The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver

Trichloroethylene (TCE) has previously been shown to be carcinogenic in mouse liver when administered by daily gavage in corn oil. The metabolism of TCE results, in part, in the formation of trichloroacetic acid (TCA) as a major metabolite and dichloroacetic acid (DCA) as a minor metabolite. These chlorinated acetic acids have not been shown to be genotoxic, although they have been shown to induce peroxisome proliferation. Therefore, we determined the ability they have been shown to induce peroxisome proliferation. Therefore, we determined the ability of TCE, TCA, or DCA to act as tumor promoters in mouse liver. Male B6C3F1 mice were administered intraperitoneally 0, 2.5, or 10 micrograms/g body wt ethylnitrosourea (ENU) on Day 15 of age. At 28 days of age, the mice were placed on drinking water containing either TCE (3 or 40 mg/liter), TCA (2 or 5 g/liter), or DCA (2 or 5 g/liter). All drinking waters were neutralized with NaOH to a final pH of 6.5-7.5. The animals were killed after 61 weeks of exposure to the treated drinking water (65 weeks of age). Both DCA and TCA at a concentration of 5 g/liter were carcinogenic without prior initiation with ENU, resulting in hepatocellular carcinomas in 81 and 32% of the animals, respectively. DCA and TCA also increased the incidence of animals with adenomas and the number of adenomas/animal in those animals that were not initiated with ENU. While 2.5 micrograms/g body wt ENU followed by NaCl in the drinking water resulted in only 5% of the animals with hepatocellular carcinomas, 2.5 micrograms/g body wt ENU followed with 2 or 5 g/liter DCA resulted in a 66 or 78% incidence of carcinoma, respectively, or, followed with 2 or 5 g/liter TCA, resulted in a 48% incidence at either concentration. None of the untreated animals had hepatocellular carcinomas. Therefore our results demonstrate that DCA and TCA are complete hepatocarcinogens in B6C3F1 mice.     

In Vitro Cytotoxicity of Mono-, Di-, and Trichioroacetate and Its Modulation by Hepatic Peroxisome Proliferation

Dichloroacetate (DCA) and trichloroacetate (TCA) are major by-productsof drinking water chlorination. Recent experiments have shownthat both of these compounds produce hepatic tumors in B6C3FImice. There was evidence that these effects may be associatedwith cytotoxic effects and/or peroxisomal proliferation. Therefore,in the present study the in vitro cytotoxicity of monochloroacetate(MCA), DCA, TCA and a metabolite, glycolate (GLY), was determinedin hepatocyte suspensions prepared from naive and clofibricacid-pretreated male Sprague-Dawley rats and B6C3F1 mice. Cytotoxicresponses, measured by release of lactic dehydrogenase and/ortrypan blue exclusion, were only observed with high concentrations(5.0 mM) of MCA and GLY in hepatocytes from naive animals (p=0.025and 0.008, respectively, Sprague-Dawley rat; p=0.033 and 0.001,respectively, B6C3F1 mouse). The cytotoxic responses to bothcompounds were observed much earlier and at much lower concentrationsin hepatocytes taken from mice and rats that had been pretreatedwith clofibric acid (p0.001, Sprague-Dawley rat and B6C3F1 mouse).DCA and TCA produced no evidence of cytotoxicity in hepatocytesfrom naive or clofibric acid-pretreated animals of either speciesat concentrations up to 5.0 mM. Increasing concentrations ofMCA and GLY resulted in dose-related depletion of intracellularreduced glutathione (GSH) that closely paralleled the cytotoxicresponses. Only GLY (0.25–5.0 mM) produced increased intracellularoxidized glutathione. Neither DCA nor TCA was found to altercellular GSH status in hepatocytes isolated from either Sprague-Dawleyrats or B6C3F1 mice. It was concluded from these in vitro observationsthat DCA and TCA are not highly cytotoxic to hepatocytes. Moreover,the rates of their conversion to MCA or GLY may be insufficientto induce cytotoxic effects in hepatocytes in vivo.     

