Dichloroacetic acid and trichloroacetic acid increase chloroform toxicity.

Dichloro- and trichloroacetic acids (DCA and TCA) and chloroform are formed during chlorination disinfection of drinking water. The effects of DCA and TCA treatment on CHCl3 toxicity were assessed in these studies. Male and female rats were gavaged with DCA or TCA (0.92 and 2.45 mmol/kg administered 3 times over 24 h). Three hours after the last dose CHCl3 was injected ip (0.75 mg/kg). Male rats experienced some weight loss (15%) and slight increases of ALT and BUN, but there were no effects of either DCA or TCA on any of these responses. In females, CHCl3 increased plasma ALT and this response was greater (up to threefold) in the DCA group, compared to saline controls. Similarly, BUN was increased by CHCl3 and this was more severe (up to threefold) in both the DCA and TCA pretreated groups. These results show that CHCl3 toxicity is increased by DCA and TCA, and this effect is gender-specific, occurring only in females. DCA increases both liver and kidney toxicity, whereas TCA affects only kidney toxicity.     

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.     

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.     

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.     

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.     

Disposition of orally and intravenously administered methyltetrahydrofuran in rats and mice

The disposition of [14C]methyltetrahydrofuran (14C-MTHF) in rats and mice was determined by following changes in the radioactivity in tissue and excreta with time after dosing. MTHF administered orally (1, 10, or 100 mg/kg) or intravenously (1 mg/kg) to either rats or mice was rapidly metabolized and excreted with <8% (mice) or 8-22% (rats) of the dose remaining in the body after 24 h (1 and 10 mg/kg doses) or 72 h (100 mg/kg dose). Based on recovery of radioactivity in excreta (other than feces) and tissues (other than the gastrointestinal [GI] tract), absorption of orally administered MTHF was essentially complete (93-100%). There were no overt signs of toxicity observed at any dose studied. The major route of excretion in mice was in urine followed by exhaled CO2. In rats the major route of excretion was exhaled CO2 followed by urinary excretion. The excretion of exhaled volatile organic compounds (VOC) was dose-dependent in both species; at lower doses exhaled VOC represented 1-5% of dose, but at the highest dose (100 mg/kg) this proportion rose to 14% (mice) and 27% (rats). Analysis of the VOCs exhaled at the high dose indicated that the increase was due to exhalation of the parent compound, 14C-MTHF. Analysis of urine showed three highly polar peaks in the mouse urine and two polar peaks in the rat urine. Because the 14C label in MTHF was in the methyl group, the polar metabolites were considered likely due to the one-carbon unit getting into the metabolic pool and labeling intermediate dietary metabolites.     

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.     

Lipid Peroxidation and Formation of 8-Hydroxydeoxyguanosine from Acute Doses of Halogenated Acetic Acids

Chlorinated, brominated, and mixed bromochloro acetates aremajor by-products of water disinfection by chlorine or ozone.The chlorinated acetates, trichloroacetate (TCA) and dichloroacetate(DCA), are carcinogenic in rodents. Brominated analogs of TCAand DCA have received little study. TCA and DCA induce lipidperoxidation in the livers of rodents when administered acutely.Oxidative stress can also result in oxidative damage to DNA,most commonly measured as increases in 8-hydroxydeoxyguanosine(8-OHdG) adducts. In this study, the ability of acute dosesof TCA, DCA, dibromoacetate (DBA), bromodichloroacetate (BDCA),and bromochloroacetate (BCA) to induce lipid peroxidation and8-OHdG formation was examined. Male B6C3F1 mice developed significantincreases in 8-OHdG/dG ratios in nuclear DNA isolated from liverswhen treated with haloacetates. The extent of 8-OHdG formationappeared to be related to the ability to induce thiobarbi-turicacid-reactive substances (TBARS). The order of potency was DBA BCA > BDCA > DCA > TCA. The induction of 8-OHdG wasfound to be generally more sensitive to treatment with haloacetatesthan the TBARS response. Significantly elevated levels of 8-OHdGwere observed at doses of DBA, BCA, and BDCA as low as 30 mg/kg.We suggest that formation of 8-OHdG by brominated haloacetatesmay contribute to their toxicological effects.     

