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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues 10 Mixtures Potential and known interactions between carcinogens and noncarcinogens in chemical mixtures in the environment have been a concern for several decades. Toxicokinetic and toxicodynamic interactions might result in decreased (antagonistic), exaggerated, additive, or unchanged toxicity relative to that of individual components. Although exaggerated toxicity is the primary concern, the antagonistic interaction resulting in decreased toxicity would also affect the assessment and management of risk. Kidney and liver are the major target organs for trichloroethylene toxicity resulting from the generation of reactive metabolites through glutathione conjugation and cytochrome P-450-mediated metabolism. Human health risks of trichloroethylene stem mainly from its carcinogenic potential. In the body, trichloroethylene is metabolized into trichloroacetic acid, chloral hydrate, 2-chloroacetaldehyde, trichloroethanol, trichloroethanol glucuronide, and perhaps dichloroacetic acid. Because trichloroethylene is readily absorbed by all routes of exposure and extensively metabolized to multiple chemical species, exposure to trichloroethylene can be considered an exposure to a toxic mixture. Information on trichloroethylene metabolite toxicity is helpful in identifying the metabolites responsible for toxicity and might influence the effect of coexposures to other toxicants, particularly if they directly or indirectly change the proportions of trichloroethylene metabolites. This chapter presents an overview of mixture toxicology, some of the important coexposure issues to consider in evaluating trichloroethylene, and possible approaches to using pharmacokinetic modeling for making predictions.
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TOXICOLOGY OF MIXTURES CONTAINING TRICHLOROETHYLENE Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species, but it cannot take into account the possible toxic effects of mixtures of compounds. For example, in a 6-month carcinogenicity assay, trichloroethylene-contaminated groundwater was found to be carcinogenic in Japanese medaka fish, after initiation with diethylnitrosamine (Gardner et al. 1998). Analysis of the groundwater indicated that contamination was not limited to trichloroethylene. No tumor promotional effect was found in a follow-up laboratory study with reagent-grade trichloroethylene added to the groundwater to simulate the exposure concentration found in the contaminated groundwater. These studies implicate other water contaminants that might synergize the tumorpromoting activity of trichloroethylene. Acute or repeated inhalation exposure to a mixture of 1,1,1-trichloroethane, 1,1-dichloroethane, trichloroethylene, and tetrachloroethylene at concentrations as low as 20 parts per million (ppm) produced neurologic impairment. Male and female weanling ICR mice were treated with a mixture of chlorinated alkanes and alkenes consisting of chloroform, 1,1-dichloroethane, 1,1-dichloroethylene, 1,1,1-trichloroethane, trichloroethylene, and tetrachloroethylene in drinking water for 16 and 18 months, respectively; male mice developed hepatocelluar neoplasms and female mice developed mammary adenocarcinoma (Wang et al. 2002). The toxicokinetics of trichloroethylene was altered in rats receiving a binary mixture of chloroform and trichloroethylene (Anand et al. 2005a). Metabolism of trichloroethylene is suppressed in humans with coexposure to tetrachloroethylene (Seiji et al. 1989). Exposure to a ternary mixture of chloroform, trichloroethylene, and allyl alcohol results in less initial liver injury in male Sprague-Dawley rats because of greater elimination of trichloroethylene (Anand et al. 2005b). A number of commonly used drugs modify the metabolism of trichloroethylene (Leibman and McAllister 1967; Carlson 1974; Moslen et al. 1977; Pessayre et al. 1979). The opposite might also occur, resulting in important modifications of the therapeutic action of the drugs (Kelley and Brown 1974). Trichloroethylene competitively inhibits the metabolism of barbiturates, producing exaggerated effects of the drugs (Kelley and Brown 1974). Sellers and Koch-Weser (1970) observed a potentiation of the anticoagulant effect of bishydroxycoumarin (warfarin) in patients after chloral hydrate ingestion, which appears to result from displacement of plasma protein binding sites by the chloral hydrate metabolite trichloroacetic acid. Trichloroacetic acid is extensively bound to plasma proteins (Templin et al. 1995), making it likely that trichloroethylene might potentiate the effects of many other drugs that normally bind to the same protein sites. Ethanol (2 g
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues in daily liquid diet for 3 weeks) pretreatments enhanced hepatic damage in male Wistar rats treated with trichloroethylene (inhalation exposures of 500 ppm for 8 hours, 2,000 ppm for 2 or 8 hours, and 8,000 ppm for 2 hours) (Okino et al. 1991). Chemical coexposures from the environment in addition to human behaviors, such as alcohol consumption, might have effects that overlap with hepatic damage in male Wistar rats in terms of toxicokinetics, pharmacodynamics, and target tissue toxicity (see also Chapters 9 and 11). Alcohol consumption is a common coexposure that has been noted to affect trichloroethylene toxicity (see discussion later in this chapter). Co-exposure to trichloroethylene might increase the toxicity of methanol and ethanol by altering their metabolism to aldehydes and also by altering their detoxification. Concomitant administration of alcohol and chloral hydrate in humans exacerbated the side effects of chloral hydrate (e.g., vasodilation, tachycardia, hypotension) (Sellers et al. 1972; Muller et al. 1975). The intolerance syndrome resulting from combined exposure to trichloroethylene and ethanol is due to increased accumulation of trichloroethylene in the central nervous system resulting from depression of trichloroethylene oxidation. Therefore, there is adequate basis for interactions to modulate the toxicity of trichloroethylene upon coexposure to other chemicals. Interaction of metals with trichloroethylene could result in altered absorption of the metals. Dermal penetration of nickel significantly increased when it was administered along with phenol, toluene, and trichloroethylene to dermatomed male pig skin samples in flow-through diffusion cells. Consequently, the potential health risk from dermal exposure to nickel is enhanced if other chemicals are present (Turkall et al. 2003). Not all metals interact with trichloroethylene in the same way. When lead carbonate and trichloroethylene were given concurrently to male rats, no additive or synergistic neurotoxicities were observed (Nunes et al. 2001). Coexposures to trichloroethylene, trichloroacetic acid, and dichloroacetic acid at environmental concentrations are not uncommon (Wu and Schaum 2000). Trichloroethylene and tetrachloroethylene share common metabolites that have similar actions and targets and, therefore, coexposures potentially increase the risk from exposure to trichloroethylene. Trichloroethylene and di-(2-ethylhexyl)phthalate, a peroxisome proliferator, were reported to synergize prenatal loss, cause a decrease in pup weight, and cause anaophthalmia in rats (Narotsky and Kavlock 1995; Narotsky et al. 1995). Veeramachaneni et al. (2001) reported effects in male rabbits exposed to drinking water containing chemicals at concentrations typical of groundwater near hazardous waste sites (the exposure mixture contained arsenic, chromium, lead, benzene, chloroform, phenol, and trichloroethylene). Even at 45 weeks after last exposure to drinking water pollutants, mating desire or ability, sperm quality, and Leydig cell function were subnormal. However, although the exposure concentrations are relevant to human environmen-
