Metabolism of chloroform by cytochrome P450 2E1 is required for induction of toxicity in the liver, kidney, and nose of male mice.

Chloroform is a nongenotoxic-cytotoxic liver and kidney carcinogen and nasal toxicant in some strains and sexes of rodents. Substantial evidence indicates that tumor induction is secondary to events associated with cytolethality and regenerative cell proliferation. Therefore, pathways leading to toxicity, such as metabolic activation, become critical information in mechanism-based risk assessments. The purpose of this study was to determine the degree to which chloroform-induced cytotoxicity is dependent on the cytochromes P450 in general and P450 2E1 in particular. Male B6C3F(1), Sv/129 wild-type (Cyp2e1+/+), and Sv/129 CYP2E1 knockout (Cyp2e1-/- or Cyp2e1-null) mice were exposed 6 h/day for 4 consecutive days to 90 ppm chloroform by inhalation. Parallel control and treated groups, excluding Cyp2e1-null mice, also received an i.p. injection (150 mg/kg) of the irreversible cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) twice on the day before exposures began and 1 h before every exposure. Cells in S-phase were labeled by infusion of BrdU via an implanted osmotic pump for 3.5 days prior to necropsy, and the labeling index was quantified immunohistochemically. B6C3F(1) and Sv/129 wild-type mice exposed to chloroform alone had extensive hepatic and renal necrosis with significant regenerative cell proliferation. These animals had minimal toxicity in the nasal turbinates with focal periosteal cell proliferation. Administration of ABT completely protected against the hepatic, renal, and nasal toxic effects of chloroform. Induced pathological changes and regenerative cell proliferation were absent in these target sites in Cyp2e1-/- mice exposed to 90 ppm chloroform. These findings indicate that metabolism is obligatory for the development of chloroform-induced hepatic, renal, and nasal toxicity and that cytochrome P450 2E1 appears to be the only enzyme responsible for this cytotoxic-related metabolic conversion under these exposure conditions.

Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice.

The weight of evidence indicates that chloroform induces cancer in the female B6C3F1 mouse liver via a nongenotoxic-cytotoxic mode of action. However, it is probable that DNA damage occurs secondary to events associated with cytolethality and regenerative cell proliferation. The purpose of the present study was to evaluate the potential mutagenic activity of chloroform in the B6C3F1 lacI transgenic mouse liver mutagenesis assay including mutagenic events that might occur secondary to cytolethality. The positive control, dimethylnitrosamine (DMN) is a DNA-reactive mutagen and carcinogen. DMN-induced mutations were anticipated to require only a brief exposure and without further treatment were predicted to remain unchanged over time at those frequencies. Chloroform-induced mutations secondary to toxicity were anticipated to require longer exposure periods and to occur only under conditions that produced sustained cytolethality and regenerative cell proliferation. Female B6C3F1 lacI transgenic mice were treated with daily doses of 2, 4, or 8 mg/kg of DMN by gavage for 4 days and then held until analysis 10, 30, 90, and 180 days postexposure. Livers from DMN-treated mice exhibited a dose-related 2- to 5-fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. Female B6C3F1 lacI mice were exposed daily for 6 hr/day 7 days/week to 0, 10, 30, or 90 ppm chloroform by inhalation, representing nonhepatotoxic, borderline, or overtly hepatotoxic chloroform exposures. Timepoints for determination of lacI mutant frequency were 10, 30, 90, and 180 days of exposure. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondary to toxicity either did not occur or were of a type not detectable by lacI mutant frequency analysis, such as large deletions.

Differential display identified changes in mRNA levels in regenerating livers from chloroform-treated mice.