Metabolomics reveals trichloroacetate as a major contributor to trichloroethylene-induced metabolic alterations in mouse urine and serum

Trichloroethylene (TCE)-induced liver toxicity and carcinogenesis is believed to be mediated in part by activation of the peroxisome proliferator-activated receptor α (PPARα). However, the contribution of the two TCE metabolites, dichloroacetate (DCA) and trichloroacetate (TCA) to the toxicity of TCE, remains unclear. The aim of the present study was to determine the metabolite profiles in serum and urine upon exposure of mice to TCE, to aid in determining the metabolic response to TCE exposure and the contribution of DCA and TCA to TCE toxicity. C57BL/6 mice were administered TCE, TCA, or DCA, and urine and serum subjected to ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based global metabolomics analysis. The ions were identified through searching metabolomics databases and by comparison with authentic standards, and quantitated using multiple reactions monitoring. Quantitative polymerase chain reaction of mRNA, biochemical analysis, and liver histology were also performed. TCE exposure resulted in a decrease in urine of metabolites involved in fatty acid metabolism, resulting from altered expression of PPARα target genes. TCE treatment also induced altered phospholipid homeostasis in serum, as revealed by increased serum lysophosphatidylcholine 18:0 and 18:1, and phosphatidylcholine metabolites. TCA administration revealed similar metabolite profiles in urine and serum upon TCE exposure, which correlated with a more robust induction of PPARα target gene expression associated with TCA than DCA treatment. These data show the metabolic response to TCE exposure and demonstrate that TCA is the major contributor to TCE-induced metabolite alterations observed in urine and serum.     

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.     

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.     

Effect of enterohepatic circulation on the pharmacokinetics of chloral hydrate and its metabolites in F344 rats.

Chloral hydrate (CH) is a commonly found disinfection by-product in water purification, a metabolite of trichloroethylene, and a sedative/hypnotic drug. CH and two of its reported metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), are hepatocarcinogenic in mice. Another metabolite of CH, trichloroethanol (TCE), is also metabolized into TCA, and the enterohepatic circulation (EHC) of TCE maintains a pool of metabolite for the eventual production of TCA. To gain insight on the effects of EHC on the kinetics of CH and on the formation of TCA and DCA, dual cannulated F344 rats were infused with 12, 48, or 192 mg/kg of CH and the blood, bile, urine, and feces were collected over a 48-h period. CH was cleared rapidly (>3000 ml/h/kg) and displayed biphasic elimination kinetics, with the first phase being elimination of the dose and the second phase exhibiting formation rate-limited kinetics relative to its TCE metabolite. The effects of EHC on metabolite kinetics were only significant at the highest dose, resulting in a 44% and 17% decrease in the area under the curve (AUC) of TCA and TCE, respectively. The renal clearance of CH, free TCE (f-TCE), and TCA of 2, 2.7, and 38 ml/h/kg, respectively, indicates an efficient reabsorption mechanism for all of these small chlorinated compounds. DCA was detected at only trace levels (<2 microM) as a metabolite of CH, TCA, or TCE.     

Disposition of Orally and Intravenously Administered Methyltetrahydrofuran in Rats and Mice

The disposition of [¹⁴C]methyltetrahydrofuran (¹⁴C-MTHF) in rats and mice was determined by following changes in the radioactivity in tissue and excreta with time after dosing. MTHF administered orally (1, 10, or 100 mg/kg) or intravenously (1 mg/kg) to either rats or mice was rapidly metabolized and excreted with <8% (mice) or 8–22% (rats) of the dose remaining in the body after 24 h (1 and 10 mg/kg doses) or 72 h (100 mg/kg dose). Based on recovery of radioactivity in excreta (other than feces) and tissues (other than the gastrointestinal [GI] tract), absorption of orally administered MTHF was essentially complete (93–100%). There were no overt signs of toxicity observed at any dose studied. The major route of excretion in mice was in urine followed by exhaled CO₂. In rats the major route of excretion was exhaled CO₂ followed by urinary excretion. The excretion of exhaled volatile organic compounds (VOC) was dose-dependent in both species; at lower doses exhaled VOC represented 1–5% of dose, but at the highest dose (100 mg/kg) this proportion rose to 14% (mice) and 27% (rats). Analysis of the VOCs exhaled at the high dose indicated that the increase was due to exhalation of the parent compound, ¹⁴C-MTHF. Analysis of urine showed three highly polar peaks in the mouse urine and two polar peaks in the rat urine. Because the ¹⁴C label in MTHF was in the methyl group, the polar metabolites were considered likely due to the one-carbon unit getting into the metabolic pool and labeling intermediate dietary metabolites.     