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues tal exposures, the design of this study precludes a conclusion about what combination of the seven toxicants, or what individual toxicant, caused the effects, exemplifying the problems associated with studying toxicology of a multicomponent mixture. Recent literature on interactions of trichloroethylene metabolites and common coexposures report the interactions of two or three chemicals at a time and use several approaches including examination of tumor phenotype, gene expression, and development of physiologically based pharmacokinetic (PBPK) models to assess possible synergy, antagonism, and additivity of effects or toxicokinetics. These studies may provide insights into possible modes of action and modulators of trichloroethylene toxicity. One area that still hampers the risk assessment is interindividual differences that lead to variation in toxic responses in human populations. Although many factors are involved and the science still does not allow us to quantitate the influence of each factor, little is known about the influence of diet and caloric intake on trichloroethylene toxicity. A diet rich in carbohydrates protects male Wistar rats from liver injury by decelerating the transformation of trichloroethylene to highly toxic intermediates (Nakajima et al. 1982). A combination of ethanol with a low-carbohydrate diet accelerates the metabolism and enhancement of hepatotoxicity of trichloroethylene in male Wistar rats (Sato et al. 1983). A dietary copper imbalance resulted in higher trichloroethylene-induced lung damage as evidenced by a larger number of vacuolated Clara cells (Giovanetti et al. 1998). Some of the important coexposures that affect the toxicity of trichloroethylene are discussed below. Contaminants of Trichloroethylene Earlier carcinogenic studies (NCI 1976) with trichloroethylene were faulted because they used commercial grade trichloroethylene as the test agent, which could contain stabilizers such as epichlorohydrin, a known carcinogen. Henschler et al. (1984) studied the effects of oral administration of trichloroethylene with and without stabilizers (epichlorohydrin and 1,2- epoxybutane) on ICR/Ha Swiss mice. They concluded that there was an increase in forestomach cancers in the mice treated with trichloroethylene containing stabilizers, but there was no effect on the induction of liver tumors. They attributed the increase in forestomach cancers to the direct alkylating properties of epichlorohydrin and epoxybutane. Interactions Between Trichloroacetic Acid and Dichloroacetic Acid A recent study (Bull et al. 2002) attempted to examine how coexposures and variations in relative concentration between two trichloroethylene
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues metabolites, dichloroacetic acid and trichloroacetic acid, might affect toxicity. Bull et al. (2002) reported that the tumor phenotype in B6C3F1 male mice depended on the proportion of the two chemicals administered after 52 weeks of exposure. Given alone, trichloroacetic acid (0.5 or 2 g/L) and dichloroacetic acid (0.1, 0.5, or 2 g/L) produced liver tumors in mice with phenotypic characteristics that are distinct in several respects, with each compound at doses that were not cytotoxic. Combinations of trichloroacetic acid (0.5 or 2 g/L) and dichloroacetic acid (0.1 or 0.5 g/L) resulted in dose-related increases in hepatic preneoplastic lesions, adenomas, and carcinomas greater than either compound alone and in an additive fashion, with the addition of dichloroacetic acid to fixed exposures to trichloroacetic acid causing an increase in adenomas but not in carcinomas. Given alone, dichloroacetic acid produces tumors in mice that display a diffuse immunoreactivity to a c-Jun antibody, whereas trichloroacetic acid-induced tumors do not stain with this antibody. When given in various combinations, dichloroacetic acid and trichloroacetic acid produced a few c-Jun+ tumors, and many that were c-Jun-, but a number with a mixed phenotype increased with the dose of dichloroacetic acid. A comparison of tumor phenotypes induced by trichloroethylene (1 g/kg) shows that such tumors also have a mixture of phenotypes, suggesting that trichloroethylene-induced tumors are not consistent with either trichloroacetic acid or dichloroacetic acid acting alone. Coexposures to Other Haloacetates Other haloacetates produced in the bromination of drinking water might affect trichloroethylene toxicity through a similarity of effects of its metabolites. Kato-Weinstein et al. (2001) reported that brominated haloacetates such as bromodichloroacetate, bromochloroacetate, and dibromoacetate appear at higher concentrations in drinking water than the chlorinated haloacetates dichloroacetic acid and trichloroacetic acid. To study the similarity in action between the brominated and chlorinated haloacetates, mice were administered dibromoacetate, bromochloroacetate, and bromodichloroacetate in drinking water at concentrations of 0.2-3 g/L for 12 weeks (Tao et al. 2005). The dihaloacetates, bromochloroacetate and dibromoacetate, caused liver glycogen accumulation similar to that of dichloroacetic acid. The authors noted possible contamination of bromochloroacetate with dichloroacetic acid and dibromoacetate in their studies. The trihaloacetates, trichloroacetic acid and low concentrations of bromodichloroacetate, produced slight decreases in liver glycogen content, especially in the centrilobular region. The high concentration of bromodichloroacetate produced a pattern of glycogen distribution similar to that in dichloroacetic acid-treated mice. All dihaloacetates reduced the amount
