4
Liver Toxicity and Cancer

This chapter reviews information on the effects of trichloroethylene and its principal metabolites (trichloroacetic acid, dichloroacetic acid, and chloral hydrate) on the liver, particularly information generated since the U.S. Environmental Protection Agency (EPA) released its draft health risk assessment (EPA 2001b). Trichloroethylene metabolism occurs primarily in the liver and is critical to understanding its toxicity and carcinogenicity. Background information on trichloroethylene metabolism is provided in Appendix C. In this chapter, hepatotoxicity and liver cancer are discussed separately, although they are not necessarily independent end points. A review of current knowledge on the proposed modes of action for trichloroethylene-induced liver cancer (peroxisome proliferator-activated receptor agonism, genotoxicity and mutagenicity) and their relevance to humans is provided.

HEPATOTOXICITY

Animal Studies

It is well documented that trichloroethylene produces hepatotoxicity in experimental animals and humans (ATSDR 1997a; EPA 2001b). Table 4-1 provides the details of some recent studies, and selected findings are discussed below.

Rodents exposed to high doses of trichloroethylene or some of its metabolites develop hepatocellular necrosis. Different studies have localized the injury to midzonal, periportal, or centrilobular hepatocytes (Buben



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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues 4 Liver Toxicity and Cancer This chapter reviews information on the effects of trichloroethylene and its principal metabolites (trichloroacetic acid, dichloroacetic acid, and chloral hydrate) on the liver, particularly information generated since the U.S. Environmental Protection Agency (EPA) released its draft health risk assessment (EPA 2001b). Trichloroethylene metabolism occurs primarily in the liver and is critical to understanding its toxicity and carcinogenicity. Background information on trichloroethylene metabolism is provided in Appendix C. In this chapter, hepatotoxicity and liver cancer are discussed separately, although they are not necessarily independent end points. A review of current knowledge on the proposed modes of action for trichloroethylene-induced liver cancer (peroxisome proliferator-activated receptor agonism, genotoxicity and mutagenicity) and their relevance to humans is provided. HEPATOTOXICITY Animal Studies It is well documented that trichloroethylene produces hepatotoxicity in experimental animals and humans (ATSDR 1997a; EPA 2001b). Table 4-1 provides the details of some recent studies, and selected findings are discussed below. Rodents exposed to high doses of trichloroethylene or some of its metabolites develop hepatocellular necrosis. Different studies have localized the injury to midzonal, periportal, or centrilobular hepatocytes (Buben

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TABLE 4-1 Hepatotoxicity of Trichloroethylene and Metabolites in Animal Stud Species (Sex) Doses/Concentrations Duration of Exposure Route/Vehicle Swiss-Cox mice (males) TCE, 0-3,200 mg/kg/day 6 wk Gavage/corn oil Sprague-Dawley rats (males) TCE, 250, 500, 1,250, 2,500 mg/kg Single dose i.p./corn oil Sprague-Dawley rats (males) TCE, 16 and 64 mg/kg, Single dose i.v. or via portal vein cannula/ vegetable oil Autoimmune prone MRL−/−mice (female) TCE, 0, 0.1, 0.5, 2.5 mg/mL 4 and 32 wk Drinking water Autoimmune prone MRL−/−mice (female) TCE, 0 or 2.5 mg/mL, in presence of diallyl sulfide (CYP2E1 inhibitor) 4 wk TCE in drinking water; diallyl sulfide via osmotic pump Autoimmune prone MRL−/−mice (female) TCA or chloral, 0, 0.1, 0.9 mg/mL 4 wk Drinking water Wistar rats (male) TCE, 376 ppm, 4 hr/day, 5 days/wk 8, 12, 24 wk Inhalation B6C3F1 mice (female) chloral hydrate, 25, 50, and 100 mg/kg, 5 days/wk 3, 6, 12 mo, 24 mo Gavage/in water Swiss-Webster mice (male) DCVC, 15, 30, 75 mg/kg Single dose i.p./in water B6CF1 mice (male) chloral hydrate, 1 g/L; DCA, 0.5 g/L 104 wk Drinking water B6C3F1 mice (male) DCA, 0.5 or 5 g/L 0, 5, 15, 20, 30 days Drinking water B6C3F1 mice (male and female) DCA or TCA, 1 or 2 g/L 52 wk Drinking water B6C3F1 mice (male and female) DCA or TCA, 1 or 2 g/L 52 wk Drinking water B6C3F1 mice (male) DCA, 0.1 to 3 g/L 1, 2 and 8 wk Drinking water

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Features of Hepatotoxicity Reference Increases in serum glutamic pyruvic transaminase evident only with the two highest doses; histologic findings: swollen hepatocytes and minimal evidence of necrosis. Buben and O’Flaherty 1985 Midzonal injury that spreads to centrilobular regions with the highest dose. Soni et al. 1998, 1999 Periportal necrosis with portal vein administration. No necrosis with i.v. administration. This is reflective of solvent-related injury of TCE to portal areas. Lee et al. 2000 CD4+ T-cell activation; mononuclear infiltration in portal regions consistent with autoimmune hepatitis; slight but significant increase of serum ALT. Griffin et al. 2000a Blockade of TCE protein adduct formation; reversal of CD4+ T-cell-mediated autoimmunity by TCE. Griffin et al. 2000b CD4+ T-cell activation by these metabolites as shown for TCE. Blossom et al. 2004 This dose (1/25 LC50) resulted in hepatomegaly and fatty infiltration; fatty changes with marked necrosis were more evident at 12 and 24 wk. No elevation in serum liver transaminases or mortality was detected. Kumar et al. 2001a No evidence of dose-dependent elevation in serum liver transaminases, only AST elevated at the 50-mg/kg dose. NTP 2002b Transient elevation in liver transaminases at the highest dose; no histologic evidence of liver injury. Vaidya et al. 2003a Increased liver weight and hepatocellular necrosis with both metabolites. Daniel et al. 1992 Dose- and time-dependent liver enlargement; morphologic evidence of focal necrosis and apoptotic bodies. Carter et al. 1995 Enlarged livers, significant glycogen accumulation, focal areas of necrosis seen with DCA. No focal necrosis with TCA, modest hyperthrophy and glycogen accumulation. Bull et al. 1990 Hepatocytes from DCA-treated mice contained large amounts of glycogen evenly distributed throughout the liver; less glycogen accumulation with TCA treatment, which was more prominent in periportal regions. Bull et al. 1990 Dose-dependent liver glycogen accumulation associated with decreased glycogen synthase activity. No effect in glycogen phosphorylase or glucose-6-phosphatase (enzymes involved glycogen metabolism). Kato-Weinstein et al. 1998