Effect of dibromoacetic acid on DNA methylation, glycogen accumulation, and peroxisome proliferation in mouse and rat liver.

Dibromoacetic acid (DBA) is a drinking water disinfection by-product. Its analogs, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are liver carcinogens in rodents. We evaluated the ability of DBA to cause DNA hypomethylation, glycogen accumulation, and peroxisome proliferation that are activities previously reported for the two other haloacetic acids. Female B6C3F1 mice and male Fischer 344 rats were administered 0, 1,000, and 2,000 mg/l DBA in drinking water. The animals were euthanized after 2, 4, 7, and 28 days of exposure. Dibromoacetic acid caused a dose-dependent and time-dependent decrease of 20%-46% in the 5-methylcytosine content of DNA. Hypomethylation of the c-myc gene was observed in mice after 7 days of DBA exposure. Methylation of 24 CpG sites in the insulin-like growth factor 2 (IGF-II) gene was reduced from 80.2% +/- 9.2% to 18.8% +/- 12.9% by 2,000 mg/l DBA for 28 days. mRNA expression of the c-myc and IGF-II genes in mouse liver was increased by DBA. A dose-dependent increase in the mRNA expression of the c-myc gene was also observed in rats. In both mice and rats, DBA caused dose-dependent accumulation of glycogen and an increase of peroxisomal lauroyl-CoA oxidase activity. Hence, DBA, like DCA and TCA, induced hypomethylation of DNA and of the c-myc and IGF-II genes, increased mRNA expression of both genes, and caused peroxisome proliferation. Again like DCA, DBA also induced glycogen accumulation. These results indicate that DBA shares biochemical and molecular activities in common with DCA and/or TCA, suggesting that it might also be a liver carcinogen.     

DNA hypomethylation induced by drinking water disinfection by-products in mouse and rat kidney.

Bromodichloromethane (BDCM), chloroform, dibromoacetic acid (DBA), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) are chlorine disinfection by-products (DBPs) found in drinking water that have indicated renal carcinogenic and/or tumor promoting activity. We have reported that the DBPs caused DNA hypomethylation in mouse liver, which correlated with their carcinogenic and tumor promoting activity. In this study, we determined their ability to cause renal DNA hypomethylation. B6C3F1 mice were administered DCA or TCA concurrently with/without chloroform in their drinking water for 7 days. In male, but not female mouse kidney, DCA, TCA, and to a lesser extent, chloroform decreased the methylation of DNA and the c-myc gene. Coadministering chloroform increased DCA but not TCA-induced DNA hypomethylation. DBA and BDCM caused renal DNA hypomethylation in both male B6C3F1 mice and Fischer 344 rats. We have reported that, in mouse liver, methionine prevented DCA- and TCA-induced hypomethylation of the c-myc gene. To determine whether it would also prevent hypomethylation in the kidneys, male mice were administered methionine in their diet concurrently with DCA or TCA in their drinking water. Methionine prevented both DCA- and TCA-induced hypomethylation of the c-myc gene. The ability of the DBPs to cause hypomethylation of DNA and of the c-myc gene correlated with their carcinogenic and tumor promoting activity in mouse and rat kidney, which should be taken into consideration as part of their risk assessment. That methionine prevents DCA- and TCA-induced hypomethylation of the c-myc gene would suggest it could prevent their carcinogenic activity in the kidney.     

Dichloroacetic acid and trichloroacetic acid-induced DNA strand breaks are independent of peroxisome proliferation