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues of serum insulin at high concentrations. Conversely, trihaloacetates had no significant effects on serum insulin concentrations. After up to 26 weeks of treatment, dibromoacetate was the only brominated haloacetate that consistently increased acyl-coenzyme A oxidase activity (a marker of peroxisome proliferator-activated receptor alpha) agonism and rates of cell replication in the liver, but these effects were limited to 2-4 weeks of treatment and at exposures > 1 g/L (Tao et al. 2004a). Coexposures to Other Solvents Promotional and gene expression effects of trichloroethylene metabolites have been investigated in a number of studies in which they were administered after initial treatment with other carcinogens. Bull et al. (2004) studied interactions of metabolites (trichloroacetic acid and dichloroacetic acid) and carbon tetrachloride, motivated by the fact that trichloroethylene and carbon tetrachloride are commonly found together at contaminated sites. B6C3F1 male mice, initially treated vinyl carbamate (3 mg/kg) at 2 weeks of age, were treated with dichloroacetic acid (0.1, 0.5, or 2.0 g/L), trichloroacetic acid (0.1, 0.5, or 2.0 g/L), and carbon tetrachloride (50, 100, and 500 mg/kg, and then reduced at week 24 to 5, 20, and 50 mg/kg due to toxicity) or pairwise combinations of the three compounds for 18-36 weeks. Histopathologically, a sample of 100 lesions was examined to verify that the criteria for the general descriptor of neoplastic and nonneoplastic lesions were satisfied. As the dose of carbon tetrachloride increased, the number of tumors per animal increased, whereas mean tumor size decreased. When administered alone in drinking water, dichloroacetic acid increased both tumor number and tumor size in a dose-related manner. With trichloroacetic acid treatment, tumor numbers plateaued by 24 weeks at a high dose. Dichloroacetic acid treatment did not produce a plateau in tumor number within the experimental period, but the numbers observed at the end of the experimental period (36 weeks) were similar to those found with trichloroacetic acid and to doses of carbon tetrachloride at 50 mg/kg. Differing combinations of the three agents in initiated animals gave more complex results between 24 and 36 weeks of observation. At 24 weeks, dichloroacetic acid produced a decrease in tumor numbers promoted by trichloroacetic acid, but the numbers were not different from those for trichloroacetic acid alone at 36 weeks. The reason for this result became apparent at 36 weeks of treatment, when dichloroacetic acid coadministration led to a dose-related decrease in the size of tumors promoted by trichloroacetic acid. However, the low dose of trichloroacetic acid decreased the number of tumors produced by a high dose of dichloroacetic acid (2 g/L), but higher doses of trichloroacetic acid (2 g/L) produced the same number that was observed with dichloroacetic acid alone. Dichloroacetic acid inhibited the
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues growth rate of carbon tetrachloride-induced tumors. Trichloroacetic acid substantially increased the number of tumors observed at early time points when combined with carbon tetrachloride, but this effect was not observed at 36 weeks. The lack of an effect at 36 weeks was attributed to the fact that more than 90% of the livers consisted of tumors and the earlier effect was masked by coalescence of the tumors. Thus, trichloroacetic acid significantly increased tumor numbers in mice treated with carbon tetrachloride. Pretreatment with trichloroethylene in drinking water at concentrations as low as 15 mM for 3 days has also been reported to increase susceptibility to liver damage to subsequent exposure to a single intraperitoneal injection (1 mL/kg) of carbon tetrachloride in Fischer 344 rats (Steup et al. 1991). Several mechanistic hypotheses offered included altered metabolism, decreased hepatic repair capability, decreased detoxification ability, or a combination of these. Simultaneous administration of trichloroethylene (0.5 mL/kg) also increased the liver injury induced by carbon tetrachloride (0.05 mL/kg) (Steup et al. 1993). The authors suggested that trichloroethylene appeared to impair the regenerative activity in the liver, thus leading to increased damage when carbon tetrachloride is given in combination with trichloroethylene. Chloroform, a chlorine disinfection by-product found in drinking water, as well as dichloroacetic acid and trichloroacetic acid, is also a mouse liver carcinogen and was the focus of another study by Pereira et al. (2001). They reported the effects of coexposure to chloroform (0, 400, 800, 1,600 mg/L) on hypomethylation and expression of the c-myc gene induced by treatment with dichloroacetic acid and trichloroacetic acid (500 mg/kg) in the livers of female B6C3F1 mice. Dichloroacetic acid, trichloroacetic acid, and to a lesser extent chloroform decreased methylation of the c-myc gene. Coadministering chloroform (at 800 and 1,600 mg/L) decreased dichloroacetic acid-induced hypomethylation but it had no effect on that of trichloroacetic acid. Expression of c-myc mRNA was increased by dichloroacetic acid and trichloroacetic acid, with the two highest doses of chloroform attenuating the actions of dichloroacetic acid but not trichloroacetic acid. In the same study, male and female B6C3F1 mice, administered N-methyl-N-nitrosourea (an initiator of liver and kidney tumors) on day 15 of age, and dichloroacetic acid (3.2 g/L) or trichloroacetic acid (4.0 g/L) with chloroform (0, 800, or 1,600 mg/L) starting at 5 weeks of age, were examined after 36 weeks for promotion of liver and kidney tumors (Pereira et al. 2001). However, the numbers of animals in the group treated with N-methyl-N-nitrosourea, dichloroacetic acid, and chloroform and in the group treated with N-methyl-N-nitrosourea, trichloroacetic acid, and chloroform were variable and small (n = 6-8 in the female group), limiting the power of the study. In female mice, coexposure to 800 and 1,600 mg/L decreased the number of adenomas induced by N-methyl-N-nitrosourea and dichloro-
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues acetic acid, with no effect on carcinomas or on adenomas and carcinomas in the liver induced by N-methyl-N-nitrosourea and trichloroacetic acid. N-methyl-N-nitrosourea and dichloroacetic acid treatment resulted in no carcinoma induction in females. Only one animal had carcinomas induced by N-methyl-N-nitrosourea, dichloroacetic acid, and chloroform treatment. In male mice, N-methyl-N-nitrosourea and dichloroacetic acid treatment induced carcinomas as well as adenomas in the liver, with chloroform coexposure (at high concentrations) having no effect on the numbers of animals with adenomas and a reduction in those with carcinomas. Only the highest concentration of chloroform appeared to decrease the number of animals with adenomas in the trichloroacetic acid-treated group. No foci of altered hepatocytes were found in N-methyl-N-nitrosourea-initiated control mice of either sex. A larger number of foci of altered hepatocytes were seen in female than in male mice after N-methyl-N-nitrosourea and dichloroacetic acid treatment, although the number of tumors per mouse was about the