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Species (Sex) Doses/Concentrations Duration of Exposure Route/Vehicle B6C3F1 mice (male) DCA, 0.1 to 2 g/L 2-10 wk Drinking water Fresh hepatocytes in culture from B6C3F1 mice (male) DCA, 10-500 μM 16-hr incubation N/A B6C3F1 mice (female) DCA, 3.2 g/L, with or without L-methionine at 4 or 8 g/kg 8 and 44 wk DCA in drinking water; L-methionine in diet Fisher 344 rats (male) and B6C3F1 mice (female) dibromoacetic acid, 0, 1, 2 g/kg 2, 4, 7, 28 days Drinking water Sprague-Dawley rats (males) TCE, 0.01, 0.1, 1, 5, and 10 mmol/kg Daily dosing for 3 days i.p./corn oil Fresh hepatocytes in culture from Sprague-Dawley rats (males) Cells dosed by vapor phase in 25-mL flasks; exposure: TCE, 0, 0.5, 1, 2, 4 μL per flask 20-min exposure N/A Fresh hepatocytes in culture from Sprague-Dawley rats (males) Cells dosed by vapor phase in 25-mL flasks; exposure: 1,1,1-trichloroethane, 0, 2, 5, 10 μL per flask (concentration range: 230-1,000 μM) 20-min exposure N/A ABBREVIATIONS: ALT, alanine aminotransferase; AST, aspartate aminotransferase; DCA, dichloroacetic acid; DCVC, S-1,2-dichlorovinyl-L-cysteine; i.p., intraperitoneal; i.v., intravenous; LC50, concentration lethal to 50% of test animals; N/A, not applicable; ppm, parts per million; TC, taurocholate; TCA, trichloroacetic acid; TCE, trichloroethylene. and O’Flaherty 1985; Soni et al. 1998, 1999; Lee et al. 2000). This lack of consistency in location of injury might reflect the routes of administration, doses, strain, or species of rodents used in the different studies. For example, Soni et al. (1999) conducted dose-response studies with trichloroethylene (250-2,500 mg/kg) to investigate the time course of liver injury and compensatory hepatocyte regeneration. Hepatocellular necrosis was evident after 24 hours at all doses. Injury was detected in midzonal areas of the liver lobule with no evidence of necrosis in hepatocytes adjacent to the central vein (centrilobular hepatocytes). This study also showed that the dose of trichloroethylene can influence the location of injury. At a trichloroethylene

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Features of Hepatotoxicity Reference Significant reduction in serum insulin levels, insulin receptor expression, and protein kinase B. Increases in liver glycogen preceded these changes. Lingohr et al. 2001 Dose- and time-dependent glycogen accumulation. Presence or absence of insulin in culture media did not affect this DCA effect. However, glycogen accumulation is dependent on phosphatidylinositol 3-kinase activity. Lingohr et al. 2002 L-Methionine prevented liver DNA hypomethylation completely, while blocking only 25% of glycogen accumulation produced by DCA. Pereira et al. 2004 Dose- and time-dependent hypomethylation of liver DNA in both species. Significant increases in liver glycogen in both species, although longer exposure is required in rats for this effect. Tao et al. 2004a Increases in total and some individual serum bile acids (TC was most sensitive). No elevation in transaminases (except for ALT at the highest dose) or morphologic evidence of injury. Separate inhalation studies showed similar elevation in serum TC levels. Wang and Stacey 1990 Dose-dependent inhibition of bile acid (TC) uptake into hepatocytes. Time-dependent inhibition of bile acid accumulation. No significant intracellular enzyme or potassium leakage. No changes in bile acid efflux from hepatocytes with TCE exposure. Bai and Stacey 1993 No ALT, lactate dehydrogenase or potassium leakage at any dose level. Concentration-dependent inhibition of TC, ouabain, and 2-aminoisobutyric acid uptake. Decrease ATP levels and activity of ATP-dependent membrane ATPases. No overt morphologic changes in TCE-exposed hepatocytes. Kukongviriyapan et al. 1990 dose of 2,500 mg/kg, centrilobular injury was clearly evident after 24 hours. High doses of trichloroethylene administered into the portal vein caused periportal liver injury via a direct solvent action rather than a mechanism dependent on activation by the enzyme cytochrome P-450 (CYP-450) (Lee et al. 2000). Reynolds and Moslen (1977) proposed that reactive intermediates of trichloroethylene generated by CYP-450 bind covalently to cellular components, resulting in cell necrosis. More recent evidence from mouse studies suggests that an autoimmune response might play a role in trichloroethylene-mediated liver disease (Griffin et al. 2000a). Administration of

