Hepatocarcinogenicity in the male B6C3F1 mouse following a lifetime exposure to dichloroacetic acid in the drinking water: dose-response determination and modes of action

Male B6C3F, mice were exposed to dichloroacetic acid (DCA) in the drinking water in order to establish a dose response for the induction of hepatocellular cancer and to examine several modes of action for the carcinogenic process. Groups of animals were exposed to control, 0.05, 0.5, 1, 2, or 3.5 g/L DCA in the drinking water for 90-100 wk. Mean daily doses (MDD) of 8, 84, 168, 315, and 429 mg/kg/d of DCA were calculated. The prevalence (percent of animals) with hepatocellular carcinoma (HC) was significantly increased in the 1-g/L (71%), 2-g/L (95%), and 3.5-g/L (100%) treatment groups when compared to the control (26%). HC multiplicity (tumors/animal) was significantly increased by all DCA treatments-0.05 g/L (0.58), 0.5 g/L (0.68), 1 g/L (1.29), 2 g/L (2.47), and 3.5 g/L (2.90)-compared to the control group (0.28). Based upon HC multiplicity, a no-observed-effect level (NOEL) for hepatocarcinogenicity could not be determined. Hepatic peroxisome proliferation was significantly increased only for 3.5 g/L DCA treatment at 26 wk. and did not correlate with the liver tumor response. The severity of hepatotoxicity increased with DCA concentration. Below 1 g/L, hepatotoxicity was mild and transient as demonstrated by the severity indices and serum lactate dehydrogenase activity. An analysis of generalized hepatocyte proliferation reflected the mild hepatotoxicity and demonstrated no significant treatment effects on the labeling index of hepatocytes outside proliferative lesions. Consequently, the induction of liver cancer by DCA does not appear to be conditional upon peroxisome induction or chemically sustained cell proliferation. Hepatotoxicity, especially at the higher doses, may exert an important influence on the carcinogenic process.     

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.     

Species and strain sensitivity to the induction of peroxisome proliferation by chloroacetic acids

B6C3F1 mice and Sprague-Dawley rats were provided drinking water containing 6-31 mM (1-5 g/liter) trichloroacetic acid (TCA), 8-39 mM (1-5 g/liter) dichloroacetic acid (DCA), or 11-32 mM (1-3 g/liter) monochloroacetic acid (MCA) for 14 days. TCA and DCA, but not MCA, increased the mouse relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxisome proliferation-associated protein, and (3) morphometric analysis of electron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 mM) increased rat liver peroxisome proliferation. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL/6) and two strains of rat (F344 and Osborne-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 mM) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL/6. A more modest enhancement in the Osborne-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 rats with 200 mg/kg TCA in water or corn oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement of liver peroxisome proliferation by TCA and DCA and suggest that if peroxisome proliferation is critical for the induction of hepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction.     

Liver tumor induction in B6C3F1 mice by dichloroacetate and trichloroacetate

Male and female B6C3F1 mice were administered dichloroacetate (DCA) and trichloroacetate (TCA) in their drinking water at concentrations of 1 or 2 g/l for up to 52 weeks. Both compounds induced hepatoproliferative lesions (HPL) in male mice, including hepatocellular nodules, adenomas and hepatocellular carcinomas within 12 months. The induction of HPL by TCA was linear with dose. In contrast, the response to DCA increased sharply with the increase in concentration from 1 to 2 g/l. Suspension of DCA treatment at 37 weeks resulted in the same number of HPL at 52 weeks that would have been predicted on the basis of the total dose administered. However, none of the lesions in this treatment group progressed to hepatocellular carcinomas. Conversely, the yield of HPL at 52 weeks when TCA treatment was suspended at 37 weeks was significantly below that which would have been predicted by the total dose administered. In this case, 3 of 5 remaining lesions were hepatocellular carcinomas. Throughout active treatment DCA-treated mice displayed greatly enlarged livers characterized by a marked cytomegaly and massive accumulations of glycogen in hepatocytes throughout the liver. Areas of focal necrosis were seen throughout the liver. TCA produced small increases in cell size and much a more modest accumulation of glycogen. Focal necrotic damage did not occur in TCA-treated animals. TCA produced marked accumulations of lipofuscin in the liver. Lipofuscin accumulation was less marked with DCA. These data confirm earlier observations that DCA and TCA are capable of inducing hepatic tumors in B6C3F1 mice and argue that the mechanisms involved in tumor induction differ substantially between these two similar compounds. Tumorigenesis by DCA may depend largely on stimulation of cell division secondary to hepatotoxic damage. On the other hand, TCA appears to increase lipid peroxidation, suggesting that production of radicals may be responsible for its effects.     