same. Chloroform decreased the number of foci of altered hepatocytes and tumors per mouse at the two highest doses of dichloroacetic acid treatment in females and at the highest doses in males. Trichloroacetic acid induced few foci in female or male mice, with chloroform having no effect on foci of altered hepatocyte formation or tumor induction. Liver tumors and foci of altered hepatocytes were characterized as basophilic or eosinophilic. In females, both foci of altered hepatocytes and tumors were eosinophilic after N-methyl-N-nitrosourea and dichloroacetic acid treatment, whereas in males only foci of altered hepatocytes were eosinophilic, with tumors being basophilic. Coexposure to chloroform increased the percentage of foci of altered hepatocytes in males that were basophilic. Liver foci of altered hepatocytes and tumors induced by N-methyl-N-nitrosourea and trichloroacetic acid treatment were basophilic in both sexes, with methyl chloroform having no effect. These results are consistent with those of Latendresse and Pereira (1997), who reported that, after initiation of N-methyl-N-nitrosourea, dichloroacetic acid-induced foci of altered hepatocytes and tumors in female mice were eosinophilic and stained positively for transforming growth factor alpha, c-jun, c-myc, CYP2E1, CYP4A1, and glutathione S-transferase (GST)-pi, while trichloroacetic acid treatment induced foci of altered hepatocytes and tumors that were predominantly basophilic, lacked GST-pi, and stained variably for other biomarkers. Pereira et al. (2001) also reported promotion of kidney tumors in male mice from dichloroacetic acid, trichloroacetic acid, and chloroform coexposures. The pattern of tumors in the kidneys were different than in the liver. No kidney tumors were initiated in male mice after treatment with N-methyl-N-nitrosourea alone or with chloroform coexposure. However, trichloroacetic acid increased the incidence (90%) and multiplicity of kidney tumors initiated by N-methyl-N-nitrosourea. Coexposure of chloroform
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues with trichloroacetic acid had no effect on tumor incidence or multiplicity. Dichloroacetic acid alone did not significantly increase the incidence (24%) or multiplicity of N-methyl-N-nitrosourea-initiated kidney tumors, but coexposure with chloroform increased the incidence of kidney tumors to 100% in male mice. In female mice, kidney tumor incidence and multiplicity after trichloroacetic acid or dichloroacetic acid treatment with or without chloroform was low after initiation with N-methyl-N-nitrosourea. Using a single-dose exposure, the toxicity of a quaternary mixture of trichloroethylene, allyl alcohol, chloroform, and thioacetamide, structurally dissimilar toxicants with dissimilar mechanisms by which they initiate liver injury, was tested and compared with the toxicity of individual components and the sum of their toxic effects in male Wistar rats (Soni et al. 1999). Also, the liver reparative responses to injury initiated by each component, and the sum of their effects, were compared with the response after exposure to the quaternary mixture. The combined toxic effects were additive, primarily because of a dose-related stimulation of liver reparative response that prevented progression and expansion of liver injury. The studies showed that the extent of injury at early time points correlates well with maximal stimulation of the liver tissue repair response suggesting that, in addition to initiation of tissue injury, the toxicodynamics of cell birth and tissue repair should be considered in evaluating the final toxic outcome. Trichloroethylene and Tetrachloroethylene Trichloroethylene and tetrachloroethylene are often found together as environmental contaminants, are metabolized by the same enzymes, and have similar metabolites (Green 1990). There are significant differences in the kinetics of metabolism of trichloroethylene and tetrachloroethylene by certain enzymes and in the chemical reactivity of certain analogous metabolites (IARC 1995a). Tetrachloroethylene metabolites are also formed in oxidative metabolism of trichloroethylene. But trichloroethanol and chloral are less important metabolites in tetrachloroethylene than in trichloroethylene metabolism. Tetrachloroethylene appears to be a much poorer substrate for cytochrome P-450 than its congener trichloroethylene (Ohtsuki et al. 1983; Volkel and Dekant 1998; Volkel et al. 1998). Hence, the various cytochrome P-450-derived metabolites from tetrachloroethylene and trichloroethylene will be produced at different rates. In vivo, tetrachloroethylene is conjugated with reduced glutathione (GSH) more extensively (1% to 2% of the dose) (Dekant et al. 1986b) than trichloroethylene (<0.05% of the dose) (Green et al. 1997a). In humans, the GSH conjugation pathway is toxicologically significant only at high doses or when the cytochrome P-450 pathway is saturated with trichloroethylene and tetrachloroethylene (Green 1990; Green et al. 1990). The glutathione pathway plays a greater role in tetra-
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues chloroethylene metabolism than in trichloroethylene metabolism. Chloral hydrate, a metabolite of both tetrachloroethylene and trichloroethylene, produces liver tumors in B6C3F1 mice (Rijhsinghani et al. 1986). Although chloral hydrate is the predominant intermediate in cytochrome P-450 metabolism of trichloroethylene, it is a minor intermediate in tetrachloroethylene metabolism (Lash and Parker 2001). Such differences in rates of metabolism of trichloroethylene and tetrachloroethylene and in mode of action imply that their risk hazards differ even though the same metabolites occur with both compounds. A nongenotoxic mode of action plays an important role in liver tumorogenesis induced by trichloroethylene and tetrachloroethylene in B6C3F1 mice. Tetrachloroethylene showed a higher degree of cytotoxicity than trichloroethylene in kidney cells isolated from male rats (Lash and Parker 2001). Low doses of trichloroethylene (5-20 μL) or tetrachloroethylene (1-5 μL) significantly enhanced the intracellular GSH concentration. However, the concentration of GSH rapidly decreased with higher doses of trichloroethylene (40-80 μL) or tetrachloroethylene (10-20 μL) (Wang et al. 2001). Trichloroethylene and Ethanol Because trichloroethylene and ethanol have common metabolic pathways and the liver is the main site of metabolism for both compounds, there is special interest in understanding whether individuals exposed to trichloroethylene who also consume alcohol regularly are at greater risk for developing target organ toxicity and cancer. Lower tolerance to the inebriating effects of alcohol among workers exposed to trichloroethylene has been well documented. A condition known as “degreasers flush” is seen in subjects exposed to trichloroethylene, where dilation of blood vessels in the skin surface occurs with consumption of small amounts of alcohol (Stewart et al. 1974). The interaction between alcohol and trichloroethylene is very complex. The outcome of this interaction depends on whether there is simultaneous or alternate exposure to the two compounds. This interaction could involve: (1) direct competition between ethanol, trichloroethylene, and its metabolites for drug-metabolizing enzymes; (2) increased expression and activity of liver CYP2E1 by alcohol consumption, which is known to affect trichloroethylene metabolism; (3) changes in availability of cofactors for enzymes catalyzing the reductive and oxidative metabolism of trichloroethylene that occurs as a result of oxidative metabolism of ethanol; and (4) abnormal generation of reactive oxygen species by induced CYP2E1, which could synergize the adverse effects of trichloroethylene metabolites. Trichloroethylene undergoes oxidation to chloral hydrate by the action of CYP2E1. Chloral hydrate then undergoes conversion to trichloroacetic