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues trichloroethylene at concentrations of 0 to 2.5 mg/mL in drinking water to autoimmune-prone MRL+/+ mice for 34 weeks resulted in an inflammatory response in the liver. Metabolic activation of trichloroethylene by CYP2E1 was demonstrated to be an obligatory step in the development of autoimmune hepatitis in the mice. The metabolites trichloroacetic acid and chloral hydrate also have the potential to induce autoimmunity in the same autoimmune-prone mice (Blossom et al. 2004). Recent studies investigated the hepatotoxicity produced by trichloroethylene in rats exposed via inhalation at 376 parts per million for 8, 12, or 24 weeks. Liver enlargement with necrotic cells and fatty infiltration was more prominent in rats in the 12- and 24-week treatment groups. The authors also detected elevated markers of lysosomal disruption. They recorded no mortality in any of the treatment groups (Kumar et al. 2001a). Human Studies Table 4-2 presents findings from human studies of hepatotoxicity. There is some evidence that occupational exposure to trichloroethylene results in several forms of noncancer liver disease such as hepatic necrosis, fatty liver, and cirrhosis. It is well established that acute occupational exposure to trichloroethylene does not produce liver injury, whereas chronic exposure does. Case reports have linked occupational exposure to trichloroethylene with Stevens-Johnson syndrome (erythema multiforme major) of abrupt onset (Phoon et al. 1984). All these cases demonstrated liver involvement ranging from mild jaundice to fatal liver failure. Another case report documented that repeated exposure to trichloroethylene in the work setting resulted in chronic cirrhosis and portal hypertension (Thiele et al. 1982). The most recent report of trichloroethylene hepatotoxicity associated with occupational exposure comes from a watch manufacturing plant in Thailand, where two female workers developed generalized skin lesions, fever, and hepatitis. One case resulted in fatal hepatic damage 2 weeks after the onset of symptoms. Both workers cleaned watch metal straps by dipping them in containers that contained trichloroethylene (Pantucharoensri et al. 2004). These case reports support data from animal studies indicating that an autoimmune response might be important in trichloroethylene-induced hepatitis. Genetic and environmental factors that influence xenobiotic metabolizing enzymes can favor the formation of trichloroethylene metabolites capable of triggering an immune response against the liver. Contribution of Metabolites to Hepatotoxicity Chloral hydrate, a metabolic intermediate of trichloroethylene, has been reported to contribute to the hepatotoxic potential of this solvent. In a 2-

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TABLE 4-2 Hepatotoxicity of Trichloroethylene and Metabolites in Human Studies Subjects Doses/Concentrations Duration of Exposure Route/Vehicle Features of Hepatotoxicity Reference Five case reports (males and females) TCE, 50-912 mg/m3 3-5 wk Inhalation of vapors in workplace Stevens-Johnson syndrome (erythema multiforme), jaundice, hepatomegaly, and hepatic encephalopathy. Other solvents besides TCE might be involved. Phoon et al. 1984 Two case reports (females) TCE, 15-45 ppm 4-5 wk Inhalation of vapors in workplace Stevens-Johnson syndrome, generalized skin eruptions, and hepatitis with no jaundice (case 1). Fulminant hepatitis (case 2). Pantucharoensri et al. 2004 Cross-sectional study (148 workers) and a 2-yr follow- up study (13 workers) Low, moderate,and high TCE exposure based on concentrations of total trichloro compounds detected in urine Duration of employment: 0.1 to 36.6 yr; average: 7 yr Ambient air; occupational Increases in high-density lipoprotein cholesterol in the absence of elevation in plasma liver transaminases, indicating that low level exposure to TCE affects cholesterol metabolism without causing hepatocellular necrosis. Alcohol intake is an influential factor in the cross-sectional study. Nagaya et al. 1993 Human workers (21 men, 1 woman) Regular exposures of TCE at <5 ppm; peak exposures for 2 workers at >250 ppm Mean duration of employment for TCE-exposed workers: 7 yr Ambient air; occupational Highly significant increases in individual and total serum bile acids in the exposed group (controlled for age and alcohol intake). No abnormalities in liver function tests. No relationship between plasma bile acid and cholesterol was detected. Driscoll et al. 1992 Human workers TCE, 8.9 ± 3.1 ppm Mean duration ofexposure: 3.4 yr Ambient air; occupational Elevation in total serum bile acids and some individual bile acids; normal hepatobiliary function tests. Neghab et al. 1997 ABBREVIATION: TCE, trichloroethylene.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues year National Toxicology Program (NTP 2002b) study, male B6C3F1 mice given chloral hydrate by gavage at 25, 50, or 100 mg/kg showed no significant changes in three serum liver transaminases, except for a significant increase in aspartate aminotransferase activity in the 50-mg/kg group. Lash et al. (1995) investigated the toxicity of trichloroethylene and its metabolites with freshly isolated rat hepatocytes in culture. The studies showed that exposure to only S-(1,2-dichlorovinyl)-L-cysteine resulted in hepatocellular damage. None of the other metabolites (CYP450 dependant or reduced glutathione dependent) or trichloroethylene produced hepatocellular injury. The metabolites tested included trichloroacetic acid, dichloroacetic acid, chloral hydrate, trichloroethanol, oxalic acid, and S-1,2-dichlorovinyl-L-glutathione. Despite this lack of cytotoxicity, trichloroethylene and its metabolites produced mitochondrial dysfunction. This in vitro study showed that S-1,2-dichlorovinyl-L-cysteine is the only trichloroethylene-derived compound cytotoxic to rat hepatocytes in culture. In contrast, in vivo studies showed that S-1,2-dichlorovinyl-L-cysteine has a very low hepatotoxic potential. Acute toxicity studies investigating the nephrotoxicity of S-1,2-dichlorovinyl-L-cysteine in male Swiss-Webster mice showed transient elevations in serum liver transaminases 12 hours after administration of the highest dose tested (75 mg/kg). This dose resulted in significant lethality due to nephrotoxicity at later times. No liver histopathology was detected at any of the doses or time points examined (Vaidya et al. 2003a). Other studies investigating the epigenetic mechanisms of dichloroaceticacid-induced carcinogenesis revealed morphologic evidence of liver injury that includes loss of cell membrane integrity, focal areas of cell debris, and appearance of apoptotic bodies in B6C3F1 mice undergoing short-term exposure to dichloroacetic acid at 0.5 or 5 g/L for up to 30 days (Carter et al. 1995). Hepatocellular necrosis was also detected in male and female Swiss-Webster mice receiving dichloroacetic acid in their drinking water at 300, 1,000, or 2,000 mg/L for up to 14 days. This was accompanied by a marked increase in liver weights. No such changes were seen with trichloroacetic acid under the same dosing regimen (Bull et al. 1990; Sanchez and Bull 1990). Exposure of male B6C3F1 mice to dichloroacetic acid (0.5 g/L) or chloral hydrate (1 g/L) via drinking water resulted in hepatocellular necrosis after 104 weeks of exposure (Daniel et al. 1992). Changes in Liver Glycogen Status Exposure to trichloroethylene produces effects in the liver other than hepatocellular injury. Treating mice with dichloroacetic acid results in marked dose-dependent accumulation of liver glycogen (Bull et al. 1990;