Interactions in the tumor-promoting activity of carbon tetrachloride, trichloroacetate, and dichloroacetate in the liver of male B6C3F1 mice

Interactions between carcinogens in mixtures found in the environment have been a concern for several decades. In the present study, male B6C3F1 mice were used to study the responses to mixtures of dichloroacetate (DCA), trichloroacetate (TCA), and carbon tetrachloride (CT). TCA produces liver tumors in mice with the phenotypic characteristics common to peroxisome proliferators. DCA increases the growth of liver tumors with a phenotype that is distinct in several respects from those produced by TCA. These chemicals are effective as carcinogens at doses that do not produce cytotoxicity. Thus, they encourage clonal expansion of initiated cells through subtle, selective mechanisms. CT is well known for its ability to promote the growth of liver tumors through cytotoxicity that produces a generalized growth stimulus in the liver that is reflected in a reparative hyperplasia. Thus, CT is relatively non-specific in its promotion of initiated cells within the liver. The objective of this study was to determine how the differing modes of action of these chemicals might interact when given as mixed exposures. The hypothesis was that the effects of two selective promoters would not be more than additive. On the other hand, CT would be selective only to cells not sensitive to its effects as a cytotoxin. Thus, it was hypothesized that neither DCA nor TCA would add significantly to the effects produced by CT. Mice were initiated by vinyl carbamate (VC), and then promoted by DCA, TCA, CT, or the pair-wised combinations of the three compounds. The effect of each treatment or treatment combination on tumor number per animal and mean tumor volume was assessed in each animal. Dose-related increases in mean tumor volume were observed with 20 and 50mg/kg CT, but each produced equal numbers of tumors at 36 weeks. As the dose of CT was increased to >/=100mg/kg substantial increases in the number of tumors per animal were observed, but the mean tumor size decreased. This finding suggests that initiation occurs as doses of CT increase to >/=100mg/kg, perhaps as a result of the inflammatory response that is known to occur with high doses of CT. When administered alone in the drinking water at 0.1, 0.5 and 2g/l, DCA increased both tumor number and tumor size in a dose-related manner. With TCA treatment at 2g/l in drinking water a maximum tumor number was reached by 24 weeks and was maintained until 36 weeks of treatment. DCA treatment did not produce a plateau in tumor number within the experimental period, but the numbers observed at the end of the experimental period were similar to TCA and doses of 50mg/kg CT. The tumor numbers observed at the end of the experiment are consistent with the assumption that the administered dose of the tumor initiator, vinyl carbamate, was the major determinant of tumor number and that treatments with CT, DCA, and TCA primarily affected tumor size. The results with mixtures of these compounds were consistent with the basic hypotheses that the responses to tumor promoters with differing mechanisms are limited to additivity at low effective doses. More complex, mutually inhibitory activity was more often observed between the three compounds. At 24 weeks, DCA produced a decrease in tumor numbers promoted by TCA, but the numbers were not different from TCA alone at 36 weeks. The reason for this result became apparent at 36 weeks of treatment where a dose-related decrease in the size of tumors promoted by TCA resulted from DCA co-administration. On the other hand, the low dose of TCA (0.1g/l) decreased the number of tumors produced by a high dose of DCA (2g/l), but higher doses of TCA (2g/l) produced the same number as observed with DCA alone. DCA inhibited the growth rate of CT-induced tumors (CT dose = 50mg/kg). TCA substantially increased the numbers of tumors observed at early time points when combined with CT, but this was not observed at 36 weeks. The lack of an effect at 36 weeks was attributable to the fact that more than 90% of the livers consisted of tumors and the earlier effect was masked by coalescence of tumors. Thus, the ability of TCA to significantly increase tumor numbers in CT-treated mice was probably real and contrary to our original hypothesis that CT was non-specific in its effects on initiated cells. It is probable that the interaction between CT and TCA is explained through stimulation of the growth of cells with differing phenotypes. These data suggest that the outcome of interactions between the mechanisms of tumor promotion vary based on the characteristics of the initiated cells. The interactions may result in additive or inhibitory effects, but no significant evidence of synergy was observed.     

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.     

Quantitative analysis of hepatocellular lesions induced by di(2-ethylhexyl)phthalate in F-344 rats

Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, has been shown to be a weak hepatocarcinogen in rats and mice. However, in previous studies no quantitative analysis of tumors was carried out. In the present study, F-344 male rats were given a diet containing 2% DEHP ad libitum for 108 wk. At necropsy livers were quantitatively analyzed for total tumor incidence and the number of lesions per liver after slicing the entire organ at 1- to 2-mm intervals. Neoplastic nodules and/or hepatocellular carcinomas were observed in 11 of 14 rats (78.5%). When evaluated according to the size, 57, 16, and 36% rats contained nodules ranging from 1 to 3, 3 to 5, and greater than 5 mm in size, respectively. The number of nodules per liver ranged from zero to four. These results indicate that DEHP induces tumors in a large number of animals at 2% dose levels. It is clear from this study that when a weak peroxisome proliferator is evaluated for carcinogenic effects, a complete and thorough gross examination of the liver is essential to obtain accurate tumor incidence.     

Dexamethasone selectively inhibits WY14,643-induced cell proliferation and not peroxisome proliferation in mice

It has been proposed that the hepatocellular proliferation induced by peroxisome proliferators may occur through an indirect mechanism involving cytokine release as opposed to direct regulation of cell growth genes by PPARalpha. We compared the induction of peroxisome proliferation and cell proliferation in C57Bl/6 mice treated with 100 mg/kg/day WY14,643 in the presence or absence of increasing doses of dexamethasone (DEX), an inhibitor of the release of proinflammatory cytokines. Biochemical markers of peroxisome proliferation, including fatty acyl-CoA oxidase activity, CYP4A content, and liver-to-body-weight ratios were markedly increased in the WY14,643-treated mice. DEX coadministration, up to a maximum dose of 50 mg/kg/day, did not prevent the induction of these parameters. Acyl-CoA oxidase mRNA levels increased 5-fold with WY14,643 treatment and 15-fold with DEX coadministration at 5 mg/kg/day. ApoCIII mRNA levels were decreased by 50% in WY14,643-treated mice. DEX alone at 5 mg/kg/day increased the ApoCIII mRNA 4-fold, but WY14,643 coadministration also inhibited this induction by greater than 50%. In addition, immunohistochemical detection of peroxisomes with anti-PMP-70 antibody demonstrated marked increase in hepatocellular peroxisomes in WY14,643-treated mice regardless of DEX treatment. In contrast, coadministration of DEX at 2 mg/kg/day partially inhibited the hepatocyte proliferation response (measured by BrdU incorporation or Ki-67 immunohistochemical detection). Moreover, DEX at doses of 5 mg/kg/day or higher completely inhibited the induction of cell proliferation and, at these higher doses, reduced the cell proliferation rate to levels below the vehicle-treated control mice. Our studies clearly demonstrate that the hepatocellular proliferation induced by a peroxisome proliferator can be modulated independently of the other pleiotropic effects usually induced by these agents, suggesting an indirect mechanism of hyperplasia.     

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.     