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues acid. This is an oxidative reaction catalyzed by aldehyde dehydrogenase, which requires oxidized nicotinamide adenine dinucleotide (NAD) as cofactor. Alternatively, chloral hydrate can be converted to trichloroethanol by alcohol dehydrogenase. This reductive reaction requires reduced nicotinamide adenine dinucleotide (NADH). Ethanol uses the same two pathways for its consecutive oxidation to acetaldehyde and acetic acid, respectively. Oxidation by CYP450 Versus GSH Conjugation Via GST: Changes in the Contribution of These Pathways to Trichloroethylene Metabolism by Ethanol Microsomal metabolism of ethanol via CYP2E1 occurs more prominently relative to alcohol dehydrogenase with chronic alcohol use (Lieber 2004). Competition for CYP2E1 during coexposure to ethanol and trichloroethylene can reduce the conversion of trichloroethylene to chloral hydrate (Muller et al. 1975). By blocking CYP2E1, less conversion of trichloroethylene to chloral hydrate can increase the narcotic and solvent effects of trichloroethylene in various tissues. This interference with CYP2E1 can also shift the metabolism of trichloroethylene into the glutathione pathway (Sato and Nakajima 1985), resulting in generation of more glutathione-derived adducts of trichloroethylene. Generation of more of these conjugates can alter the susceptibility of exposed subjects to the adverse effects of trichloroethylene in kidneys because greater generation and delivery of S-1,2-dichlorovinyl-L-cysteine to this organ can be detrimental. This metabolite has been linked to both acute tubular necrosis (Gandolfi et al. 1981; Vaidya et al. 2003a) and cancer formation by trichloroethylene. These findings are based primarily on animal studies. Additional experimentation is needed to determine whether this shift in trichloroethylene metabolism occurs with ethanol coexposure and what its toxicologic significance is in humans. Glutathione-mediated metabolism of trichloroethylene in humans is considered a minor pathway compared wtih its oxidative metabolism. This is in contrast to rodents, which are considered more susceptible to the acute nephrotoxicity and nephrocarcinogenicity of trichloroethylene. Accordingly, conjugation of trichloroethylene with glutathione is more prominent in rodents than in humans (Green et al. 1997a; Lash et al. 2000a). Bernauer et al. (1996) analyzed the urine of rats and humans for the presence of the N-acetylated metabolite of S-1,2-dichlorovinyl-L-cysteine after trichloroethylene exposure via inhalation. Urinary excretion of this metabolite was compared with that of products of the oxidative metabolites of trichloroethylene. The results showed that the urinary content of N-acetylated S-1,2-dichlorovinyl-L-cysteine in humans was 1,000-7,000 times lower than that for trichloroethanol and trichloroacetic acid (Bernauer et al. 1996). The existing data suggest that GSH conjugation is a minor pathway for
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues the metabolism of trichloroethylene in humans, but there is no indication of whether this pathway becomes more prominent during coexposure to ethanol and trichloroethylene, when ethanol metabolism impairs the oxidative metabolism of trichloroethylene via CYP2E1. By contrast, exposure to trichloroethylene alone in alcohol users is expected to have contrasting effects on the ability of the liver to metabolize trichloroethylene. With CYP2E1 induction, chloral hydrate formation during abstinence from alcohol consumption is expected to be higher, which should lead to enhanced generation of oxidative and conjugative trichloroethylene metabolites. Several animal studies have shown that to be the case. On the other hand, human studies documenting this finding are scarce. Shift in Reducing Equivalents During Alcohol Metabolism Ethanol metabolism is also known to shift the balance of reducing equivalents in hepatocytes (Kalant et al. 1970). A shift in the ratio of NAD+ to NADH in favor of the reduced pyridine nucleotide takes place during alcohol metabolism. This higher reducing environment in hepatocytes is known to affect the oxidative metabolism of trichloroethylene, as illustrated in studies by Larson and Bull (1989) where coadministration of ethanol and trichloroethylene to male Sprague-Dawley rats resulted in decreased blood concentrations of trichloroacetic acid compared with animals receiving trichloroethylene alone. Generation of trichloroacetic acid depends on NAD+ availability, which is lower during alcohol metabolism. Ethanol coexposure also increases the urinary excretion ratio for trichloroethanol/trichloroacetic acid (Larson and Bull 1989). The authors of the study pointed out that this effect was pronounced only when very high doses of trichloroethylene and ethanol were used. Nevertheless, the study shows that a larger supply of reducing equivalents by alcohol metabolism favors the formation of trichloroethanol over trichloroacetic acid, which was highly predictable based on the form of NADH needed to catalyze the different chloral hydrate biotransformation reactions. The effect of ethanol on trichloroethylene metabolism has also been investigated in isolated perfused rat livers (Watanabe et al. 1998). The results of these liver perfusion studies are similar to those reported by Larson and Bull (1989). The change in the NAD+-to-NADH ratio produced by ethanol oxidation has another implication for exposure to mixtures beyond the changes in activity of metabolic pathways for trichloroethylene just described. This shift in reducing equivalents resulting from NADH accumulation also increases mitochondrial superoxide production by accelerating the flow of electrons down the respiratory electron transport chain (Koch et al. 2004). This, along with a higher production of reactive oxygen species under conditions of CYP2E1 induction, can enhance the susceptibility of the liver and