The nongenotoxic-cytotoxic carcinogen chloroform induces liver necrosis, regenerative cell proliferation, and, eventually, liver tumors in female B6C3F1 mice when administered by gavage at doses of 238 or 477 mg/kg/d. Administration of 1800 ppm of chloroform in the drinking water results in similar daily doses but does not produce liver toxicity or cancer. The differential-display technique was used to compare the expression of a subset of mRNAs in normal (control) and regenerating liver after chloroform-induced toxicity to define the proportion of genes whose expression changes under hepatotoxic conditions and to identify the genes that might play a role in regeneration and perhaps cancer. RNA was purified from the livers of female B6C3F1 mice after 4 d or 3 wk of gavage treatment with 3, 238, or 477 mg/kg/d of chloroform or treatment with 1800 ppm chloroform in drinking water. There was a remarkably high degree of consistency of gene expression among the animals and across dose and treatment groups as visualized by the differential-display technique. Of the 387 bands observed, only four (about 1%) changed expression in regenerating liver. The genes were assigned locus names by GenBank after sequence submission. The genes with increased mRNA levels as confirmed by northern blot analysis were MUSTIS21, a mouse primary response gene induced by growth factors and tumor promoters; MUSMRNAH, a gene highly homologous to a human gene isolated from a prostate carcinoma cell line; and MUSFRA, a novel gene. The novel gene MUSFRB exhibited decreased mRNA levels. No change in expression was seen among control mice given the nontoxic regimens of 3 mg/kg/d chloroform or 1800 ppm chloroform in drinking water, indicating that changes in expression were associated with toxicity and regeneration rather than chloroform per se. These genes and others that may be identified by expanding this approach may play a role in regeneration and perhaps in the process of chloroform-induced carcinogenesis in rodent liver.

Chloroform-induced cytotoxicity and regenerative cell proliferation in the kidneys and liver of BDF1 mice.

In a 2-year chloroform inhalation bioassay, an increased incidence of tumors was observed in the kidneys of male BDF1 mice and the liver of female BDF1 mice exposed to the highest exposure concentration of 90 ppm. To investigate the role of cytotoxicity and regenerative cell proliferation in tumor formation, male and female BDF1 mice were exposed to chloroform vapor concentrations of 0, 0.3, 5, 30, or 90 ppm 6 h/day for 4 days. Bromodeoxyuridine (BrdU) was administered via osmotic pumps implanted 3.5 days prior to necropsy, and the labeling index (LI), or percentage of cells in S-phase, was quantified using BrdU immunohistochemistry. To assess longer-term responses, additional male mice were exposed 5 days/week for 2 weeks to 0, 30, or 90 ppm. Degenerative lesions and an increase in the LI of seven- to ten-fold over controls were observed in the kidneys of male but not female mice exposed to 30 or 90 ppm. Liver lesions and increased hepatocyte LI were observed in male mice exposed to 30 or 90 ppm and in female mice exposed to 90 ppm. In the 2-week exposure groups 40% of the 30 ppm group and 80% of the 90 ppm group died with severe kidney damage, indicating that both 30 and 90 ppm exceed a maximum tolerated dose. Thus, in the 2-year bioassay chloroform concentrations had to be stepped-up over a period of weeks in order for the male mice exposed to 30 or 90 ppm to survive. The extrapolation of tumor data from such an unusual procedure is questionable. These observations are consistent with a substantial database that indicates that tumor induction by chloroform occurs via a non-genotoxic-cytotoxic mode of action and is secondary to organ-specific toxicity. These data further support the premise that doses that do not induce regenerative cell proliferation do not present an increased risk of cancer.

Levels of myc, fos, Ha-ras, met and hepatocyte growth factor mRNA during regenerative cell proliferation in female mouse liver and male rat kidney after a cytotoxic dose of chloroform.

Chloroform is a liver carcinogen in mice and a kidney carcinogen in rats. It is thought to act through a non-genotoxic-cytotoxic mode of action. Changes in expression of growth control genes accompanying chloroform-induced cytolethality and regeneration may play a part in the development of chloroform-induced tumors. In this experiment, we examined the levels of the myc, fos, Ha-ras, met and hepatocyte growth factor mRNA in livers of female B6C3F(1) mice and kidneys of male F-344 rats to detect changes in gene expression following a single, cytotoxic gavage dose of chloroform in corn oil. Poly A+ RNA was purified from homogenates of livers of mice treated with 350mg/kg chloroform and kidneys of rats treated with 180 mg/kg chloroform and used for Northern blot analysis. Livers of female mice showed large transient increases in levels of myc and fos mRNA while levels of Ha-ras, met and the hepatocyte growth factor gene mRNA remained near control levels. In the male rat kidney, levels of myc mRNA increased after treatement, while levels of mRNA of all other genes examined remained near control levels. This pattern of gene expression is consistent with that induced by other cytotoxic carcinogens and suggest that alteration of the myc and fos genes could be involved in the regenerative cell proliferation that ultimately could play a role in chloroform-induced tumors.