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Kato-Weinstein et al. 1998). This dose-response relationship parallels that for the development of hepatocellular carcinomas. Furthermore, patients with glycogen storage disorders have a greater propensity for developing liver tumors (Labrune et al. 1997). These observations have prompted investigators to study in depth the relationship between increased liver glycogen storage and carcinogenesis. Studies have been carried out to assess the effect of dichloroacetic acid treatment on insulin secretion, insulin receptor expression, and activity and expression of protein kinases controlled by insulin receptor signaling due to the role of these gene products in glycogen synthesis and homeostasis (Lingohr et al. 2001). Mice treated with dichloroacetic acid in drinking water at 0.1 to 2.0 g/L for 2-10 weeks showed a significant reduction in expression of the insulin receptor in the liver. As early as 2 weeks after the initiation of dichloroacetic acid treatment, insulin concentrations were significantly reduced. Dichloroacetic acid similarly reduced the expression of protein kinase B (an insulin-sensitive enzyme involved in glycogen homeostasis). Because dichloroacetic-acid-induced glycogen accumulation precedes down-regulation of the insulin receptor and insulin-dependent signaling pathways, these changes in gene expression for insulin and related genes are considered to be compensatory responses to changes in glycogen homeostasis. Lingohr et al. (2002) investigated whether the changes in glycogen accumulation brought about by dichloroacetic acid were insulin dependent. Freshly isolated mouse hepatocytes exposed to dichloroacetic acid accumulated more glycogen than control hepatocytes. This response was dose dependent. Omitting insulin from the culture media did not prevent the enhanced accumulation of glycogen produced by dichloroacetic acid. By contrast, dichloroacetic-acid-induced glycogen deposition was fully blocked by inhibition of the enzyme phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase participates in the signal transduction pathway leading to glycogen synthesis initiated by activation of the insulin receptor. This suggests that dichloroacetic-acid-induced glycogen accumulation involves a phosphatidylinositol 3-kinase-dependent pathway downstream of the insulin receptor (Lingohr et al. 2002). In addition to regulating glycogen synthesis, phosphatidylinositol 3-kinase has been implicated in the proliferative and antiapoptotic effects of peroxisome proliferators (Mounho and Thrall 1999), which further establishes an association between abnormal liver glycogen status and the carcinogenic effect of trichloroethylene and its metabolites. Other halogenated solvents such as bromochloroacetate and dibromoacetate induce glycogen accumulation in the liver to a similar degree as dichloroacetic acid (Kato-Weinstein et al. 2001). Another potential link between aberrant liver glycogen homeostasis and the carcinogenicity of trichloroethylene is provided by the effect of