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues other target organs to lipid peroxidation and oxidative damage to DNA produced by trichloroethylene and its metabolites. This shift in reducing equivalents produced by alcohol metabolism and NADH accumulation has also been implicated in some pathologic findings of alcoholic liver disease, including inhibition of fatty acid oxidation and steatosis. CYP2E1 Induction and Oxidative Stress The effect of alcohol use on microsomal metabolism and CYP2E1 expression deserves more in depth attention. The biochemical and toxicologic features of CYP2E1, as they relate to alcohol metabolism and toxicity, were recently reviewed by Caro and Cederbaum (2004). A decade ago, Cederbaum’s group developed a human hepatoma HepG2 cell line with constitutive expression of CYP2E1. The parental cell line lacks any detectable CYP2E1. Overexpression of CYP2E1 in HepG2 cells results in a 50% increase in production of reactive oxygen species compared with untransfected cells. Associated with this, CYP2E1-expressing cells also exhibited increased lipid peroxidation and a significant decrease in cell proliferation that is possibly due to mitochondrial damage inflicted by CYP2E1-induced oxidative stress. It is worth noting that the enhanced oxidative stress in transfected cells occurs in the absence of added toxicant, which indicates that CYP2E1 expression by itself is responsible for this effect. Although ethanol oxidation by liver alcohol dehydrogenase is the ratelimiting step in the total oxidation of this alcohol, ethanol oxidation also occurs via CYP450. This alternative metabolic pathway for ethanol is more pronounced with chronic alcohol consumption due to the well-documented CYP2E1 induction that occurs with chronic exposure. Multiple reviews have described this phenomenon and the mechanism involved in CYP2E1 induction (Lieber 2004). Normal CYP2E1 enzymatic activity generates reactive oxygen species such as superoxide and hydrogen peroxide in higher amounts than other CYP450 isoforms (Gorsky et al. 1984). With ethanol induction of hepatic CYP2E1, the enhanced formation of reactive oxygen species resulting from normal CYP2E1 catalysis has been linked to development of chronic alcoholic liver disease. Most importantly, in vivo and in vitro studies with freshly isolated hepatocytes have also demonstrated that ethanol exposure can produce oxidative stress and hepatocellular injury. CYP2E1 induction by ethanol has dual implications to toxicity resulting from exposure to mixtures consisting of ethanol and other xenobiotics. Enhanced expression of CYP2E1 influences not only the toxicologic potency of xenobiotics by altering the profile of metabolites that are generated but also the formation of reactive oxygen species that occurs with CYP2E1 induction can potentiate the toxic effects of xenobiotics that work by generating reactive oxygen species. These considerations are highly relevant to
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues trichloroethylene because alcohol consumption has been reported to affect trichloroethylene metabolism and also its hepatotoxicity (Nakajima et al. 1988; Okino et al. 1991). In summary, two sources of potentially damaging reactive oxygen species have been presented in relation to alcohol consumption: (1) one coming from NADH accumulation, which stimulates mitochondrial superoxide generation, and (2) a second one originating from induced CYP2E1 enzymatic activity. Reactive nitrogen species is another category of damaging intermediates produced in response to alcohol consumption (see below). Ethanol and Nitric Oxide Production: Changes in Blood Flow and Peroxynitrite Formation The enhanced production of nitric oxide that occurs in association with alcohol consumption can also affect the toxicity of trichloroethylene and its metabolites. Ethanol increases blood flow to selected organs, such as the kidney and liver, without affecting perfusion to other tissues like the brain and lungs. This effect appears to be mediated by a stimulation of nitric oxide production (Baraona et al. 2002a). However, there are conflicting reports on the effect of ethanol on the activity of inducible nitric oxide synthase. Some investigations indicate that ethanol induces nitric oxide synthase activity (Baraona et al. 2002a,b), but a recent study in rats showed that consuming a liquid diet containing 3% ethanol (vol/vol) for 12 weeks reduced hepatic inducible nitric oxide synthase activity significantly (Wang and Abdel-Rahman 2005). These results are inconsistent with higher nitric oxide generation. Regardless of the mechanism involved, increased production of nitric oxide by ethanol has dual implications for the toxicity of other xenobiotics. Changes in blood perfusion rates to selected organs can lead to changes in pharmacokinetic and pharmacodynamic parameters for xenobiotics in alcohol users. Second, increased production of nitric oxide leads to secondary production of peroxynitrite. This reactive nitrogen intermediate has been shown to cause protein nitration and tissue injury (Jaeschke et al. 2002). The combined effect of peroxynitrite and reactive oxygen species generated in response to alcohol consumption can synergize the cytotoxic potential of trichloroethylene. Implications Ethanol coexposure can change the biotransformation and disposition of trichloroethylene through three distinct mechanisms: (1) by direct competition between chloral hydrate and ethanol or its oxidative product acetaldehyde for alcohol or aldehyde dehydrogenase, (2) by changing the ratio of pyridine dinucleotide cofactors needed to convert chloral hydrate
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues to trichloroacetic acid or trichloroethanol, and (3) by direct competition between trichloroethylene and ethanol for the active site of CYP2E1. Greater availability of NADH favors the conversion of chloral hydrate to trichloroethanol, which is considered to be a noncarcinogenic metabolite of trichloroethylene. The significance to human health of this shift in metabolism is not known because most reports documenting this effect were generated with rodents. Simultaneous metabolism of trichloroethylene and ethanol by CYP2E1 can shift more of the trichloroethylene metabolism into the GST-conjugation pathway. In turn, higher generation of GSH-derived adducts of trichloroethylene (including S-1,2-dichlorovinyl-L-cysteine) can alter the susceptibility of the kidneys to acute necrosis and cancer. Once again, the significance of this interaction in human health is unknown. On the other hand, exposure to trichloroethylene after alcohol consumption in habitual drinkers represents another chemical interaction with mechanistic features that are distinct from the coexposure situation. As a result of CYP2E1 induction in alcohol users, trichloroethylene metabolism to chloral hydrate proceeds faster when ethanol is not present. This has been documented in rat studies in which pretreatment with ethanol resulted in increased urinary excretion