This study examined whether the induction of single strand breaks in hepatic DNA by dichloroacetic acid (DCA) and trichloroacetic acid (TCA) depends upon peroxisome proliferation. Male B6C3F1 mice were given a single oral dose of either DCA or TCA. At varying times, between 1 and 24 h after administration of the compounds, breaks in DNA were measured using an alkaline unwinding assay. Peroxisome proliferation was monitored at the same time intervals in a parallel experiment by measuring peroxisomal B-oxidation of [14C]palmitoyl-CoA in liver homogenates. Both DCA and TCA significantly increased breaks in DNA at 1, 2, and 4 h post-treatment, with a return to control levels after 8 h. No evidence for an increase in peroxisomal beta-oxidation was produced by either chemical up to 24 h after administration. In a separate experiment, mice were treated with DCA or TCA for 10 days and their livers examined for evidence of peroxisome proliferation. An increase in liver weight was observed, particularly with DCA. Both TCA and DCA increased peroxisomal beta-oxidation in liver homogenates, with TCA-treated animals showing more activity than those treated with DCA. Electron microscopy revealed that the number of peroxisomes were approximately the same in DCA- and TCA-treated animals. However, peroxisomes induced by DCA treatment frequently lacked nucleoid cores. These data indicate that peroxisomes induced by these compounds differ in their concentration of peroxisomal enzymes. Except for a slight hypertrophy, repeated doses of TCA do not produce significant degenerative changes in the liver of mice. Repeated doses of DCA produce multifocal, subcapsular necrotic regions, and a marked hypertrophic response in the liver. Mice treated with TCA for 10 days and sacrificed 24 h after the last dose did not display increased strand breaks in hepatic DNA. This indicates that peroxisomal proliferation does not contribute to the induction of DNA strand breaks.     

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.     

Chlorinated hydrocarbon-induced peroxisomal enzyme activity in relation to species and organ carcinogenicity

Trichloroethylene (TCE), perchloroethylene (PER), and pentachloroethane (PENT) are widely used industrial chemicals that cause an increased incidence of hepatocellular carcinoma in mice and a very low incidence of renal tubular adenocarcinoma in rats. A recent study (C. R. Elcombe, M. S. Rose, and I.S. Pratt (1985), Toxicol. Appl. Pharmacol. 79, 365-376) suggested that the species difference in the hepatocarcinogenicity of TCE seen between rats and mice was due to a species difference in peroxisomal proliferation and cell proliferation. The purpose of the present investigation was to understand better the association of peroxisome proliferation in the species-specific hepatocarcinogenicity, and nephrocarcinogenicity of TCE, PER, and PENT. TCE (1000 mg/kg body wt), PER (1000 mg/kg body wt), PENT (150 mg/kg body/wt), the metabolite trichloroacetic acid (TCA; 500 mg/kg body wt) or the potent peroxisome proliferating agent Wy-14,643 (WY; 50 mg/kg body wt) was administered by gavage to male F-344 rats and B6C3F1 mice for 10 days. Cyanide-insensitive palmitoyl CoA oxidation activity (PCO) was used to measure the peroxisome proliferation response. Of the chlorinated hydrocarbons, TCE and PER elevated PCO activity in mouse liver whereas only TCE elevated rat liver and kidney PCO. All agents increased PCO activity in the kidneys of mice. None of the chlorinated hydrocarbons induced a PCO response stronger than WY. These results support an association between peroxisome proliferation and hepatic tumors in mice following TCE and PER, but not PENT, administration and suggest that chlorinated hydrocarbon-induced peroxisome proliferation does not correlate with species-specific renal carcinogenicity.     

Effect of Dichloroacetic Acid and Trichloroacetic Acid on DNA Methylation in Liver and Tumors of Female B6C3F1 Mice