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues this chemical and its metabolites on DNA methylation status. Exposure to dichloroacetic acid results in hypomethylation of protooncogenes and other genes involved in cell growth, such as insulin-like growth factor II in mouse liver (Tao et al. 2000a, 2004b; Ge et al. 2001). This response has been causally linked to the development of hepatocellular adenomas and carcinomas induced by these chemicals. Dibromoacetic acid, a related haloacetic acid, similarly induced hepatic DNA hypomethylation in female mice and male rats receiving 1 or 2 g/L in their drinking water for up to 28 days. Specifically, hypomethylation of c-myc and insulin-like growth factor II was detected along with increases in mRNA expression of both genes in mouse liver. Only c-myc mRNA expression was increased in rat liver. Glycogen accumulation and induction of markers of peroxisome proliferation were also observed in mice and rats receiving dibromoacetic acid (Tao et al. 2004a). These results further support the relationship between glycogen accumulation and liver tumors induced by trichloroethylene and its metabolites, making it more uncertain whether trichloroethylene-induced glycogen accumulation can be considered a non-cancer liver effect or an early event in carcinogenesis. More recently, Pereira et al. (2004) showed that methionine treatment not only prevents the DNA hypomethylation induced by dichloroacetic acid and related solvents, but it also prevents liver tumor formation in mice. Interestingly, methionine did not prevent glycogen accumulation completely. Dichloroacetic-acid-induced glycogen storage was reduced by only 25% with methionine treatment. This new information suggests dissociation between glycogen accumulation and the carcinogenic effects of dichloroacetic acid. Clearly, more studies are needed to clarify the relationship between altered glycogen storage and liver cancer in response to trichloroethylene. Elevation of Serum Bile Acids Occupational exposure to trichloroethylene has been reported to increase serum bile acid and cholesterol concentrations. Chronic occupational exposure to low concentrations of trichloroethylene appears to alter cholesterol metabolism in the absence of noticeable hepatocellular damage, as evidenced by lack of increase in serum liver transaminases (Nagaya et al. 1993). Similarly, serum concentrations of total and individual bile acids were significantly elevated in a group of workers exposed to trichloroethylene (Driscoll et al. 1992; Neghab et al. 1997). Because no association was observed between elevated plasma bile acids and conventional markers of liver injury, it was concluded that this perturbation in bile acid homeostasis could be indicative of early changes in liver function independent of hepatocellular damage.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Similar alterations in bile acid status have been observed in experimental animals exposed to trichloroethylene and its metabolites. Exposure to trichloroethylene via inhalation or intraperitoneal injection resulted in elevation of serum bile acid concentrations at doses that did not produce changes in markers of liver function, such as serum liver transaminases and bilirubin concentrations (Wang and Stacey 1990). To determine whether this increase in serum bile acids after low exposure to trichloroethylene is indicative of early liver dysfunction, the mechanism(s) responsible for these changes was investigated by examining the effect of trichloroethylene on bile acid transport in freshly isolated rat hepatocytes in culture (Bai and Stacey 1993). The uptake of the bile acids taurocholic and cholic acids into rat hepatocytes was inhibited in a dose-dependent manner by trichloroethylene at concentrations up to 1.84 mM. These concentrations did not result in significant leakage of transaminases or intracellular potassium into the culture media. Trichloroethylene inhibition of bile acids uptake was determined to be noncompetitive. These results indicate that the increase in serum bile acids produced by trichloroethylene occurs through interference with uptake into hepatocytes. The same study also determined that trichloroethylene does not affect the efflux of bile acids from hepatocytes. These observations clearly show that alterations in transport processes for bile acids (primarily by inhibiting uptake) occur in the absence of pathologic evidence of liver dysfunction. The fact that inhibition of bile acid uptake by trichloroethylene was determined to be noncompetitive strongly argues against direct competition between trichloroethylene and bile acids, such as taurocholic and cholic acids, for the main basolateral carrier in hepatocytes for bile acids, the sodium taurocholate transporter polypeptide. This effect on bile acids is not unique for trichloroethylene, because occupational exposure to a mixture of other organic solvents including toluene, xylene, acetone, n-butanol, and ethylacetate similarly increases serum bile acids (Franco et al. 1986). These results also argue against competition for common transport as it is very unlikely that the uptake of all these solvents into hepatocytes requires the same transport process. Because of their high lipid solubility, these solvents can readily partition into the plasma membrane of hepatocytes by diffusion, which does not require transport protein function. A decrease in hepatic bile acid uptake by trichloroethylene can be the result of changes in or disruption of plasma membrane lipids and changes in membrane fluidity. Changes in the fluidity of the plasma membrane lipid bilayer are known to affect the function of uptake transporters and other transmembrane proteins. Again, this is supported not only by the noncompetitive nature of the inhibition of the uptake of bile acids by trichloroethylene but also by its reversibility with time. In contrast, the lack of changes

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TABLE 4-10 Chloral Hydrate and PPARα Mode of Action Event Comments References Causal Events     PPARα activation Has not been studied.   Regulation of growth regulatory genes Has not been studied.   Cell proliferation or apoptosis Although hepatocelluar carcinoma was observed in mice and not rats, there was no increase in cell proliferation in either species. Chloral hydrate had no effect on hepatocyte intercellular communication in either rat or mouse cells. Klaunig et al. 1989; George et al. 2000 Clonal expansion Has not been studied.   Associative Events     Peroxisome proliferation and regulation of lipid metabolism genes Although hepatocellular carcinoma was observed in mice and not rats, there was no increase in palmitoyl CoA oxidation in either species. In diet-controlled mice, peroxisome proliferation and an increase in CYP4A protein and enzyme activity were seen. George et al. 2000; NTP 2002b Oxidative stress Has not been studied.   by Cohen et al. (2003) and illustrated for PPARα agonists by Klaunig et al. (2003). Briefly, strong weight of evidence is defined as several studies that support the mode of action, and weak weight of evidence is defined by having a single study from a single laboratory or a significant amount of contradiction in the literature. Dose-Response Relationships The dose-response relationships for the key events in the PPARα mode of action are shown in the Table 4-12. Note the results from the comparison of wild-type with PPARα null mice that show the role of this receptor in the toxicity of trichloroethylene or its metabolites (discussed earlier in this chapter). PPARα activation per se is reserved for transactivation assays, as that is the most direct and definitive way to examine nuclear receptor agonism.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TABLE 4-11 Strength of the Weight of Evidence for PPARα Mode of Action for Trichloroethylene and Its Metabolites Chemical Weight of Evidencea Comments TCE Strong TCE activates PPARα at high concentrations and regulates a variety of target genes. Studies with PPARα null mice show that most responses are dependent on this receptor, including peroxisome proliferation, cell proliferation, and target gene expression. More evidence could be provided by a long-term bioassay in this model system. TCA Strong TCA activates PPARα at high concentrations. Less extensive characterization of PPARα dependence on cell proliferation is provided than is known for TCE. However, the evidence of clonal expansion and phenotypic characteristics of tumors is strong and shows similarity to peroxisome proliferators. DCA Strong DCA activates PPARα at high concentrations. As is the case with TCA, PPARα dependence of DCA’s effects on cell proliferation is less than for trichloroethylene. Increasingly, it appears that DCA-induced clonal expansion is dissimilar to that of TCA and TCE. The reason for this discrepancy is not known but may require examination of tumors from PPARα null mice. Also, liver weight changes (and presumably cell proliferation) are not dependent on PPARα, indicating a potential for other modes of action that would be different than that of TCA. Chloral hydrate Weak There is no evidence of PPARα activation by chloral hydrate aside from it being a weak peroxisome proliferator. aA strong weight of evidence is defined as evidence from several studies which support the mode of action, while a weak weight of evidence is defined as having a single study from a single laboratory or a significant amount of contradiction in the literature (Klaunig et al. 2003). ABBREVIATIONS: DCA, dichloroacetic acid; TCA, trichloroacetic acid; TCE, trichloroethylene. FINDINGS AND RECOMMEDATIONS Hepatotoxicity The existing data clearly demonstrate that trichloroethylene produces hepatotoxicity in experimental animals and humans that is dependent on generation of reactive intermediates by CYP-450 in the liver. Besides its hepatotoxic potential, trichloroethylene and its metabolites produce liver effects categorized as independent of hepatotoxicity. These effects include elevations in plasma bile acids and accumulation of liver glycogen in the absence of subclinical evidence of liver dysfunction. The absence of liver dysfunction in rodents has been documented in studies using serum markers