同位素标记示踪法测定小鼠G蛋白抑制肽GCIP-27血药浓度

目的:考察小鼠静脉注射G蛋白抑制肽GCIP-27后药动学情况。方法:125I-GCIP采用氯甘脲法标记,血浆中GCIP的浓度采用同位素示踪法结合三氯醋酸(TCA)沉淀法或低分子量SDS-PAGE电泳法测定。结果:GCIP-27在小鼠体内按一级动力学代谢,并呈三室开放模型;静脉注射125I-GCIP90μg.kg-1后,电泳法和酸沉法测得t1/2α、t1/2β、t1/2γ分别为0.009、0.245、2.054h和0.025、0.306、2.323h;tmax均为0.0333h,平均血浆清除率分别为0.295、0.322L.h-1.kg-1,表观分布容积分别为0.559、1.29L.kg-1,体内平均滞留时间分别为2.353、2.515h。结论:GCIP-27在小鼠体内血药浓度下降快,分布快,自中央室迅速向周边室分布;消除、代谢缓慢。本研究结果可为GCIP-27的剂型设计和药效学研究提供重要依据。     

Contribution of trichloroacetic acid to liver tumors observed in perchloroethylene (perc)-exposed mice

Perchloroethylene (perc), a solvent used in dry cleaning operations and industrial applications, has been found to produce increases in hepatocellular carcinomas and/or adenomas in mice in chronic inhalation bioassays. Perc is metabolized primarily to trichloroacetic acid (TCA), which is also a mouse hepatocarcinogen. The fractional conversion of perchloroethylene to TCA by mice was determined from physiologically based pharmacokinetic (PBPK) modeling of TCA in mouse blood at the conclusion of inhalation exposure of male and female B6C3F1 mice to 10, 50, 100, or 200 ppm perc for 6 h/day for 5 days. The dose-dependent bioavailability of TCA in B6C3F1 mice exposed to TCA in drinking water was estimated by optimizing the fit of time course blood, plasma, and liver TCA concentrations for TCA doses ranging from 12 to 800 mg/(kg day) to predictions of a previously published TCA PBPK model. Using the PBPK models, the area under the liver TCA concentration vs. time curve (liver TCA AUC) was calculated for TCA and perc bioassays. Benchmark dose analyses were conducted to determine the dose–response relationship between liver TCA AUC and the additional risk of hepatocellular adenomas or carcinomas (combined) in mice ingesting TCA. Using the dose–response relationships derived for the TCA-exposed mice, the contribution of TCA produced by metabolism to the additional risk of liver adenomas and carcinomas in mice exposed to perchloroethylene by inhalation was computed. The analysis indicated that the levels of TCA observed in perchloroethylene-exposed mice are sufficient to explain the incidence of liver adenomas and carcinomas.     

Lack of carcinogenicity of phenytoin in (C57BL/6 x C3H)F1 mice

Groups of 50 B6C3F1 mice of each sex were given 0.012% or 0.006% phenytoin in their powdered diet for 78 wk and were then fed a basal diet for 8 wk. Control groups of 50 mice of each sex were fed powdered basal diet for 86 wk. Mean total intakes of phenytoin per mouse were 301 and 150 mg in males, and 292 and 154 mg in females, respectively. The survival rates of each group at week 86 were 72-86% in males, and 86-94% in females. Liver-cell tumors, alveolar tumors, and Harderian-gland adenomas in male mice, malignant lymphomas and/or leukemias in female mice, and a few tumors in other organs of both sexes were found. The total number of hepatocellular tumors in mice treated with the high dose of phenytoin was significantly smaller than that of control mice in males (p less than 0.05). However, hepatocellular carcinomas developed 15 to 3 wk earlier in a few mice of phenytoin-treated males than in the controls. In other organs, no significant increase of any particular tumor type was observed in the treated groups of both sexes. Thus, phenytoin was not carcinogenic in B6C3F1 mice in this study.     

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.     

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.     