Dichloroacetic acid (DCA) and trichloroacetic add (TCA) arefound in drinking water and are metabolites of trichloroethylene.They are carcinogenic and promote liver tumors in B6C3F1 mice.Hypomethylation of DNA is a proposed nongenotoxic mechanisminvolved in carcinogenesis and tumor promotion. We determinedthe effect of DCA and TCA on the level of DNA methylation inmouse liver and tumors. Female B6C3F1 mice 15 days of age wereadministered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeksstarted to receive 25 mmol/liter of either DCA or TCA in theirdrinking water until euthanized 44 weeks later. Other animalsnot administered MNU were euthanized after 11 days of exposureto either DCA or TCA. DNA was isolated from liver and tumors,and after hydrolysis 5-methylcytosine (5MeC) and the four baseswere separated and quantitated by HPLC. In animals exposed toeither DCA or TCA for 11 days but not 44 weeks, the level of5MeC in DNA was decreased in the liver. 5MeC was also decreasedin liver tumors from animals exposed to either chloroaceticacid. The level of 5MeC in TCA-promoted carcinomas appearedto be less than in adenomas. Termination of exposure to DCA,but not to TCA, resulted in an increase in the level of 5MeCin adenomas to the level found in noninvolved liver. Thus, hypomethylatedDNA was found in DCA and TCA promoted liver tumors and the differencein the response of DNA methylation to termination of exposureappeared to support the hypothesis of different mechanisms fortheir carcinogenic activity.     

Prevention by methionine of dichloroacetic acid-induced liver cancer and DNA hypomethylation in mice.

Dichloroacetic acid (DCA) is a liver carcinogen that induces DNA hypomethylation in mouse liver. To test the involvement of DNA hypomethylation in the carcinogenic activity of DCA, we determined the effect of methionine on both activities. Female B6C3F1 mice were administered 3.2 g/l DCA in their drinking water and 0, 4.0, and 8.0 g/kg methionine in their diet. Mice were sacrificed after 8 and 44 weeks of exposure. After 8 weeks of exposure, DCA increased the liver/body weight ratio and caused DNA hypomethylation, glycogen accumulation, and peroxisome proliferation. Methionine prevented completely the DNA hypomethylation, reduced by only 25% the glycogen accumulation, and did not alter the increased liver/body weight ratio and the proliferation of peroxisomes induced by DCA. After 44 weeks of exposure, DCA induced foci of altered hepatocytes and hepatocellular adenomas. The multiplicity of foci of altered hepatocytes/mouse was increased from 2.41 +/- 0.38 to 3.40 +/- 0.46 by 4.0 g/kg methionine and decreased to 0.94 +/- 0.24 by 8.0 g/kg methionine, suggesting that methionine slowed the progression of foci to tumors. The low and high concentrations of methionine reduced the multiplicity of liver tumors/mouse from 1.28 +/- 0.31 to 0.167 +/- 0.093 and 0.028 +/- 0.028 (i.e., by 87 and 98%, respectively). Thus, the prevention of liver tumors by methionine was associated with its prevention of DNA hypomethylation, indicating that DNA hypomethylation was critical for the carcinogenic activity of DCA.     

Biochemical, histological, and ultrastructural changes in rat and mouse liver following the administration of trichloroethylene: possible relevance to species differences in hepatocarcinogenicity

Trichloroethylene (TRI), administered by gavage for 10 consecutive days, at doses of 500 to 1500 mg/kg body wt increased liver weight (175% of control), decreased hepatic DNA concentration (66% of control), and increased the synthesis of DNA (500% of control; as measured by [3H]dT incorporation) in B6C3F1 mice and Alderley Park mice. Similar treatment of Osborne-Mendel rats or Alderley Park rats resulted in smaller increases in liver weight (130% of control) and decreases in DNA concentration (83% of control). No effect of TRI on DNA synthesis was seen in rats. The increased DNA synthesis in the mouse was not apparently due to regenerative hyperplasia since no signs of necrosis were seen. Furthermore the increased [3H]dT incorporation probably represented semiconservative replication of DNA and not repair, since a parallel increase of mitotic figures was observed. Hence, the liver growth noted after TRI administration appears to be due to liver cell enlargement (hypertrophy) in the rat, but both hypertrophy and hyperplasia (cell proliferation) in the mouse. An important observation has been that TRI induced the peroxisomal enzyme activities, catalase, and cyanide-insensitive palmitoyl-CoA oxidation (147 and 786% of control, respectively), in mice but not in rats. Furthermore, increases in peroxisome volume density (up to 1110% of control) were observed in mice receiving TRI. These observations lead us to suggest that the species difference in hepatocarcinogenicity of TRI, seen between the rat and mouse, is possibly due to a species difference in peroxisome proliferation and cell proliferation, the peroxisome proliferation leading to increased reactive oxygen species and DNA damage, and the cell proliferation then acting to promote this lesion.     