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues TABLE 4-12 PPARα Mode-of-Action Dose-Response Relationships Effect Dose/Concentrationa Route Vehicle Duration Gender Species/Strain Reference Causal Events               PPARα Activation                 TCA, 1.0 mM; 5 mM In vitro DMSO 24 hr N/A Cos-1 cells transfected with human and mouse PPARα Maloney and Waxman 1999   DCA, 1.0 mM, 5 mM In vitro DMSO 24 hr N/A Cos-1 cells transfectedwith human and mouse PPARα Maloney and Waxman 1999   TCA, 4 mM (DCA at 4 mM, no effect) In vitro Unknown 24 hr N/A HL8.5 cells transfected with mouse PPARα Walgren et al. 2000b Regulation of Growth Regulatory Genes c-jun, c-myc TCE, 1,000 mg/kg Oral gavage, 5 days/wk Corn oil 33 days Female B6C3F1 mice Tao et al. 1999 c-jun, c-myc (in tumors) DCA, 20 mmol/L Drinking water Water 46 wk Female B6C3F1 mice Tao et al. 1999 c-jun, c-myc (in tumors) TCA, 20 mmol/L Drinking water Water 46 wk Female B6C3F1 mice Tao et al. 1999 c-jun, c-myc TCE, 1,000 mg/kg Oral gavage Corn oil 5 days Female B6C3F1 mice Tao et al. 2000b c-jun, c-myc TCA, 500 mg/kg Oral gavage Water 5 days Female B6C3F1 mice Tao et al. 2000b

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Effect Dose/Concentrationa Route Vehicle Duration Gender Species/Strain Reference c-jun, c-myc DCA, 500 mg/kg Oral gavage Water 5 days Female B6C3F1 mice Tao et al. 2000b Growth regulatory genes via microarray TCE, 1,500 mg/kg Oral gavage Methyl cellulose (0.1%) 3 days Male SV129 wild-type and PRARα null mice Laughter et al. 2004 Cell Proliferation or Apoptosis BrdU labeling TCE, 500 and 1,000 mg/kg/day (not observed in PPARAα null) Oral gavage Methyl cellulose (0.1%) 3 wk Male SV129 wild-type and PPARAα null mice Laughter et al. 2004 [3H]Thymidine incorporation TCE, 500 mg/kg Oral gavage Corn oil 7, 14 days Male B6C3F1 mice Klaunig et al. 1991 [3H]Thymidine incorporation TCE, no effect Oral gavage Corn oil 3, 7, 14 days Female B6C3F1 mice Klaunig et al. 1991 [3H]Thymidine incorporation TCE, no effect Oral gavage Corn oil 3, 7, 14 days Male F344 rat Klaunig et al. 1991 [3H]Thymidine incorporation TCE, no effect Oral gavage Corn oil 3, 7, 14 days Male F344 rat Klaunig et al. 1991 BrdU labeling (not within tumors) DCA, 2 g/L Drinking Water Water 14 days Male B6C3F1 mice Stauber and Bull 1997 BrdU labeling (not within tumors) TCA, 2 g/L Drinking water Water 14, 28 days Male B6C3F1 mice Stauber and Bull 1997

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues [3H]Thymidine incorporation TCE, 250-2,500 mg/kg Intraperitoneal Corn Oil 24 hr (500, 1,250) 36 hr (250-1,250) 48 hr (all doses) 72 hr (250, 1,250, 2,500) 96 hr (none) Male Sprague-Dawley rats Soni et al. 1998 Anchorage-independent growth TCA, DCA, 0.5-2 mM In vitro Media 10-25 days Male Hepatocytes from B6C3F1 mice Stauber et al. 1998 [3H]Thymidine incorporation TCA, DCA, 0.1-1 mM. Varied based on individual In vitro Media 72 hr Male and Female Human primary cultures Walgren et al. 2000a PCNA labeling TCA, DCA, 500 mg/kg Oral gavage Saline 72-96 hr Female B6C3F1 mice Ge et al. 2001 Clonal Expansionb Associative Events Peroxisome Proliferation and Regulation of Lipid Metabolism Genes CYP 4a12 mRNA TCE, 1,500 mg/kg/day (not observed in PPARAα null) Oral gavage Methyl cellulose (0.1%) 3 days Male SV129 wild-type and PPARAα null mice Laughter et al. 2004 CYP4A, ACO protein TCE, 125, 500, 1,000 mg/kg/day (not observed in PPARAα null) Oral gavage Methyl cellulose (0.1%) 3 wk Male SV129 wild-type and PPARAα null mice Laughter et al. 2004 Palmitoyl CoA oxidase TCE, 1,500 mg/kg/day (not observed in PPARAα null) Oral gavage Methyl cellulose (0.1%) 3 wk Male SV129 wild-type and PPARα null mice Laughter et al. 2004