of CYP450-derived metabolites of trichloroethylene, trichloroacetic acid, and trichloroethanol (Nakajima et al. 1988). This was associated with more pronounced hepatotoxicity. There is a large volume of data documenting this interaction between ethanol and trichloroethylene, where both metabolism and pattern of toxicity by trichloroethylene are changed. However, the bulk of this information is limited to studies using laboratory animals. Although some of the human data suggest that this interaction can occur in the workplace, its significance to alterations in patterns of trichloroethylene toxicity and cancer is unknown and deserves further attention. POTENTIAL MECHANISMS OF INTERACTION Bartonicek and Teisinger (1962) showed that disulfiram markedly inhibits the terminal enzymatic steps (detoxification) of trichloroethylene metabolism, resulting in enhanced trichloroethylene toxicity. Trichloroethylene induces CYP2E1 and inhibits alcohol dehydrogenase (Wang et al. 1999). Chloroform, when coadministered with dichloroacetic acid and trichloroacetic acid (metabolites of trichloroethylene), promoted kidney tumors in male mice by preventing hypomethylation of DNA and increasing mRNA expression of the c-myc gene (Pereira et al. 2001). The inductive and inhibitory effects of trichloroethylene on CYP2E1 and alcohol dehydrogenase, respectively, might result in different effects on the metabolism of other chemicals when coadministered with trichloroethylene. Besides a toxic response, tissue repair, a simultaneous biological compensatory response
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues that accompanies chemical-induced injury, also plays an important role in mixture toxicity (Anand et al. 2005a,b). Trichloroethylene potentiates the hepatotoxicity of carbon tetrachloride by increasing carbon tetrachlorideinduced lipid peroxidation (Pessayre et al. 1982). Recent studies (Vaidya et al. 2003b,c; Korrapati et al. 2005) suggest another potential mechanism of altered toxicity upon coexposure to other toxicants. Renal tissue repair was inhibited by a high dose of S-(1,2-dichlorovinyl)-L-cysteine (75 mg/kg, intraperitoneally) due to down-regulation of the IL-6/STAT-3 or the IL-6/ERK1/2 pathways causing cell cycle arrest at the beginning of the G1- to S-phase transition (Vaidya et al. 2003c). Downstream of the ERK1/2 pathway, a high dose of S-(1,2-dichlorovinyl)L-cysteine inhibits phosphorylation of IkBα, resulting in limited nuclear translocation of NF-kB. A cdk4/cdk6 system-mediated phosphorylation of retinoblastoma protein was down-regulated due to overexpression of p16 (Korrapati et al. 2005). Prior administration of a low priming dose of S-(1,2-dichlorovinyl)-L-cysteine (15 mg/kg) protects mice from a later lethal dose of S-(1,2-dichlorovinyl)-L-cysteine (75 mg/kg) (Vaidya et al. 2003b). A low dose of S-(1,2-dichlorovinyl)-L-cysteine exhibits prompt renal tubular regeneration by timely and adequate stimulation of IL-6, TGF-α, HB-EGF, EGFr, IGF-1Rβ, and phosphorylated ERK1/2, leading to recovery from a lethal dose challenge (Vaidya et al. 2003c). A priming dose led to higher expression of cyclin D1/cdk4-cdk6 downsteam, resulting in increased phosphorylation of retinoblastoma protein (Korrapati et al. 2005). Coexposure to other toxicants may result in interactions with the cellular signaling mechanisms affecting the response to trichloroethylene and, conversely, trichloroethylene (or its metabolites) might interfere with the cellular signaling mechanisms and cellular responses. Effects of chronic exposure to trichloroethylene or its metabolites on cellular signaling mechanisms and how they might be altered upon coexposure to other toxicants are not known. EFFECTS OF ALTERED OR SPECIAL PHYSIOLOGIC STATES Studies of exposure to trichloroethylene suggest a concern about reproductive issues and congenital heart defects (see Chapter 5 for complete discussion). For mixtures containing trichloroethylene, an increase in miscarriages has been reported among nurses exposed to unspecified concentrations of trichloroethylene and other chemicals in operating rooms (Corbett et al. 1974). Early exposure of male rabbits to a mixture of arsenic, chromium, lead, benzene, chloroform, phenol, and trichloroethylene in drinking water caused acrosomal dysgenesis, nuclear malformations, lower testosterone secretion, subnormal mating desire and ability, lower sperm quality, and decreased Leydig cell function (Veeramachaneni et al. 2001). Simultaneous oral administration of trichloroethylene (0.5 mL/kg) resulted in a marked
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues potentiation of liver injury caused by an oral dose of chloroform (0.05 mL/ kg) due to delayed hepatic regeneration (Steup et al. 1993). Pretreatment with drinking water solutions containing trichloroethylene or chloroform enhances the hepatotoxicity of carbon tetrachloride in Fischer 344 rats (Steup et al. 1991). Inhalation of small concentrations of petroleum and trichloroethylene caused degenerative changes in the hepatic parenchyma cells in pregnant female Wistar rats (Duricic and Duricic 1991). COEXPOSURE PREDICTIONS USING PBPK MODELS An important issue is whether and the degree to which modulation of toxicity by coexposures can be quantified. PBPK models have been developed to predict possible synergy, antagonism, and additivity of effects on pharmacokinetics. Given that trichloroethylene, tetrachloroethylene, and methyl chloroform are often found together in contaminated groundwater, Dobrev et al. (2001) attempted to investigate the pharmacokinetic interactions among the three solvents to calculate defined “interaction thresholds” for effects on metabolism and expected toxicity. Their null hypothesis was defined as competitive metabolic inhibition being the predominant result for trichloroethylene given in combination with other solvents. They used gas uptake inhalation studies to test different inhibition mechanisms. A PBPK model was developed with the gas uptake data to test multiple mechanisms of inhibitory interactions (competitive, noncompetitive, or uncompetitive) with the authors reporting competitive inhibition of trichloroethylene metabolism by methyl chloroform and tetrachloroethylene in simulations of pharmacokinetics in rats. Occupational exposures to chemical mixtures of the three solvents within their threshold limit value or time-weighted average limits were predicted to result in a significant increase (22%) in trichloroethylene blood concentrations compared with single exposures. Dobrev et al. (2002) extended this work to humans by developing an interactive human PBPK model to explore the general pharmacokinetic profile of two common biomarkers of exposure: peak trichloroethylene blood concentrations and total trichloroethylene metabolites generated in rats and humans. Increases in the trichloroethylene blood concentrations were predicted to lead to greater availability of the parent compound for glutathione conjugation, a metabolic pathway that may be associated with kidney toxicity or carcinogenicity. A fractional change in trichloroethylene blood concentration of 15% for a combined threshold limit value for exposure to the three chemicals (25, 50, and 350 ppm of tetrachloroethylene, trichloroethylene, and methyl chloroform, respectively) resulted in a 27% increase in S-(1, 2-dichlorovinyl)-L-cysteine metabolites, indicating a nonlinear risk increase due to combined exposures to trichloroethylene. Binary combinations of the solvents produced glutathione-mediated metabolite