Impact of repeated exposure on toxicity of perchloroethylene in Swiss Webster mice

The aim was to study the subchronic toxicity of perchloroethylene (Perc) by measuring injury and repair in liver and kidney in relation to disposition of Perc and its major metabolites. Male SW mice (25-29g) were given three dose levels of Perc (150, 500, and 1000 mg/kg day) via aqueous gavage for 30 days. Tissue injury was measured during the dosing regimen (0, 1, 7, 14, and 30 days) and over a time course of 24-96h after the last dose (30 days). Perc produced significant liver injury (ALT) after single day exposure to all three doses. Liver injury was mild to moderate and regressed following repeated exposure for 30 days. Subchronic Perc exposure induced neither kidney injury nor dysfunction during the entire time course as evidenced by normal renal histology and BUN. TCA was the major metabolite detected in blood, liver, and kidney. Traces of DCA were also detected in blood at initial time points after single day exposure. With single day exposure, metabolism of Perc to TCA was saturated with all three doses. AUC/dose ratio for TCA was significantly decreased with a concomitant increase in AUC/dose of Perc levels in liver and kidney after 30 days as compared to 1 day exposures, indicating inhibition of metabolism upon repeated exposure to Perc. Hepatic CYP2E1 expression and activity were unchanged indicating that CYP2E1 is not the critical enzyme inhibited. Hepatic CYP4A expression, measured as a marker of peroxisome proliferation was increased transiently only on day 7 with the high dose, but was unchanged at later time points. Liver tissue repair peaked at 7 days, with all three doses and was sustained after medium and high dose exposure for 14 days. These data indicate that subchronic Perc exposure via aqueous gavage does not induce nephrotoxicity and sustained hepatotoxicity suggesting adaptive hepatic repair mechanisms. Enzymes other than CYP2E1, involved in the metabolism of Perc may play a critical role in the metabolism of Perc upon subchronic exposure in SW mice. Liver injury decreased during repeated exposure due to inhibition of metabolism and possibly due to adaptive tissue repair mechanisms.     

Toxicity and carcinogenicity of the water disinfection byproduct, dibromoacetic acid, in rats and mice

Dibromoacetic acid (DBA) is a water disinfection byproduct formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromide. Male and female F344/N rats and B6C3F₁ mice were exposed to DBA in drinking water for 2 weeks (N = 5), 3 months (N = 10), or 2 years (N = 50). Concentrations of DBA in drinking water were 0, 125, 250, 500, 1000, and 2000 mg/L in the 2-week and 3-month studies, and 0, 50, 500, and 1000 mg/L in the 2-year studies. Toxic effects of DBA in the prechronic studies were detected in the liver (hepatocellular cytoplasmic vacuolization in rats and mice) and testes (delayed spermiation and atypical residual bodies in male rats and mice, and atrophy of the germinal epithelium in rats). In the 2-year studies, neoplasms were induced at multiple sites in rats and mice exposed to DBA; these included mononuclear cell leukemia and abdominal cavity mesothliomas in rats, and neoplasms of the liver (hepatocellular adenoma or carcinoma and hepatoblastoma) and lung (alveolar adenoma or carcinoma) in mice. The increase in incidence of hepatocellular neoplasms in male mice was significant even at the lowest exposure concentration of 50 mg/L, which is equivalent to an average daily dose of approximately 4 mg/kg. These studies provide critical information for future re-evaluations of health-based drinking water standards for haloacetic acids.     

Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate.