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Effect Dose/Concentrationa Route Vehicle Duration Gender Species/Strain Reference CYP4A, ACO protein TCA, 1.0, 2.0 M (not observed in PPARα null) Drinking water Water 1 wk Male SV129 wild-type and PPARα null mice Laughter et al. 2004 Palmitoyl CoA oxidase TCA, 2 M (not observed in PPARα null) Drinking water Water 3 wk Male SV129 wild-type and PPARα null mice Laughter et al. 2004 CYP4A protein DCA, 2 M (not observed in PPARα null) Drinking water Water 3 wk Male SV129 wild-type and PPARα null mice Laughter et al. 2004 Palmitoyl CoA oxidase TCA, DCA, 600 mg/kg In vitro Media 24 hr Male and Female B6C3F1 homogenate and primary culture. No effects seen with LEH rat or human cells/cultures mouse liver Walgren et al. 2000a Laural CoA oxidase DCA, 3.2 g/L Unaffected by methionine Drinking water Water 8 and 44 wk Female B6C3F1 mice Pereira et al. 2004 Lauric acid oxidase; CYP4A protein CH, 100 mg/kg Gavage (5 days/week) Water 104 wk Male B6C3F1/Nctr mice Leakey et al. 2003a Palmitoyl CoA oxidase activity TCA, 5 g/L MCA, no effect Drinking water Water 15-104 wk Male F344 rats DeAngelo et al. 1997 Palmitoyl CoA oxidase activity Perc, 200, 400 ppm Inhalation Air 28 days Male and Female B6CF1 mice F344 rats Odum et al. 1988

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Peroxisome number Perc, 200, 400 ppm Inhalation Air 28 days Male and Female B6CF1 mice Odum et al. 1988 Palmitoyl CoA oxidase activity MCA, 500 μMM DCA, 1000 μMM TCA, 500 μMM In vitro Media 72 hr Male Long Evans rat primary hepatocytes Walgren et al. 2004 Peroxisome proliferation TCE, 0.75 g/kg Gavage (daily) Corn oil 2 wk Male and Female Sv/129 (not seen in PPARα null) Nakajima et al. 2000 Peroxisome proliferation TCE, 0.75 g/kg Gavage (daily) Corn oil 2 wk Male Sv/129 (not seen in PPARα null or in female wt or null) Nakajima et al. 2000 Palmitoyl CoA oxidase activity DCA, 0.5 mM (mouse); 1.0 mM (rat) In vitro Media 72 hr Male B6C3F1 mice, Long Evans rat primary hepatocytes Everhart et al. 1998 Palmitoyl CoA oxidase activity TCE, 100 mg/kg TCA, 500 mg/kg Perc, 1,000 mg/kg (1/4- 1/2 the effect seen with Wy14,643 at 50 mg/kg) Gavage (10 days) DMSO/corn oil 10 days Male B6C3F1 mice, F344 rats Goldsworthy and Popp 1987 aDoses where statistically significant effects were observed. bClonal expansion is pertinent only in reference to tumor formation and phenotyping of foci and nodules. ABBREVIATIONS: BrdU, bromodeoxyuridine; CH, chloral hydrate; DCA, dichloroacetic acid; DMSO, dimethyl sulfoxide; MCA, monochloroacetic acid; Perc, tetrachloroethylene; TCA, trichloroacetic acid; TCE, trichloroethylene.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues of liver injury and is further supported by histopathologic examinations that showed no ultrastructural changes. However, the absence of liver dysfunction in humans has been based entirely on measures of serum markers of liver injury (e.g., plasma transaminases, bilirubin concentrations). Therefore, the possibility that humans might have discrete ultrastructural changes in the liver that can affect bile acid homeostasis cannot be ruled out. There are some mechanistic data addressing the nature of the elevation in bile acids in plasma but the precise mode of action remains unknown. Elevation of plasma bile acids could result in their accumulation in other tissues, which could conceivably have detrimental effects on those organs. In addition to their lipid-solubilizing effect, bile acids are also signaling molecules that regulate gene transcription. The farnesoid X receptor functions as a bile acid nuclear receptor which regulates transcription of multiple genes responsible for maintaining cholesterol and bile acid homeostasis. Thus, accumulation of bile acids might contribute to the adverse effects of trichloroethylene exposure in organs other than the liver through a detergent effect or altered cellular signaling. However, it is not clear whether or not this is a significant effect. The human relevance of liver glycogen accumulation observed in rodents exposed to dichloroacetic acid remains unclear. There are no studies documenting this effect in humans. Furthermore, all research on glycogen accumulation has been carried out using dichloroacetic acid, and not trichloroethylene. In light of this, it is not known whether exposure to trichloroethylene at environmentally relevant concentrations results in glycogen accumulation in rodents or humans. Investigators have been able to dissociate the glycogen deposition effect from the peroxisome proliferation produced by trichloroethylene and its metabolites because dichloroacetic acid, which produces significant liver enlargement and no peroxisome proliferation, induces a marked accumulation of liver glycogen. In contrast, exposure to trichloroacetic acid produces only modest glycogen accumulation while stimulating considerable peroxisome proliferation. Data from studies with autoimmune-prone mice also suggest that trichloroethylene and its metabolites are capable of triggering an immunemediated reaction against the liver. This observation is highly relevant to humans because there are multiple case reports of workers exposed to trichloroethylene with Stevens-Johnson syndrome who developed generalized skin reactions often accompanied by hepatitis of acute onset. In summary, data generated since EPA (2001b) released its draft health risk assessment have not significantly advanced understanding of whether some of the noncancer liver effects of trichloroethylene and its metabolites are independent of early ultrastructural and discrete pathologic changes.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Also, the relationship of these effects to hepatocarcinogenesis remains unclear. Liver Cancer Data on trichloroethylene indicate that relatively high doses are needed to induce liver cancer, even in susceptible strains of mice. The three major metabolites of trichloroethylene—trichloroacetic acid, dichloroacetic acid, and chloral hydrate—can contribute to liver cancer in mice. None of the three is directly mutagenic or genotoxic as the principal mode of action. Trichloroacetic acid and dichloroacetic acid have been shown to promote liver cancer in classic initiation-promotion experimental protocols. The concentrations of trichloroacetic acid in blood required to induce liver cancer approach the millimolar range, whereas dichloroacetic acid concentrations in blood associated with carcinogenesis are in the submicromolar range. The carcinogenic activity of chloral hydrate is largely dependent on its conversion to trichloroacetic acid and dichloroacetic acid. Dichloroacetic acid and trichloroacetic acid adequately account for the hepatocarcinogenic responses to trichloroethylene. There is sufficient weight of evidence to conclude that the mode of action of trichloroacetic acid as a rodent liver carcinogen is principally as a liver peroxisome proliferator in a specific strain of mouse, B6C3F1. This strain also has a particularly high background incidence of liver tumors. Moreover, F344 rats in which peroxisome proliferation is not induced do not show induction of liver cancer at the same doses at which B6C3F1 mice do. Altered cellular metabolism leading to transient changes in cell proliferation and cell regulation is related to induction of peroxisome proliferation in rodents. Dichloroacetic acid produces liver tumors with a different phenotype than trichloroacetic acid. Its tumorigenic effects are closely associated with differential effects on cell replication rates in tumors, normal hepatocytes, and suppression of apoptosis. There is sufficient weight of evidence to conclude that the mode of action of dichloroacetic acid at high doses in rodents includes hepatomegaly and marked cytomegaly, which are closely associated with its activity as a differential promoter with effects on increased cell replication rates in tumors and normal hepatocytes and suppression of apoptosis. High-dose treatments alter activities of key enzymes in metabolism and cell growth. Dichloroacetic acid induces liver tumorigenesis in both mice and rats by this mode of action. However, dichloroacetic acid is a minor metabolite of trichloroethylene and whether it is formed in humans has not been clearly established. The mode of action of chloral hydrate as a weak rodent liver carcinogen is dominated by induction of peroxisome proliferation activity in male