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues amounts almost twice as high as the expected rates of increase in the parent compound blood concentrations. The authors suggested that using parent blood concentrations (a less sensitive biomarker) would result in two to three times higher (less conservative) estimates of potentially safe exposures. For detecting metabolic inhibition from tetrachloroethylene and methyl chloroform, the simulations showed trichloroethylene blood concentrations to be the more sensitive dose metric in rats, but the total of trichloroethylene metabolites was a more sensitive dose measure in humans. Finally, interaction thresholds were predicted to occur at lower concentrations in humans than in rats. Thrall and Poet (2000) investigated the pharmacokinetic impact of lowdose coexposures to toluene and trichloroethylene in male F344 rats in vivo using a real-time breath analysis system coupled with PBPK modeling. The authors reported that, using the binary mixture to compare the measured exhaled breath concentrations from high- and low-dose exposures with the predicted concentrations under various metabolic interaction simulations (competitive, noncompetitive, or uncompetitive inhibition), the optimized competitive metabolic interaction description yielded an interaction parameter Ki value closest to the Michaelis-Menten affinity parameter (Km) of the inhibitor solvent. They suggested that competitive inhibition is the most plausible type of metabolic interaction between these two solvents. Isaacs et al. (2004) reported gas uptake coexposure data for chloroform and trichloroethylene. They questioned whether it was possible to use inhalation data in combination with PBPK modeling to distinguish between different metabolic interactions using sensitivity analysis theory. They reported that chloroform and trichloroethylene act as competitive inhibitors of each other’s metabolism. Recommendations were made for the design of efficient experiments aimed at determining the type of inhibition mechanisms resulting from a binary coexposure protocol. Even though, as stated by Dobrev et al. (2002), other solvents inhibit trichloroethylene metabolism, it is possible to quantify the synergistic interaction of trichloroethylene on other solvents with techniques such as gas uptake inhalation exposures. Haddad et al. (2000) developed a theoretical approach to predict the maximum impact that a mixture consisting of coexposure to dichloromethane; benzene; trichloroethylene; toluene; tetrachloroethylene; ethylbenzene; m-, p-, and o-xylene; and styrene would have on venous blood concentration due to metabolic interactions in Sprague-Dawley rats. They conducted two sets of experimental coexposures. The first study evaluated the change in venous blood concentration after a 4-hour constant inhalation exposure to the 10-chemical mixture. The second study was designed to examine the impact of possible enzyme induction by using the same inhalation coexposure after a 3-day pretreatment with the same 10-chemical mixture. The resulting venous concentration measurements for trichloroethylene from the first
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues study were consistent with metabolic inhibition. The 10-chemical mixture was the most complex coexposure used in this study. The authors stated that resulting parent concentration time courses change less as mixture complexity increases, an observation consistent with metabolic inhibition. For the pretreatment study, the authors found a systematic decrease in venous concentration (due to higher metabolic clearance) for all chemicals except tetrachloroethylene. Overall, these studies suggest a complex metabolic interaction between trichloroethylene and other solvents. A PBPK model for trichloroethylene including all its metabolites and their interactions can be considered a mixture model in which all metabolites have a common starting point in the liver. An integrated approach is needed after taking into account trichloroethylene metabolites and their interactions with each other, including inhibition of metabolites. FINDINGS AND RECOMMENDATIONS Although the available data indicate that toxic effects of trichloroethylene and its metabolites are likely to change in the presence of exposure to other chemicals, including its metabolites and similar metabolites of other toxicants, a definitive understanding of whether and which of the toxic effects might be increased, decreased, or unchanged is lacking. Much of this must come from research in animals or other biosystems, because in humans, exposures to other compounds and factors would be difficult to obtain accurately and reliably in humans. The present state of knowledge does allow identifying the major potential mechanisms as the basis of such interactions at the biophase, but to what extent and how they could influence the toxicity outcomes cannot be predicted. Examples of such mechanisms are altered xenobiotic metabolizing enzymes, toxicokinetic factors (absorption, distribution, and elimination), toxic metabolite accumulation in target and nontarget tissues, and toxicodynamic factors, such as cell death, proliferation, expression of survival factors, and epigenetic and genotoxic mechanisms. Recommendations: Toxicokinetic and toxicodynamic studies are needed with mixtures to evaluate the effect of coexposures to other chemicals on toxic outcomes of trichloroethylene and on the toxicity of other coexposed toxicants including metabolites of trichloroethylene. Important toxic outcomes of trichloroethylene might be selected as end points for these studies. Species differences should be investigated. Testing large numbers and doses of compounds is not practical.
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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Studies designed to learn more about mechanisms and modes of action in the presence of the most commonly occurring toxicants are likely to yield the most meaningful results. Testing to evaluate the impact of lifestyle factors, such as alcohol consumption, smoking, chronic drug intake, and diet (e.g., nutrition, caloric restriction) should be performed. Testing of mixtures to evaluate the impact of disease (e.g., diabetes) and special physiologic states (e.g., pregnancy, aging) should be performed.
Representative terms from entire chapter: