whose lungs are generally in a state of inflammation. Asthmatics are, however, clearly more sensitive to the bronchoconstrictive effects of both SO2 and sulfuric acid aerosols. Other factors that play a role in the pulmonary response to O3 include age and obesity. Animal studies have demonstrated that O3-induced lung injury and inflammation is greater in neonatal compared with that in adult rabbits, rats, and mice (Vancza et al., 2009). In addition to age as a susceptibility factor, a series of studies has shown that obesity predisposes mice to the adverse effects of O3. Because obesity is associated with ongoing systemic inflammation, it suggests that a preexisting physiologic or disease state enhances the adverse effects of O3.
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Both human and animal studies have demonstrated that sensitivity to O3 appears to have a genetic component as well. Studies in inbred stains of mice have shown that O3-induced pulmonary neutrophilia, airway hyperresponsiveness, and permeability are significantly affected by SNPS in a single gene linked to the Toll-like receptor 4 (Tlr4) locus that has been associated with endotoxin sensitivity (Kleeberger et al., 2000). Further research has determined that the involvement of an innate immunity factor such as Tlr4 in the multifaceted response to O3 involves the release of hyaluronan fragments that appear to directly activate the Tlr4 cascade in mice (Li et al., 2011). The role of genetic polymorphisms in the pulmonary response to O3 is complicated, however, and many other pathways including TNF, IL-1, Nrf-2, and NF-κB can modulate the response to O3 in animal models (Bauer and Kleeberger, 2010).
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The growth of genomic and proteomic technologies has made it possible to begin studies in humans to define genetic linkages to O3 sensitivity. These studies have typically focused on genes involved in oxidative stress pathways. For example, again using Mexico City as a backdrop, it was found that a polymorphism in GSTM1 (as noted above with regard to PM) appears to convey O3 sensitivity as well (Romieu et al., 2004). Similarly, functional polymorphisms in the catalase and the myeloperoxidase genes have been associated with enhanced response to ambient ozone. More detailed controlled O3 chamber exposures have confirmed that genetic polymorphisms in these and other genes, including NAD quinone oxidoreductase (NQO), GSTP1, heme oxygenase-1, and TNF, are associated with responsiveness to ozone and that gene–gene interactions are involved.
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Because of the strong epidemiological evidence that O3 can exacerbate asthma (eg, increased hospital admissions) and the inability of epidemiology studies to demonstrate causality, investigators have utilized chamber studies to test the hypothesis that O3 enhances the immune response to allergens. The potential for O3 to influence allergic sensitization or challenge–responses has received a significant amount of investigation in animal studies. Enhanced response to allergen challenge has been noted in a number of animal species exposed to ozone. Biochemical, cellular, and pulmonary function parameters of allergic response have all been observed to increase in sensitized animals exposed to O3 prior to antigen challenge. Ozone’s effect on allergic asthma also occurs at the sensitization stage in which repeated exposure to O3 before or during the sensitization process exacerbates a range of allergic end points. Thus, in general, animal studies have shown the ability of O3 to enhance the allergic process under different conditions, but evidence of this in humans is less strong. Controlled studies of heightened antigen responsiveness in allergic subjects have only been suggestive, with enhancement of allergic rhinitis after 0.5 ppm for four hours. However, diary studies of asthmatic nurses reported worsened allergy symptoms, as well as durations thereof, at concentrations of O3 near the NAAQS (Schwartz, 1992). Similarly, eosinophils and IL-4, central components of allergic asthma, were increased in some but not all human chamber studies of O3 exposure (U.S. EPA ISA, 2012).
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Early research showed that exposure to O3 before a challenge with aerosols of infectious agents produces a higher incidence of infection than is seen in control animals (Coffin and Blommer, 1967). Studies have demonstrated that this type of effect can be a direct result of altered phagocytosis by macrophages in O3-exposed mice previously infected with an aerosol of Streptococcus (group C) bacteria (Gilmour et al., 1993). The host resistance model has shown responsiveness to an exposure as low as 0.08 ppm for three hours. The susceptibility of mice and hamsters to Klebsiella pneumoniae aerosol is also increased by prior exposure to O3. In the rat, altered microbe-killing may relate to membrane or receptor damage in macrophages, thus impairing the production of bactericidal agents such as superoxide anions. The rat appears less susceptible than mice because it has a more vigorous PMN response to bacteria than do mice, which seems to compensate for macrophage impairments. This is yet another example of where susceptibility lies more in the inability to compensate than in the initial responsiveness to a given challenge. It is not clear whether O3’s impairment of bacterial infections in the lung also extends to viral infections. While there is epidemiological evidence of ambient O3 being associated with increased hospital admissions for viral and bacterial infections, animal studies have shown mixed results for O3’s effect on viral infections, although some of the contradictory findings may be a result of experimental design differences (eg, O3 exposure occurring before or after viral infection).
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Morphometric studies of the centriacinar region of rats exposed for 12 hours per day for six weeks to 0.12 or 0.25 ppm O3 have shown hyperplasia and hypertrophy of type 1 alveolar cells (smaller and thicker cells) coupled with damage and alterations in ciliated and Clara cell populations in small airways (Barry et al., 1988). A collective cross-protocol analysis of type 1 cell hypertrophy was conducted; type 1 cell thickness appeared to be linearly related to the O3 C × T (Chang et al., 1991, 1992). This finding suggested that over a season, the impact of O3 in the distal lung may be cumulative and perhaps more importantly may be without threshold. The biological significance of this change is unclear—it may be part of a compensatory response to “thicken” that part of the alveolar duct junction that receives the greatest dose and is most affected. This response may be protective since the thickened cells were also smaller, offering therefore a smaller exposure surface to the incoming O3. This changed morphology could limit membrane damage. When returned to clean air, most of the epithelial morphologic changes regressed, but there was evidence of residual interstitial remodeling below the epithelium in the alveolar duct region. Examination of autopsied lung specimens from young smokers shows many analogous tissue lesions that come to be described as the “smoldering” precursor of emphysema. A long series of studies at the University of California, Davis, has examined the structural and functional changes produced in nonhuman primates exposed chronically to O3. Because of the greater similarity of monkey respiratory tract to that of humans, the burden of extrapolation from rodent study results to risk assessment for humans is lessened, and these studies have proven important in establishing the causality of many of the long-term effects of O3 observed in epidemiology studies. Episodic exposure of adult monkeys to 0.25 ppm O3 for 18 months produced physiologic, cellular, and biochemical changes in the lung. Exposure to as little as 0.2 ppm O3 for up to 90 days was found to produce cellular changes throughout the respiratory tract. Nasal lesions were accompanied by changes in cell populations in the respiratory bronchioles. In the second phase of these studies, infant rhesus monkeys were cyclically exposed to 0.5 ppm O3 for eight hours per day for five days followed by several days of exposure to air. While the study design did not permit a comparison of the responsiveness of infant versus adult monkeys, the infant monkeys showed evidence of structural changes that parallel epidemiology studies linking lung growth and development with exposure to air pollution. The infant monkeys also showed a significant interaction between sensitization to house dust mite antigen and ozone. While antigen or ozone alone produced small changes in baseline airway resistance and airway responsiveness, antigen plus ozone produced more than additive effects on both parameters.
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Studies involving episodic exposures of rats and monkeys using a pattern of alternating months of O3 (0.25 ppm) for 18 months indicate that there may be carryover effects, notably thickening of interstitial fibrous matrix (Tyler et al., 1988, 1991). These interstitial changes were quantitatively similar regardless of the twofold difference in the cumulative exposure dose (ie, C × T). This would imply that a pattern of exposure resembling seasonal O3 patterns might result in more serious lesions than predicted by dose alone—indeed more than would have occurred had the exposure been continuous. Hence, the concept of “more dose = more effect” may not hold in chronic episodic scenarios for O3, as it appears to do with uninterrupted exposures. The number of episodes experienced may well be more significant to long-term outcomes than total dose—a phenomenon not unlike that of repeated sunburning and deterioration of the skin.
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Studies of lung function in rodents exposed chronically to O3 have been conducted, but have yielded mixed results at relevant exposure concentrations. Generally, the dysfunction is reflective of stiffened or fibrotic lungs, particularly at higher concentrations. There have been two prominent chronic O3 studies—the EPA 18-month chronic study fashioned after a realistic urban exposure profile with a peak exposure of 0.25 ppm (Chang et al., 1992; Costa et al., 1995) and the National Toxicology Program (NTP)–Health Effects Institute (HEI Report, 1994/1995) study of 0.125 to 1 ppm for 20 months (square wave; six hours per day; five days per week). From an environmental relevance perspective, the C × T doses for these studies were similar, but the urban profile study produced evidence for centriacinar interstitial fibrosis suggesting a possible influence of the exposure pattern. There was no general biochemical evidence for fibrosis (Last et al., 1994), as reported in monkey and rat studies that had been conducted at higher O3 concentrations. If one attempts to compare these results with the Cincinnati beagle study, one finds that the synthetic smog atmosphere showed degenerative and not fibrotic lung lesions. However, it should be noted that the air pollutant mixture used in the beagle study both was more complex and involved considerably higher concentrations than more recent studies.
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The ability of O3 to induce tolerance to itself is a curious phenomenon that has implications for both episodic and chronic exposures. Classic O3 tolerance takes the form of protection against a high or even lethal dose in animals that received a very low initial challenge or challenges several days before. This term, tolerance, is sometimes used to describe “adaptation” or acclimatization over time to near-ambient levels of O3, and, as such, has led to some confusion. However, with regard to “adaptation” to O3, the process begins during and immediately after the initial exposure and progresses to completion in at most two to four days. This adaptive phenomenon has been well established in humans with regard to lung function and has been correlated with several inflammatory end points (Devlin et al., 1993). Lavage lactate dehydrogenase (LDH; a marker of cell injury) and elastase (enzymatically active against lung matrix material), interestingly, do not appear to adapt in humans based on the Devlin study. An analogous pattern of adaptation of functional and biochemical end points (including LDH and elastase) in rodents also takes place with repeated exposures up to a week. But to date, the linkages between acute, adaptive, and long-term process remain unclear, since over longer periods of exposure both morphologic and functional effects do appear to develop. The precise mechanism for O3 adaptation is not known and several theories abound, including changes in cell profiles, lung surface fluids, and induced antioxidants. Few studies have tackled the problem but in rats the adaptation of the neutrophilic response appears to be related to the induction of an endogenous acute-phase response (McKinney et al., 1998). On the other hand, adaptation to lung function changes in rats after chronic exposure appears linked to lung antioxidants such as ascorbic acid (Wiester et al., 2000). The significance of this finding in humans is uncertain because ascorbic acid is not endogenously synthesized as it is in the rat. However, self-administration of ascorbate has been shown to reduce O3-induced lung function decrements in adults (Mudway et al., 1999), and in children, supplementation with ascorbate and α-tocopherol lessened nasal inflammatory responses to O3 in Mexico City (Sienra-Monge et al., 2004). Despite these interesting findings, it remains unclear if antioxidant supplements can protect humans from long-term O3 effects given the many mechanisms that may be involved in the various responses. How these interplay with long-term adaptation and the likelihood of degenerative disease is unclear.
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Ozone Interactions with Copollutants
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An approach simplifying the complexity of synthetic smog studies, yet addressing the issue of pollutant interactions involves the exposure of laboratory animals or humans to binary or more complex synthetic mixtures of pollutants that occur together in ambient air. The most frequent combination involves interactions of O3 and NO2 or O3 and PM (eg, sulfuric acid or diesel particles). Not surprisingly, study design adds a level of complexity in interpretation such that evidence exists supporting either augmentation or antagonism of lung function impairments, lung pathology, and other indices of injury. This apparent conflict in the findings only emphasizes the need to carefully consider the myriad of factors that might affect studies involving multiple determinants and the nature of the exposure that is most relevant to reality.
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When O3 and NO2 (1 and 14 ppm, respectively) were administered to rats from a premixed retention chamber, the resulting damage evident in bronchoalveolar lavage exceeded that of either toxicant alone, regardless of the temporal sequence of exposure (Gelzleichter et al., 1992). Biochemical and histological indices of fibrogenesis also were increased in related studies (Last et al., 1994). In retrospect, it was hypothesized that the two oxidants formed relatively stable intermediate nitrogen radicals that were more toxic than either gas alone. At lower, more realistic concentrations (0.3 ppm O3 and 3.0 ppm NO2), where this reaction would not be favored, the impact of these irritants on rabbits was only additive (Schlesinger et al., 1991). This contrast in response serves to illustrate that the tenets of dose dependency that hold for any single-toxicant response may be of equal or more importance when two or more pollutants coexist and have the potential to interact.
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Studies of O3 mixed with acid aerosols also have shown enhanced or antagonistic responses that were time-dependent during the period of exposure. On the one hand, as noted above, field studies of children in camps and studies of asthma admissions in the Northeast and in Canada suggested an interaction of acid and O3 underlying responses to summer haze. Yet, in an experimental setting with rabbits exposed over an extended period, there was exposure duration–specific evidence of enhanced as well as antagonized secretory cell responses with combined O3 (0.1 ppm) and sulfuric acid (125 μg/m3) over the course of the one-year exposure (Schlesinger et al., 1992). As the number of interacting variables increases, so does the difficulty in interpretation. Studies of complex atmospheres involving acid-coated carbon combined with O3 at near-ambient levels also show varied evidence of interaction on lung function and macrophage receptor activity (Kleinman et al., 1999). As such, the platform of any multicomponent study is its statistical design and the ability to either separate or determine the nature of the interacting variables. However, it is indeed the complex mixture to which people are exposed that we wish to evaluate. Creative approaches to understanding mixture responses are a likely part of the new agenda that toxicologists will need to address in the future (Mauderly, 2006).
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Nitrogen dioxide, like O3, is a deep lung irritant that can produce pulmonary edema if it is inhaled at high concentrations. It is a much less potent irritant and oxidant than O3, but NO2 can pose clear toxicological problems. Potential life-threatening exposure is a real-world problem for farmers, as near-lethal high levels of NO2 can be liberated from fermenting fresh silage. Being heavier than air, the generated NO2 and CO2 displace air and oxygen at the base of silo and diffuse into closed spaces where workers can inadvertently get exposed to very high concentrations perhaps with depleted oxygen. Typically, shortness of breath rapidly ensues with exposures nearing 75 to 100 ppm NO2, with delayed edema and symptoms of pulmonary damage. Not surprisingly, the symptoms are collectively termed “silo-filler’s disease.” Nitrogen dioxide is also an important indoor pollutant, especially in homes with unventilated gas stoves or kerosene heaters (Spengler and Sexton, 1983) or, in developing countries, with the unvented burning of biomass fuels (Kumie et al., 2009). Under such circumstances, very young children and their mothers who spend considerable time indoors may be especially at risk. In general, indoor environments with NO2 sources achieve concentrations far in excess of those observed outdoors at central monitoring sites. Peak levels of NO2 near high-traffic-volume roadways can be several-fold higher than away from roadways and are thus similar to peak levels observed indoors with unvented stoves (ie, 0.2–0.5 ppm). Interestingly, protocols that simulate an urban (rush-hour) or household (cooking) patterns of two daily peaks superimposed on a low continuous background concentration have elicited effects in experimental animals that continuous exposure to NO2 did not evoke, suggesting an important dependency on exposure profile. Among the common air pollutants, empirical studies of NO2 have frequently shown greater effects with higher peak concentrations at equivalent C × T steady-state concentrations.
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Although the distal lung lesions produced by acute NO2 are similar among species, there exist differences in species sensitivity. Where direct comparison is possible, guinea pigs, hamsters, and monkeys appear more sensitive than rats, although comparative dosimetry information might explain some of this difference. As in the case of O3, theoretical dosimetry studies indicate that NO2 is deposited along the length of the respiratory tree, with preferential deposition being in the distal airways. Not surprisingly, the pattern of damage to the respiratory tract reflects this profile: damage is most apparent in the terminal bronchioles, just a bit more proximal in the airway than is seen with O3. At high concentrations, the alveolar ducts and alveoli are also affected, with type 1 cells again showing their sensitivity to oxidant challenge. In the airways of these animals there is also damage to epithelial cells in the bronchioles, notably with loss of ciliated cells, as well as a loss of secretory granules in Clara cells. Thus, the pattern of injury of NO2 is quite similar to that of O3, although its potency is about an order of magnitude lower.
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Pulmonary Function Effects
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Exposure of normal human subjects to concentrations of <4 ppm NO2 for up to three hours produces no consistent effects on spirometry. However, a study has shown slightly enhanced airway reactivity with 1.5 to 2.0 ppm. Interestingly, ascorbic acid pretreatment of human subjects appeared to protect them from this hyperreactivity (Mohsenin, 1987). Whether asthmatics have a particular sensitivity to NO2 is a controversial issue. A number of factors appear to be involved (eg, exercise, inherent sensitivity of the asthmatic subject, exposure method). Some studies have reported effects in some individuals at 0.2 ppm, which approximates an indoor level in a household with an unvented gas stove. Recent meta-analyses, which have incorporated the findings of many studies to achieve a weight-of-evidence perspective, support an effect of NO2 on asthmatics, but it is not clear if the small but statistically significant changes in airway hyperresponsiveness in asthmatics exposed to NO2 below 0.6 ppm are adverse. As for an appropriate animal model, only very high concentrations (10 ppm NO2) invoke an irritancy response in guinea pigs (tachypnea); these levels are well above those a person probably would encounter in everyday life. However, NO2 has been found to be associated with mortality in some time-series studies of air pollution attempting to tease out specific pollutant effects (focusing mainly on PM) (Gold et al., 2000; Samoli et al., 2006). These epidemiology studies have demonstrated that each 20 to 30 ppb increase in NO2 results in a small but significant increase in acute cardiovascular and respiratory mortality. Similarly, NO2 has been associated with increased hospital admissions and emergency room visits, but these epidemiology studies are confounded by the coexposure to multiple pollutants. Thus, as stressed elsewhere, it is important that epidemiology studies results be interpreted with caution when considering causality and that controlled human exposure and animal studies be utilized, within ethical limits, to confirm the biological plausibility of the effect of NO2, or any other pollutant, on the cardiopulmonary system. This caution obviously extends to many recent studies that have consistently linked adverse health outcomes with living near high-traffic-volume roadways. Because of the many constituents present in the exhaust of both gas and diesel combustion engines, as well as their ever changing chemistry, it is unclear from these studies whether NO2 is acting as a marker or surrogate for vehicular traffic rather than as an indication of a specific NO2 effect. These studies have found correlates with cardiovascular deaths, which have raised new questions of the mechanisms by which pollutants might affect health in susceptible subgroups (Rosenlund et al., 2006).
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Inflammation of the Lung and Host Defense
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Unlike O3, NO2 does not induce significant neutrophilic inflammation in humans at exposure concentrations encountered in the ambient outdoor environment. There is some evidence for bronchial inflammation after four to six hours at 2.0 ppm, which approximates the highest transient peak indoor levels of this oxidant. Exposures at 2.0 to 5.0 ppm have been shown to affect T lymphocytes, particularly CD8+ cells and natural killer cells that function in host defenses against viruses. Although these concentrations may be high, epidemiological studies variably show effects of NO2 on respiratory infection rates in children, especially in indoor environments. Animal models, by contrast, have for years shown associations between NO2 and bacterial infection (Gardner, 1984). As noted for other effects, the incidence of infection in exposed models appears to be governed more by the peak exposure concentration than by exposure duration. The effects are ascribed to suppression of macrophage function and clearance from the lung, in the form of suppressed bactericidal and/or motility functions of macrophages from rabbits exposed to 0.3 ppm for three days (Schlesinger, 1987). Similar effects have been reported in humans exposed to 0.10 ppm for 6.6 hours (Devlin et al., 1991).
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Toxicological studies of the interaction of NO2 with viruses also suggest enhanced infectivity. Squirrel monkeys infected with nonlethal levels of A/PR-8 influenza virus and then exposed continuously to 5 or 10 ppm NO2 suffered high mortality rates; 6/6 monkeys exposed to 10 ppm died within three days, while only 1/3 exposed to 5 ppm died (Henry et al., 1970). Other experiments suggest that exposure of squirrel monkeys for five months to 5 ppm NO2 depresses the formation of protective antibodies against the A/PR-8 influenza virus. Controlled human studies with virus challenges, however, have been inconclusive, perhaps because of low subject numbers. One study showed decreased virus inactivation by alveolar macrophages recovered from four of nine subjects when cultured and exposed for 3.5 hours to 0.6 ppm NO2 in vitro. The responsive macrophages produced interleukin-1, a known cytokine modulator of immune cell function (Frampton et al., 1989). Thus, the potential for augmented risk of viral infection associated with NO2 exposure remains unclear and suggests a role for underlying host susceptibility. This concern would be greatest for children, especially during seasonal use of unvented gas heaters, who have less mature pulmonary immune function.
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Concern about the chronic effects of NO2 stems from early observations that a continuous 30-day, 30-ppm exposure produces emphysema in hamsters. Whether the result of this exposure scenario relates to cyclic human exposures at 1/100th that level is unclear. On the one hand, an 18-month study in rats exposed to an urban pattern of NO2 in which a daily background of 0.5 ppm peaked at 1.5 ppm for 4 hours each day showed little ultrastructural damage to the distal lung (Chang et al., 1988). On the other hand, mice exposed for a year to a base level of 0.2 ppm NO2 with a one-hour spike of 0.8 ppm twice a day, five days per week (Miller et al., 1987) displayed effects that differed between the base-only and peak-only exposure groups. The base level produced no effects, while the overlaid peaks induced slight functional impairment and augmented susceptibility to bacterial infection. Early studies (Ehrlich and Henry, 1968) showed that clearance of bacteria from the lungs is suppressed with 0.5 ppm NO2 through 12 months of exposure. Interestingly, studies with a similar double diurnal peak design for NO2, with NO used as a negative control, showed more pronounced effects of NO on alveolar septal remodeling than did NO2 (Mercer et al., 1995). These and similar studies utilizing varied peak-plus-baseline NOx exposures indicate the importance of exposure profile for at least this pollutant.
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While a number of other reactive oxidants have been identified in photochemical smog, most are short-lived because of their reaction with copollutants. PAN, which is thought to be responsible for much of the eye-stinging activity of smog, is known to exist in smog situations. It is more soluble and reactive than O3, and hence rapidly decomposes in mucous membranes before it can penetrate into the respiratory tract. The cornea is a sensitive target and is prominent in the burning/stinging discomfort often associated with oxidant smogs. A few studies with high levels of PAN have shown that it can cause lung damage and have mutagenic activity in bacteria, but it is not likely that these scenarios are relevant to ambient levels of PAN.
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Carbonyl compounds, notably short-chained (2-4 C) aldehydes, are common photo-oxidation products of unsaturated hydrocarbons. Two aldehydes are of major interest by virtue of their concentrations and irritancy: formaldehyde (HCHO) and acrolein (H2C=CHCHO). They contribute to the odor as well as eye and sensory effects of smog. Formaldehyde accounts for about 50% of the estimated total aldehydes in polluted air, while acrolein, the more irritating of the two, accounts for about 5% of the total. Acetaldehyde (C3HCHO) and many other longer-chain aldehydes make up the remainder, but they are not as intrinsically irritating, exist at low concentrations, and have less solubility in airway fluids. Formaldehyde and particularly acrolein are also found in mainstream tobacco smoke (~90 and ~8 ppm, respectively, per puff) and are likely to be found at lower levels in sidestream smoke as well. Formaldehyde is also an important indoor air pollutant and can often achieve higher concentrations indoors than outdoors due to off-gassing by new upholstery or other furnishings. In the United States, the formaldehyde off-gassing issue was highlighted in the controversy surrounding the trailer homes provided to hurricane-displaced home owners in Katrina-New Orleans-2005.
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Empirical studies have shown that formaldehyde and acrolein are competitive agonists for similar irritant receptors in the airways. Thus, irritation may be related not to “total aldehyde” concentration but to specific ratios of acrolein and formaldehyde. Their relative difference in solubility, with formaldehyde being somewhat more water-soluble and thus having more nasopharyngeal uptake, may distort this relationship under certain exposure conditions (eg, exercise). On the other hand, acrolein is very reactive and may interact easily with many tissue macromolecules and, for example, can form DNA adducts.
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Formaldehyde is a primary sensory irritant. Because it is very soluble in water, it is absorbed in mucous membranes in the nose, upper respiratory tract, and eyes. The dose–response curve for formaldehyde is steep: 0.5 to 1 ppm yields a detectable odor, 2 to 3 ppm produces mild irritation, and 4 to 5 ppm is intolerable to most people. Formaldehyde is thought to act via sensory C-fibers that signal locally as well as through the trigeminal nerve to reflexively induce bronchoconstriction through the vagus nerve. In guinea pigs, a one-hour exposure to about 0.3 ppm of formaldehyde induces an increase in airflow resistance accompanied by a smaller decrease in compliance (Amdur, 1960). Respiratory frequency and minute volume also decreased, but these changes were not statistically significant until >10 ppm. The no observed effect level (NOEL) using these lung function criteria is about 0.05 ppm. The general pattern of the irritant response and its rapid recovery is similar to that produced by higher concentrations of SO2. Like SO2, breathing through a tracheal cannula to bypass nasal scrubbing greatly augments the irritant response, indicating that deep lung irritant receptors can also be activated by this vapor.
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The irritancy of inhaled formaldehyde vapor, again like SO2, has been shown to be potentiated by water-soluble salt aerosols. Irritancy appears to be augmented in proportion to the aerosol concentration, but the potentiation could not be accounted for by a simple aerosol “carrier” effect (Amdur, 1960). Moreover, reversal of bronchoconstriction was slower than had been observed with SO2. Thus, it appeared that the vapor-aerosol itself constituted a new irritant species, the product of a chemical transformation of formaldehyde—perhaps methylene hydroxide. In addition to interactions with water-soluble particles, formaldehyde has been shown to interact with carbon-based particles (Jakab, 1992) to augment bacterial infectivity in a murine model. In this case, the potentiation appears to correlate with the surface carrying capacity of the inhaled particle.
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Two aspects of formaldehyde toxicology have brought it from relative obscurity to the forefront of attention in recent years. One is its near ubiquitous presence in indoor atmospheres as an off-gassed product of construction materials such as plywood, furniture, or improperly polymerized urea-formaldehyde foam insulation (Spengler and Sexton, 1983). Complaints of formaldehyde irritation in industry have been reported at 50 ppb (Horvath et al., 1988). In studies relating household formaldehyde to chronic effects, children were found to have significantly lower peak expiratory flow rates (about 22% in homes with 60 ppb) than did unexposed children and asthmatic children were affected below 50 ppb. Thus, this irritant vapor can cause respiratory effects, and perhaps act as an allergen, at commonly experienced exposure levels (Krzyzanowski et al., 1990). Also, there is epidemiological evidence, sometimes inconsistent, that formaldehyde is associated with asthma (McGwin, 2010) and lower respiratory tract infections in children (Roda et al., 2011).
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A longtime concern regarding formaldehyde has been its potential carcinogenicity. In 2004, IARC concluded, based on a thorough review of the published data, that formaldehyde was a probable human carcinogen; IARC deemed that there was “sufficient” epidemiological evidence that formaldehyde causes nasopharyngeal cancer, “strong but not sufficient” evidence of leukemia, and limited evidence of sinonasal cancer, along with “sufficient” evidence that formaldehyde causes nasal cancer in animals (Cogliano, 2005). Nasal cancer had been induced empirically with formaldehyde vapor in a two-year study where rats were exposed to 2, 6, or 14 ppm six hours per day, five days per week. The incidence of nasal squamous cell carcinomas was zero in the control and 2-ppm groups, 1% in the 6-ppm group, and 44% in the 14-ppm group. Mice were much less sensitive; only one carcinoma was seen at 14 ppm. The detection of DNA adducts in the two species paralleled the difference in the incidence of tumors as well as regional dosimetry. Formaldehyde, with its large and diverse database and potential public health impact, has remained the focus of considerable debate among modelers and risk assessors. The arguments behind this debate crosses both cancer and noncancer considerations and is beyond that which can be discussed in this chapter. The reader is encouraged to sample the recent literature (eg, Conolly et al., 2004; Nielsen et al., 2010).
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Because acrolein is an unsaturated aldehyde, it is more reactive than formaldehyde. It penetrates a bit deeper into the airways and may not have the same degree of sensory irritancy but it may cause more damage. Concentrations below 1 ppm cause irritation of the eyes and the mucous membranes of the respiratory tract. Exposure of guinea pigs to <0.6 ppm reversibly increased pulmonary flow resistance and tidal volume and decreased respiratory frequency (Murphy et al., 1963). With irritants of this type, flow resistance is increased by concentrations below those that cause the classic decrease in frequency seen with sensory irritants. This suggests that increases in flow resistance would be produced by far lower concentrations of acrolein than were tested. The mechanism of increased resistance appears to be mediated through both a local C-fiber and centrally mediated cholinergic reflexes (Bessac and Jordt, 2010). Ablation of the C-fiber network and atropine (muscarinic blocker) block this response.
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Exposures of rats to 0.4, 1.4, or 4.0 ppm for six hours per day, five days per week for 13 weeks resulted in paradoxical effects on lung function (Costa et al., 1986). The lowest concentration resulted in hyperinflation of the lung with an apparent reduction in small-airway flow resistance, while the highest concentration resulted in airway injury and peribronchial inflammation and fibrosis. The intermediate concentration was functionally not different from the control, although airway pathology was evident. It appears that the high-concentration response reflected the cumulative irritant injury and remodeling as a result of the repeated acrolein, while the low-concentration group had little overt damage and appeared to have slightly stiffened airways, perhaps a result of the protein cross-linking action of acrolein. The pathology in these rats contrasts with that found in formaldehyde studies of similar duration where more upper airway involvement was observed. Ambient exposure to acrolein probably would be about 10% to 20% of the low concentration used in the subchronic study discussed above. Because of a lack of sufficient human data, these animal data were used to perform a risk assessment analysis that showed an excess risk to humans for the adverse effect of ambient levels of acrolein on pulmonary function (Woodruff et al., 2007). However, ambient concentrations of acrolein are well below those found in mainstream tobacco smoke and the occupational exposure levels. As reviewed elsewhere (Bein and Leikauf, 2011), acrolein produces a wide range of adverse pulmonary effects including lung cancer that may be linked mechanistically to p53 (a tumor suppressor gene) DNA adducts and mutations.