The role of trichloracetic acid and peroxisome proliferation in the differences in carcinogenicity of perchloroethylene in the mouse and rat

Fischer 344 rats and B6C3F1 mice of both sexes were exposed to 400 ppm perchloroethylene (PER) by inhalation, 6 hr/day for 14, 21, or 28 days or to 200 ppm for 28 days. Increased numbers of peroxisomes were seen under the electron microscope and increased peroxisomal cyanide-insensitive palmitoyl CoA oxidation was measured (3.6-fold increase in males and 2.1-fold increase in females) in the livers of mice exposed to PER. Hepatic catalase was not increased. Peroxisome proliferation was not observed in rat liver or in the kidneys of either species. Trichloracetic acid (TCA), a known carcinogen and hepatic peroxisome proliferating agent, was found to be a major metabolite of PER. Blood levels of this metabolite measured in mice and rats during and for 48 hr after a single 6-hr exposure to 400 ppm PER showed that peak blood levels in mice were 13 times higher than those seen in rats. Comparison of areas under the curves over the time course of the experiment showed that mice were exposed to 6.7 times more TCA than rats. The difference in metabolism of PER to TCA in mice and rats leads to the species difference in hepatic peroxisome proliferation which is believed to be the basis of the species difference in hepatocarcinogenicity. Peroxisome proliferation does not appear to play a role in the apparent carcinogenicity of PER in the rat kidney.     

The Extent of Dichloroacetate Formation from Trichloroethylene, Chloral Hydrate, Trichloroacetate, and Trichloroethanol in B6C3F1 Mice

Conflicting data have been published related to the formationof dichloroacetate (DCA) from trichloroethylene (TRI), chloralhydrate (CH), or trichloroacetic acid (TCA) in B6C3F1 mice.TCA is usually indicated as the primary metabolic precursorto DCA. Model simulations based on the known pharmacokineticsof TCA and DCA predicted blood concentrations of DCA that were10- to 100-fold lower than previously published reports. BecauseDCA has also been shown to form as an artifact during sampleprocessing, we reevaluated the source of the reported DCA, i.e.,whether it was metabolically derived or formed as an artifact.Male B6C3F1 mice were dosed with TRI, CH, trichloroethanol (TCE),or TCA and metabolic profiles of each were determined. DCA wasnot detected in any of these samples above the assay LOQ of1.9 µM of whole blood. In order to slow the clearanceof DCA, mice were pretreated for 2 weeks with 2 g/liter of DCAin their drinking water. Even under this pretreatment condition,no DCA was detected from a 100 mg/kg iv dose of TCA. Althoughthere is significant uncertainty in the amount of DCA that couldbe generated from TRI or its metabolites, our experimental dataand pharmacokinetic model simulations suggest that DCA is likelyformed as a short-lived intermediate metabolite. However, itsrapid elimination relative to its formation from TCA preventsthe accumulation of measurable amounts of DCA in the blood.     