Metabolism of 3-deoxy-4-sulphohexosulose, a reaction product of sulphite in foods, by rat and mouse

The excretion of single intragastric doses of 14C-labelled 3-deoxy-4-sulphohexosulose (DSH) was studied in male CF1 mice and male and female Wistar albino rats. Urine and faeces were collected 6, 12, 24, (36), 48 and 72 hr after administration of 2100 mg [14C]DSH/kg body weight (to mice), 1700 mg/kg (to male rats) and 100 and 500 mg/kg (to male and female rats). After 72 hr, plasma and total carcass levels were determined in some experiments. In mice 29% of the administered radioactivity was excreted in the urine, 50% in the faeces and some 13% in cage washings. In rats, faecal excretion varied between 58.5 and 73%. Urinary excretion varied between 16.5 and 31% and was slightly higher in male than in female rats. No radioactivity was detected in expired air of rats, and carcass levels in rats and mice after 72 hr were less than 0.1% of the dose. TLC analysis of urine extracts revealed only unchanged [14C]DSH. In similar studies, male rats and mice were given 35S-labelled DSH in a dose of 6500 mg/kg or 10,700 mg/kg, respectively. Urinary activity accounted for 19.5% of the dose in rats and 27.5% in mice by 72 hr and no 35S-labelled sulphate was detectable in the urine. Organ analyses at nine intervals from 0.25 to 24 hr after intragastric administration of 1600 and 1800 mg [14C]DSH/kg to male rats and mice, respectively, showed that at all times most of the 14C activity was associated with the gastro-intestinal tract in both species. Maximum tissue levels were 2.16% of the dose in the rat liver 0.5 hr after dosing and 1.57% in the mouse kidney after 0.25 hr. Significant amounts of activity (greater than 0.25% of the dose) occurred transiently also in the pancreas and lungs of both species, in the rat testes and in the mouse bladder. Maximum plasma levels were 0.09% of the dose/ml in rats 0.5 and 1 hr after dosing and 0.34%/ml in mice at 0.25 hr.     

Development of an updated PBPK model for trichloroethylene and metabolites in mice, and its application to discern the role of oxidative metabolism in TCE-induced hepatomegaly

Trichloroethylene (TCE) is a lipophilic solvent rapidly absorbed and metabolized via oxidation and conjugation to a variety of metabolites that cause toxicity to several internal targets. Increases in liver weight (hepatomegaly) have been reported to occur quickly in rodents after TCE exposure, with liver tumor induction reported in mice after long-term exposure. An integrated dataset for gavage and inhalation TCE exposure and oral data for exposure to two of its oxidative metabolites (TCA and DCA) was used, in combination with an updated and more accurate physiologically-based pharmacokinetic (PBPK) model, to examine the question as to whether the presence of TCA in the liver is responsible for TCE-induced hepatomegaly in mice. The updated PBPK model was used to help discern the quantitative contribution of metabolites to this effect. The update of the model was based on a detailed evaluation of predictions from previously published models and additional preliminary analyses based on gas uptake inhalation data in mice. The parameters of the updated model were calibrated using Bayesian methods with an expanded pharmacokinetic database consisting of oral, inhalation, and iv studies of TCE administration as well as studies of TCE metabolites in mice. The dose-response relationships for hepatomegaly derived from the multi-study database showed that the proportionality of dose to response for TCE- and DCA-induced hepatomegaly is not observed for administered doses of TCA in the studied range. The updated PBPK model was used to make a quantitative comparison of internal dose of metabolized and administered TCA. While the internal dose of TCA predicted by modeling of TCE exposure (i.e., mg TCA/kg-d) showed a linear relationship with hepatomegaly, the slope of the relationship was much greater than that for directly administered TCA. Thus, the degree of hepatomegaly induced per unit of TCA produced through TCE oxidation is greater than that expected per unit of TCA administered directly, which is inconsistent with the hypothesis that TCA alone accounts for TCE-induced hepatomegaly. In addition, TCE-induced hepatomegaly showed a much more consistent relationship with PBPK model predictions of total oxidative metabolism than with predictions of TCE area-under-the-curve in blood, consistent with toxicity being induced by oxidative metabolites rather than the parent compound. Therefore, these results strongly suggest that oxidative metabolites in addition to TCA are necessary contributors to TCE-induced liver weight changes in mice.     