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Thus, as a class the aldehydes can be very irritating and may constitute a significant fraction of the discomfort and sensation experienced during an oxidant pollution episode, especially in mixed atmospheres containing particles.
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Carbon monoxide is classed toxicologically as a chemical asphyxiant because its toxic action stems from its formation of carboxyhemoglobin, preventing oxygenation of the blood for systemic transport. The fundamental toxicology of CO and the physiologic factors that determine the level of carboxyhemoglobin attained in the blood at various atmospheric concentrations of carbon monoxide are detailed in Chap. 11.
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The normal concentration of carboxyhemoglobin (COHb) in the blood of nonsmokers is about 0.5%. This is attributed to endogenous production of CO from heme catabolism. Blood COHb is a function of the concentration in air, the length of exposure, and the ventilation of the exposed individual. Uptake is said to be ventilation-limited, implying that virtually all the CO inspired in a breath is absorbed and bound to the available hemoglobin. Thus, continuous exposure of human subjects to 30 ppm CO leads to an equilibrium value of 5% COHb. The Haldane equation is used to compute the COHb equilibrium under a given exposure situation. The equilibrium values generally are reached after eight hours or more of exposure, but the time required to reach equilibrium can be shortened by physical activity.
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Carbon monoxide emissions from automobiles have progressively decreased since the 1970s. Increased combustion efficiency and catalytic converters have reduced emissions at the tailpipe by more than 90%. However, motor vehicles still account for two thirds of urban CO. Depending on the location in a community, traffic density, vehicle types and age, and the urban structure, CO concentrations can vary widely. Concentrations predicted inside passenger compartments of motor vehicles in downtown traffic were almost three times those for central urban areas and five times those expected in residential areas. Occupants of vehicles traveling on expressways had CO exposures somewhere between those in central urban areas and those in downtown traffic and concentrations in underground garages, tunnels, and buildings over highways can still reach high levels. But it must be kept in mind that there are many sources of CO—anywhere combustion is ongoing. Certainly both main and sidestream tobacco smoke are a source. Home heating systems and mobile auxiliary heating sources emit CO that when used inappropriately (ie, unventilated) can create life-threatening circumstances.
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No overt clinical human health effects have been demonstrated for COHb levels below 2%, while levels above 40% cause fatal asphyxiation. However, there is evidence that in some people COHb in the range of 2.0% to 2.4% can elicit acute cardiovascular effects. A 90-minute exposure to about 50 ppm CO would likely result in COHb levels of ~2.5%, with a resultant impairment of time-interval discrimination even in healthy subjects; at approximately 5% COHb, there is an impairment of other psychomotor faculties. At 5% COHb in nonsmokers (the median COHb value for smokers is about 5%), however, maximal exercise duration and maximal oxygen consumption are reduced (Aronow, 1981). Clear cardiovascular changes also may be produced by exposures sufficient to yield COHb in excess of 5%. These include increased cardiac output, arteriovenous oxygen difference, and coronary blood flow in patients without coronary disease. Decreased coronary sinus blood PO2 occurs in patients with coronary heart disease, and this would impair oxidative metabolism of the myocardium. In the early 1990s, a series of studies in subjects with cardiovascular disease was conducted in several laboratories under the sponsorship of the Health Effects Institute (HEI) to determine the potential for angina pectoris when they exercised moderately with COHb levels in the range of 2% to 6% (Allred et al., 1989). The results of these studies indicate that premature angina can occur under these conditions but that the potential for the induction of ventricular arrhythmias remains uncertain.
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Carbon monoxide has been implicated as a potentially important copollutant of PM with potential human health effects at low ambient levels. Independent CO effects have been reported in some epidemiological studies of ambient air pollution, but its singular role versus indicator role (for traffic) continues to be debated. The fact that effects noted in these studies are cardiovascular and involve angina and effects on EKG patterns lends credence to a role for CO in epidemiological health outcomes. Other data from epidemiological studies have shown a linkage between CO and some general CNS indices as well as birth outcomes. The sensitivity of newer analysis methods for these population studies is revealing previously unappreciated health effects. Among these are preterm births, cardiac birth defects, and infant mortality in the postneonatal period. Moreover, toxicological studies with animals also suggest potential developmental risks.
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Reducing CO emissions is a major goal because of its high hazard—a colorless and odorless nonirritant—so technical combustion engineering advances are being pursued as well as changes in fuel formulations. The introduction of gasoline oxygenates such as MTBE in the 1990s and other ether derivatives constitutes attempts to enhance fuel combustion and reduce CO emissions. The experience with MTBE (as noted earlier) has led to such formulations being approached with some caution with greater attention being placed on assessments of potential unexpected consequences.
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Hazardous Air Pollutants
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HAPs (so-called air toxics) represent an inclusive classification for air pollutants of anthropogenic origin that are generally of measurable quantity in the air, and are not covered in the Criteria Pollutant list. They are covered under Section 112 of the CAA. Selected regulatory issues of the HAPs were discussed above. Most exposure estimates for these pollutants are derived from emission inventories that are modeled into the National Air Toxics Assessment (www.epa.gov/ttn/atw/nata/sitemap.html). The most recent NATA assessment (2005) used 2005 data on 177 of the 188 HAPs, plus diesel PM. The diverse nature of even 33 of the 188 HAPs (the so-called dirty 30 noted above) complicates a general discussion of their toxicology because the group includes various classes of organic chemicals (by structure, eg, acrolein, benzene), minerals (eg, asbestos), polycyclic hydrocarbon particulate material (eg, benzo(a)pyrene), and various metals and metal compounds (eg, mercury, beryllium compounds) and pesticides (eg, carbaryl, parathion).
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The focus to date on the HAPs has been on their potential carcinogenicity, as shown in chronic bioassays, mutagenicity tests in bacterial systems, structure–activity relationships, or—in a few special cases (eg, benzene, asbestos)—their known carcinogenicity in humans. These cancers need not be, and generally are not, pulmonary. Noncancer issues frequently relate to direct lung toxicants that, on fugitive emissions or accidental release, might risk those with preexisting diseases (eg, asthma) or might lead to chronic lung disease. The assessment of noncancer risk by air toxics to any organ system is based on the computation of long-term risk reference exposure concentrations (RfCs) to which individuals may be exposed over a lifetime without adverse, irreversible injury. This approach to HAP assessment is discussed in detail by Jarabek and Segal (1994). An analogous short-term RfC method has been developed for exposures up to 30 days. Of the noncancer risks deemed highest in this analysis, acrolein ranks at the top based on emission inventories, the potential for exposure, and its inherent irritancy. Discussion of this topic and an approach to mixed exposures to noncancer HAPs has been reviewed (Costa, 2004).
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Accidental versus “Fence-Line” Exposures
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The relationship between the effects associated with an accidental release of a large quantity of a volatile chemical into the air from a point source such as a chemical plant and the effects associated with a chronic low-level exposure over many years or a lifetime is not clear. With regard to cancer, which defaults to a linearized model of dose and effect (though some alternative models can be used if there are appropriate data), the issue is fairly straightforward. Any exposure must be minimized if not eliminated if cancer risk is to be kept as close to zero as possible. With noncancer risks, the roles of nonspecific or specific host defenses, thresholds of response, and repair and recovery after exposure complicate the assessment of risk. In large part, the issue here relates to C × T. Can we better relate disease or injury to cumulative dose or peak concentration for protracted exposures? Is there an exposure peak beyond which a cumulative approach fails (ie, the effect is concentration-driven), or is concentration always the dominant determinant? Many of these questions have yet to be answered, not to mention their specificity with regard to individual compounds and tissues affected.
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Methyl isocyanate provides a contrast between the effects of a large accidental release and those produced by cyclic or continuous small fugitive vapor releases. The reactive nature of methyl isocyanate with aqueous environments is of such magnitude that on inspiration, almost immediate mucous tissue corrosion can be perceived. The vapor undergoes hydrolysis within the mucous lining of the airways to generate hydrocyanic acid, which destroys the airway epithelium and causes acute bronchoconstriction and edema. The damage is immediately life-threatening at concentrations above 50 ppm; at 10 ppm, it is damaging in minutes. These concentrations are in the range of the dense vapor cloud that for several hours enshrouded the village of Bhopal bordering the Union Carbide pesticide plant. Studies in guinea pigs showed the immediate irritancy of this isocyanate, which in just a few minutes also resulted in significant pathology (Alarie et al., 1987). Rats exposed to 10 or 30 ppm for two hours also showed severe airway and parenchymal damage, which did not resolve in surviving rats; transient effects were seen at 3 ppm. Even six months after exposure, the airway and lung damage remained, having evolved into patchy, mostly peribronchial fibrosis with associated functional impairments (Stevens et al., 1987). There was also cardiac involvement secondary to the damage to the pulmonary parenchyma and arterial bed. As a result, there was pulmonary hypertension and right-sided heart hypertrophy. This same spectrum of health effects has resulted in disability and deaths of thousands of initial survivors since the incident.
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In the United States, methyl isocyanate has been measured in Katawba Valley, Texas, as a result of small but virtually continual fugitive releases of the vapor into the community air (“fence-line”) from an adjoining region with several chemical plants. While these levels of methyl isocyanate are not sufficient to cause the damage seen in Bhopal, there is concern that low-level exposure over many years may have more diffuse, chronic effects. Residents complain of odors and a higher frequency of respiratory disorders, but clear evidence of injury or disease is lacking.
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Phosgene is best known for its use as a war gas, but it is also a common intermediate reactant used in the chemical industry, particularly in pesticide formulation. It is also a constituent of photochemical smog. Because of its direct pulmonary reactivity, it lends itself to use as a model pulmonary toxicant for studies addressing C × T relationships. These studies suggest that there may be a threshold below which compensatory and other bodily defenses (eg, antioxidants) may be able to cope with long-term low-level exposure (tolerance). For phosgene, this appears to be at or below the current threshold limit value of 0.1 ppm for eight hours. At higher concentrations, however, concentration appears to be the primary determinant of injury or disease regardless of duration. Thus, even though there is some adaptation with time, there continues to be a concentration-driven response that exceeds that predicted by C × T. This relationship appears to be different from that of O3 at ambient levels, which can be approximated acutely by the C × T paradigm.