Ninety Day Toxicity Study of Chloroacetic Acids in Rats

Ninety Day Toxicity Study of Chloroacetic Acids in Rats. BHAT, H. K, KANZ, M. F., CAMPBELL G. A., AND ANSARI. G. A. S.(1991).Fundam. Appl Toxicol. 17, 240–253. Chloroacetic acidsare produced in drinking water as a result of disinfection processes.Chloroacetic acids are also metabolites of widely usad and toxichalogenated hydrocarbons. Thus, chronic human exposure to thesechemicals is likely to occur. The objective of the present studywas to examine the toxic effects of monochloroacetic acid (MCA),dichloroacetic acid (DCA), and trichloroacetic acid (TCA) ina 90-day subchronic study in rats via oral exposure by drinkingwater. Chloroacetic acid solutions were prepared at concentrationswhich provided an approximate intake of ? the LD50 dose perday: MCA, 1.9 mM; DCA, 80.5 mM; TCA, 45.8 mM. Control rats receiveddistilled water only. After 90 days, major organs were removed,fixed, paraffin embedded, and stained. Light microscopic examinationof the major organs revealed variable degrees of alterationsin the lung and liver of all three treated groups. In the liver,morphological changes were predominantly localized to the portaltriads, which were mildly to moderately enlarged with randombile duct proliferation, extension of portal veins, fibrosis,edema, and occasional foci of inflammation. In the lungs, minimalalterations were observed as foci of perivascular inflammationon small pulmonary veins. Morphological changes in the testesand brain were seen only in the DCA treated group. Testes wereatrophic with few spermatocytes and no mature spermatozoa. Focalvacuolation and gliosis were present in the forebrain and brainstem.The results of these studies indicate that, relative to theirrespective LD50 values DCA given at 80.5 mM is more toxic thanTCA given at 45.8 mM and MCA at 1.9 mM is least toxic.     

Peroxisome proliferation due to di (2-ethylhexyl) adipate, 2-ethylhexanol and 2-ethylhexanoic acid

The dose-response relationships for peroxisome proliferation due to Di (2-ethylhexyl) adipate (DEHA), 2-ethylhexanol (EH), 2-ethylhexanoic acid (EHA) have been investigated in rats and mice. Linear dose-response relationships were observed for induction of cyanide-insensitive palmitoyl CoA oxidation (PCO), used as a enzyme marker of peroxisome proliferation, by DEHA, EH and EHA in both species. Relative liver weights were also increased in a dose related manner. On a molar basis, DEHA was twice as potent as EH or EHA which were equipotent and PCO was stimulated to a greater extent in male mice than in rats or female mice. At doses above 8 mmol/kg/day, EH was toxic to rats (both sexes) and similarly EHA at 13.5 mmol/kg/day lead to the death of female rats. In a attempt to explain the species difference in carcinogenicity of DEHA previously reported, we also used Fischer 344 rats and B6C3F1 mice. DEHA administration (2.5 g/kg/day) to Fischer 344 rats and B6C3F1 mice lead to toxicity in female rats. Relative liver weights were increased in a dose related fashion by DEHA administration to both rats and mice, PCO but not catalase was markedly increased (up to 15 fold in male rats). Light microscopy examination indicated some glycogen loss, a dose related hypertrophy and increased eosinophilia in both rats and mice. Electron microscopy confirmed peroxisome proliferation accompanied by a marked reduction of lipid in the centrilobular hepatocytes. These data suggest EHA to be the proximate peroxisome proliferator derived from DEHA. These data indicate a higher sensitivity for Fischer 344 rats than B6C3F1 mice to hepatic peroxisome proliferation due to DEHA and ratio of PCO activity and catalase activity data suggest that more hydrogen peroxide (H₂O₂) could escape from peroxisomes in male Fischer 344 rats than B6C3F1 mice. These data obtained with B6C3F1 mice and Fischer 344 rats are not agreement with the carcinogenicity bioassays previously reported showing an incidence of hepatic tumours only in B6C3F1 mice.     

The induction of hepatocellular neoplasia by trichloroacetic acid administered in the drinking water of the male B6C3F1 mouse

The prevalence (percent of animals with a tumor) and multiplicity (number of tumors per animal) of hepatocellular neoplasia in the male B6C3F1 mouse exposed to trichloroacetic acid (TCA) in the drinking water were determined. Male mice were exposed to 0.05, 0.5, and 5 g/L TCA for 60 wk (Study 1), to 4.5 g/L TCA for 104 wk (Study 2) and to 0.05 and 0.5 g/L TCA for 104 wk (Study 3). Time-weighted mean daily doses measured for the low, medium, and high dose groups were consistent over the three studies, 6-8, 58-68, and 572-602 mg/kg-d for the 0.05, 0.5, and the 4.5-5 g/L treatment groups, respectively. No significant changes in animal survival were noted across the studies. A significant increase in the prevalence and multiplicity of hepatocellular tumors was found in the 58-68 and 572-602 mg/kg/d TCA dose groups. Nonhepatoproliferative changes (cytoplasmic alterations, inflammation, and necrosis) in mice treated with TCA were mild and dose related. A TCA-induced increase in liver palmitoyl CoA oxidase activity, a marker of peroxisome proliferation, correlated with tumor induction. A linear association was found between peroxisome proliferation and tumor induction. Sporadic increases in the labeling index of nuclei outside of proliferative lesions were observed at carcinogenic doses throughout the studies. Given that there are no compelling data demonstrating genotoxic activity of either TCA or any metabolite, data are consistent with an epigenetic mode of action. The studies provide dose-response data on the development of hepatocellular neoplasia in male mice over a lifetime exposure to TCA. A no-observed-effect-level (NOEL) of 6 mg/kg/d was calculated for neoplastic and nonproliferative liver pathology.     

Differential effects of dihalogenated and trihalogenated acetates in the liver of B6C3F1 mice

Haloacetates are produced in the chlorination of drinking water in the range 10--100 microg l(-1). As bromide concentrations increase, brominated haloacetates such as bromodichloroacetate (BDCA), bromochloroacetate (BCA) and dibromoacetate (DBA) appear at higher concentrations than the chlorinated haloacetates: dichloroacetate (DCA) or trichloroacetate (TCA). Both DCA and TCA differ in their hepatic effects; TCA produces peroxisome proliferation as measured by increases in cyanide-insensitive acyl CoA oxidase activity, whereas DCA increases glycogen concentrations. In order to determine whether the brominated haloacetates DBA, BCA and BDCA resemble DCA or TCA more closely, mice were administered DBA, BCA and BDCA in the drinking water at concentrations of 0.2--3 g l(-1). Both BCA and DBA caused liver glycogen accumulation to a similar degree as DCA (12 weeks). The accumulation of glycogen occurred in cells scattered throughout the acinus in a pattern very similar to that observed in control mice. In contrast, TCA and low concentrations of BDCA (0.3 g l(-1)) reduced liver glycogen content, especially in the central lobular region. The high concentration of BDCA (3 g l(-1)) produced a pattern of glycogen distribution similar to that in DCA-treated and control mice. This effect with a high concentration of BDCA may be attributable to the metabolism of BDCA to DCA. All dihaloacetates reduced serum insulin levels. Conversely, trihaloacetates had no significant effects on serum insulin levels. Dibromoacetate was the only brominated haloacetate that consistently increased acyl-CoA oxidase activity and rates of cell replication in the liver. These results further distinguish the effects of the dihaloacetates from those of peroxisome proliferators like TCA.     

Metabolism and lipoperoxidative activity of trichloroacetate and dichloroacetate in rats and mice.

Trichloroacetate (TCA) and dichloroacetate (DCA) have been shown to be hepatocarcinogenic in mice when administered in drinking water. However, DCA produces pathological effects in the liver that are much more severe than those observed following TCA treatment in both rats and mice. To identify potential mechanisms involved in the liver pathology, the biotransformation of TCA and DCA was investigated in male Fischer 344 rats and B6C3F1 mice. Rodents were administered 5, 20, or 100 mg/kg [14C]TCA or [14C]DCA as a single oral dose in water. Elimination was examined by counting radioactivity in urine, feces, exhaled air, and carcass. Blood concentration over time curves were constructed for both TCA and DCA at the 20 and 100 mg/kg doses. Analysis of the data reveals two significant differences in the systemic clearance of TCA relative to DCA. First, DCA was much more extensively metabolized than TCA. More than 50% of any single dose of TCA was excreted unchanged in the urine of both rats and mice. In contrast, less than 2% of any dose of DCA was recovered in the urine as the parent compound. Second, while the blood concentration over time curves for TCA were similar in rats and mice, the blood concentrations of DCA were markedly greater in rats compared to those in mice, both when DCA was administered and when DCA resulted from metabolism of TCA. DCA was detected in the urine of TCA-treated animals and chloroacetate was found in the urine of DCA-treated animals. These metabolic products would be expected to arise from a free radical-generating, reductive dechlorination pathway. To evaluate the ability of acute doses of TCA and DCA to elicit a lipoperoxidative response, additional groups of mice were administered 0, 100, 300, 1000, and 2000 mg/kg TCA or DCA and thiobarbituric acid-reactive substances (TBARS) measured in liver homogenates. Both TCA and DCA enhanced the formation of TBARS in a dose-dependent manner, thereby providing further evidence of a reductive metabolic pathway. DCA was found to be the more potent of the chlorinated acetates in increasing TBARS formation in the livers of both rats and mice. In view of these data, it appears that the more extensive metabolism and rapid rate of elimination of DCA relative to TCA and the more potent lipoperoxidative activity of DCA may be important factors in the pathological effects associated with DCA treatment.     