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues B6C3F1 mice. Female mice and rats are resistant to the carcinogenic effects of chloral hydrate. Because the metabolites of chloral hydrate are trichloroacetic acid and dichloroacetic acid, the contribution to liver tumor induction of the specific modes of action of each of these metabolites is also likely; however, an overall lack of potency for chloral hydrate in the carcinogenic response is notable. Induction of peroxisome proliferation in human liver is not a prominent feature; therefore, this key event related to trichloroacetic acid liver carcinogenesis is not likely to occur in humans. The promotional activity of dichloroacetic acid includes a significant effect on cellular metabolism, differentiation function, and proliferation that encompass a mitogenic mode of action. Repeated exposure to dichloroacetic acid results in an inhibition of both mitosis and apoptosis and eventual formation of focal eosinophilic hyperplastic lesions. Assuming that the underlying mode of action for dichloroacetic acid as a liver carcinogen in rodents is promotional events affecting and culminating in mitogenesis, genotypic species differences between mice (one transforming growth factor type-β growth factor receptor allele) and humans with two functional copies of the gene suggest that humans would be phenotypically much less susceptible to liver carcinogenesis from agents that demonstrate a mitogenic mode of action (Andersen et al. 1995). The weak carcinogenic activity of chloral hydrate in the liver of male B6C3F1 mice (with no liver cancer induction in female mice and rats) combined with lower rates of oxidation and higher rates of conjugation in humans compared with mice indicates that the mode of action in mice is not likely to be relevant to humans. Exposure to trichloroethylene at concentrations relevant to the general public is not likely to induce liver cancer in humans. However, it is possible that much higher exposures to trichloroethylene, such as in certain high-risk occupations or in heavily contaminated locales, could result in increased risks of liver toxicity and cancer. In addition, the existence of sensitive populations due to genetics, disease, or life stage cannot be discounted. The epidemiologic evidence for an association between liver cancer and trichloroethylene exposure is inconclusive. Excess liver cancer incidence was observed in most of the cohort studies that examined this outcome. However, cohort studies of mortality and population-based case-control studies yielded mixed results. Of particular interest is a recent case-control study that found an association between liver cancer mortality and trichloroethylene concentrations in well water in a community that was downstream from a Taiwanese factory. Although this study suffers from several methodologic weaknesses, it is the first to show an association between environmental exposures to trichloroethylene and liver cancer mortality.

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Assessing the Human Health Risks of Trichloroethylene: Key Scientific Issues Recommendations: Additional laboratory studies are needed to establish the significance of increased bile acids in relation to the hepatotoxic potential of trichloroethylene, as well as in relation to other systemic effects. Such studies will help clarify whether elevation of serum bile acids is an early indicator of changes in liver function or is a marker of exposure to trichloroethylene (or other halogenated solvents which induce this effect). More research is also needed to assess whether increases in serum bile acids in humans exposed to trichloroethylene are independent of discrete pathologic changes in the liver. Because histopathologic assessment would be difficult to perform in human subjects, new, highly sensitive, and noninvasive toxicologic parameters are needed to clarify the toxicologic importance of these effects of trichloroethylene. Additional studies of the effects of trichloroethylene on glycogen accumulation, perhaps using cultured human hepatocytes, should shed some light on the significance of this effect and its relevance to humans. More research is needed to determine whether an autoimmune response might play a role in trichloroethylene-mediated liver disease. Adducts formed between metabolites of trichloroethylene and liver proteins can result in the formation of neoantigens. These neoantigens can lead to antibody-dependent hepatocellular injury. The same process has been reported with chemicals such as halothane, which is well known to produce immune-mediated hepatotoxicity. Studies similar to those carried out with halothane could be instrumental in elucidating whether autoimmunity is a causal factor in the hepatotoxicity of trichloroethylene. Studies are needed to determine the metabolic pathway and yield for forming dichloroacetic acid from trichloroethylene either via trichloroacetic acid or via other pathway(s). If dichloroacetic acid is found to be a metabolite of concern, additional studies may be needed to understand its role in the toxicities associated with trichloroethylene.