Differing rates and extent of trichloroethylene (TCE) metabolism have been implicated as being responsible for varying sensitivities of mice and rats to the hepatocarcinogenic effects of TCE. Recent data indicate that the induction of hepatic tumors in mice may be attributed to the metabolites trichloroacetate (TCA) and/or dichloroacetate (DCA). The present study was directed at determining whether mice and rats varied in (1) the peak blood concentrations, (2) the area under the blood concentration over time curves (AUC) for TCE and metabolites in blood, and (3) the net excretion of TCE to these metabolites in urine in the dose range used in the cancer bioassays of TCE, and to contrast the kinetic parameters observed for TCE-derived TCA and DCA with those obtained following direct administration of TCA and DCA. Blood and urine samples were collected over 72 hr from rats and mice after a single oral dose of TCE of 1.5 to 23 mmol/kg. The AUC values from the blood concentration with time profiles of TCE, TCA, and trichloroethanol (TCOH) were similar for Sprague-Dawley rats and B6C3F1 mice. Likewise, the percentages of initial TCE dose recovered as the urinary metabolites TCA and TCOH were comparable. Nevertheless, the peak blood concentrations of TCE, TCA, and TCOH observed in mice were much greater than those in rats, while the residence time of TCE and metabolites was prolonged in rats relative to that of mice. DCA was detected in the blood of mice but not in rats. The blood concentrations of DCA observed in mice given a carcinogenic dose of TCE (15 mmol/kg) were of the same magnitude as those observed with carcinogenic doses of DCA. In conclusion, the net metabolism of TCE to TCA and TCOH was similar in rats and mice. The initial rates of metabolism of TCE to TCA, however, were much higher in mice, especially as the TCE dose was increased, leading to greater concentrations of TCA and DCA in mice approximated those produced by carcinogenic doses of the chlorinated acetates makes it highly likely that both compounds play a role in the induction of hepatic tumors in mice by TCE.     

Inhibition by wine of tumorigenesis induced by ethyl carbamate (urethane) in mice

Groups of 18-21 male weanling C3H mice were given, as drinking fluid, tap-water, 12% ethanol solution, one of two commercial white wines, or red wine, ad lib. for 41 wk. Ethyl carbamate was added to each of the drinking liquids at levels adjusted to provide average daily ethyl carbamate intakes of 0, 10 or 20 mg/kg body weight. After 41 wk the cumulative survival of the mice given 20 mg ethyl carbamate/kg in water was depressed compared with the mice drinking wines or ethanol solution with this ethyl carbamate level. Both ethanol and wine treatments reduced the incidence of lung Clara-cell adenomas in mice given 10 mg ethyl carbamate/kg and reduced the frequency (number of specific tumours/number of tumour-bearing mice) of both Clara-cell adenomas in mice given 10 mg ethyl carbamate/kg and of alveolar adenomas in mice given 20 mg ethyl carbamate/kg. Wine treatments also reduced the frequency of hepatocellular adenomas compared with those of other treatment groups, and no hepatocellular carcinomas developed in any of the groups given wine, even with the 20-mg/kg ethyl carbamate dose. The incidence of hepatocellular adenomas in the groups given 10 mg ethyl carbamate/kg was, as shown by chi-square analysis, significantly reduced by the ethanol and wine treatments. The mean weight gains of mice on all the wine treatments were lower than those of water-treated mice and this may have been a factor in tumour inhibition; however, it is also possible that wine components other than ethanol may play a role in the inhibition of tumour development.     

Evaluation of dichloroacetate treatment in a murine model of hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease severely affecting liver and kidney and is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Administration of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) improves the HT1 phenotype but some patients do not respond to NTBC therapy. The objective of the present study was to evaluate whether administration of dichloroacetate, an inhibitor of maleyl acetoacetate isomerase (MAAI) to FAH-knockout mice could prevent acute pathological injury caused by NTBC withdrawal. DCA (0.5 and 5g/L) was given in combination with a standard diet or with a tyrosine-restricted diet. With the low-tyrosine diet body weight loss and most of hepatic and renal injuries were prevented regardless the DCA dose. The administration of DCA with a standard diet did not prevent damage nor the oxidative stress response nor the AFP induction seen in FAH-knockout mice. DCA was shown to inhibit hepatic MAAI activity to 86% (0.5g/L) and 94% (5g/L) of untreated wild-type mice. Interestingly, FAH(-/-) mice deprived of NTBC (NTBC-OFF) and NTBC-treated FAH-knockout mice had similar low hepatic MAAI activity levels, corresponding to 10-20% of control. Thus the failure of DCA treatment in FAH(-/-) mice seems to be attributed to the residual MAAI activity, high enough to lead to FAA accumulation and HT1 phenotype.