Amino acid and protein targets of 1,2-diacetylbenzene,a potent aromatic gamma-diketone that induces proximal neurofilamentous axonopathy

Determining the key events in the induction of liver cancer in mice by trichloroethylene (TRI) is important in the determination of how risks from this chemical should be treated at low doses. At least two metabolites can contribute to liver cancer in mice, dichloroacetate (DCA) and trichloroacetate (TCA). TCA is produced from metabolism of TRI at systemic concentrations that can clearly contribute to this response. As a peroxisome proliferator and a species-specific carcinogen, TCA may not be important in the induction of liver cancer in humans at the low doses of TRI encountered in the environment. Because DCA is metabolized much more rapidly than TCA, it has not been possible to directly determine whether it is produced at carcinogenic levels. Unlike TCA, DCA is active as a carcinogen in both mice and rats. Its low-dose effects are not associated with peroxisome proliferation. The present study examines whether biomarkers for DCA and TCA can be used to determine if the liver tumor response to TRI seen in mice is completely attributable to TCA or if other metabolites, such as DCA, are involved. Previous work had shown that DCA produces tumors in mice that display a diffuse immunoreactivity to a c-Jun antibody (Santa Cruz Biotechnology, SC-45), whereas TCA-induced tumors do not stain with this antibody. In the present study, we compared the c-Jun phenotype of tumors induced by DCA or TCA alone to those induced when they are given together in various combinations and to those induced by TRI given in an aqueous vehicle. When given in various combinations, DCA and TCA produced a few tumors that were c-Jun+, many that were c-Jun-, but a number with a mixed phenotype that increased with the relative dose of DCA. Sixteen TRI-induced tumors were c-Jun+, 13 were c-Jun-, and 9 had a mixed phenotype. Mutations of the H-ras protooncogene were also examined in DCA-, TCA-, and TRI-induced tumors. The mutation frequency detected in tumors induced by TCA was significantly different from that observed in TRI-induced tumors (0.44 vs 0.21, p < 0.05), whereas that observed in DCA-induced tumors (0.33) was intermediate between values obtained with TCA and TRI, but not significantly different from TRI. No significant differences were found in the mutation spectra of tumors produced by the three compounds. The presence of mutations in H-ras codon 61 appeared to be a late event, but ras-dependent signaling pathways were activated in all tumors. These data are not consistent with the hypothesis that all liver tumors induced by TRI were produced by TCA.     

Disposition of 14C-oltipraz in animals. Pharmacokinetics in mice, rats and monkeys. Comparison of the biotransformation in the infected mouse and in the schistosomes

14C-labelled 4-methyl-5(2-pyrazinyl)-1,2-dithiole-3-thione (14C-oltipraz, 35 972 R.P.) was orally administered to rhesus monkeys (20 mg/kg), rats (50 mg/kg) and female mice infected with Schistosoma mansoni (100 and 250 mg/kg). The absorption of oltipraz varied with the animal species and the dose administered. In each species, the pharmacokinetics of oltipraz in the plasma and red blood cells were generally similar. 40 to 57% of the radioactive dose was excreted in urine, depending on the animal species and dose levels. In the mouse, there was negligible elimination of radioactivity as 14CO2. Whole-body autoradiographic studies in mice showed that, during the first 24 h, radioactivity was present mainly in the gastro-intestinal tract, bile, urine, liver and kidneys. In the male and female worms, the nature and amounts of radioactive products present differed.     

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.     

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.