Bioactivation of chloroform in hepatic microsomes from rodent strains susceptible or resistant to CHCl3 carcinogenicity.

The dependence of adduct formation on oxygen concentration and glutathione (GSH) presence was used to characterize the bioactivation of chloroform in hepatic microsomes of Sprague-Dawley and Osborne-Mendel rats and B6C3F1 and C57Bl/6J mice. Both oxidative and reductive pathways were present in all the animals tested. Oxidative activation, very sensitive to oxygen withdrawal, was the major pathway responsible for the covalent binding to microsomal proteins and lipids at 0.1 mM CHCl3. The relative contribution of either pathway to the covalent binding to microsomal lipids at 5 mM CHCl3 was dependent on the oxygen concentration. At 1% pO2, i.e., in the range of the hepatic physiological oxygenation level, B6C3F1 mouse hepatic microsomes showed an oxidative activation distinctly higher than that of hepatic microsomes of other rodents; on the other hand, reductive activation was present only in B6C3F1 mouse and Osborne-Mendel rat liver microsomes. The reductive intermediates were the only contributors to the covalent binding of CHCl3 equivalents to lipids in the presence of GSH; indeed the reactive intermediates produced by the oxidative pathway were fully scavenged by this compound. These results are discussed with respect to the species specificity of CHCl3 hepatocarcinogenesis.     

Physiologically based pharmacokinetic modeling with trichloroethylene and its metabolite, trichloroacetic acid, in the rat and mouse

The uptake and metabolism of trichloroethylene (TCE), and the stoichiometric yield and kinetic behavior of one of its major metabolites, trichloroacetic acid (TCA), were compared in Fischer 344 rats and B6C3F1 mice using a physiological model. Physiologically based pharmacokinetic (PB-PK) model parameters (metabolic rate constants and tissue partition coefficients) were determined in male and female B6C3F1 mice and were taken from the literature for the male and female Fischer 344 rats. The kinetic behavior of TCA was described by a classical one-compartment model linked to a PB-PK model for TCE. The TCE blood/air partition coefficients for male and female mice, determined by vial equilibration, were 13.4 and 14.3. The Vmaxe values for male and female mice, using gas uptake techniques, were 32.7 +/- .06 and 23.2 +/- 0.1 mg/kg/hr and the Km was 0.25 mg/liter. The PB-PK model for TCE adequately described the uptake and clearance of TCE in male and female rats exposed to a single, constant concentration of TCE vapor, but failed to describe the uptake and clearance of TCE in male and female mice exposed to a wide range TCE vapor concentrations. Computer-predicted blood concentrations of TCE were generally greater than observed blood concentrations of TCE. The stoichiometric yield of TCA in mice exposed to these TCE vapors was concentration dependent. The capacity for oxidation of TCE was much greater in B6C3F1 mice than in Fischer 344 rats, and as a result the systemic concentration of TCA was greater in these mice than rats. An increased body burden of TCA in B6C3F1 mice may be related to the formation of hepatocellular carcinomas in B6C3F1 mice exposed to TCE.