Expression of myc, fos and Ha-ras associated with chemically induced cell proliferation in the rat liver.

Events secondary to induced cell proliferation may play a role in the carcinogenic process. These studies investigated the expression of genes associated with growth control in response to two types of cell proliferation stimuli in the livers of male F344 rats. Regenerative hepatocyte proliferation after partial hepatectomy or a single dose of carbon tetrachloride, and mitogenic liver hyperplasia induced by a single dose of phenobarbital or WY-14,643 were assessed by thymidine incorporation and quantitative autoradiography. The expression of myc, fos, and Ha-ras was evaluated by Northern blot analysis of liver derived poly(A)+ mRNA from these same animals. After each treatment, the level of hepatocyte proliferation (labelling index 4-32%) was observed to peak between 24 and 48 h and return to control values by 8 days. In every case, a peak in myc expression was seen between 0.5 and 18 h depending on the proliferative stimulus treatment. A large peak in fos expression was seen at 0.5-2 h but only with the cytotoxic and regenerative proliferative treatments partial hepatectomy or carbon tetrachloride. A broad peak in Ha-ras expression was observed 12 to 36 h after each treatment. These data demonstrate transient expression of these genes following the synchronous induction of hepatocyte proliferation. The increased expression of fos upon treatment with cytotoxicants, but not mitogens, suggests different modes of growth regulation that may be important in understanding the induction of cell proliferation by these two types of agents.

Lack of chloroform-induced DNA repair in vitro and in vivo in hepatocytes of female B6C3F1 mice.

Chloroform has been shown to induce hepatocellular carcinomas in female B6C3F1 mice when administered by gavage, but not when given in drinking water. When administered in corn oil at the carcinogenic doses of 238 and 477 mg/kg, chloroform induced necrosis and sustained regenerative cell proliferation in the liver. To investigate the mode of action of tumor induction in the target cells, the ability of chloroform to induce unscheduled DNA synthesis (UDS) was examined in the in vitro and in vivo hepatocyte DNA repair assays. In the in vitro assay, primary hepatocyte cultures from female B6C3F1 mice were incubated with concentrations from 0.01 to 10 mM chloroform in the presence of 3H-thymidine. UDS was assessed by quantitative autoradiography. No induction of DNA repair was observed at any concentration. In the in vivo assay, animals were treated by gavage with 238 and 477 mg/kg chloroform in corn oil. Primary hepatocyte cultures were prepared 2 and 12 hr later, incubated with 3H-thymidine, and assessed for induction of UDS as above. No DNA repair activity was seen at either dose or at either timepoint. These negative results in the target organ are consistent with the concept that neither chloroform nor its metabolites are directly DNA reactive and that the carcinogenicity of chloroform is secondary to induced cytolethality and regenerative cell proliferation.

Expression of myc, fos, and Ha-ras in the livers of furan-treated F344 rats and B6C3F1 mice.

Furan administered by gavage for 2 yr has been reported to induce hepatocellular carcinomas in male and female B6C3F1 mice and in male but not female F344 rats. Chronic exposure studies in our laboratory using bioassay conditions showed extensive hepatocellular toxicity and sustained increases in regenerative cell proliferation after 1, 3, and 6 wk of treatment in male and female rats and male mice. Altered expression of growth-control genes associated with this hyperproliferative state may enhance the susceptibility of these genes to mutation or may provide a selective growth advantage to preneoplastic cells. Quantitative northern blot analysis of mRNA was used to examine the expression of the oncogenes myc, fos, and Ha-ras in the livers of animals treated with furan. In male rats, a single administration of 30 mg/kg furan produced necrosis and a subsequent wave of cell proliferation 48 h after treatment and induced transient peaks in the expression of myc, fos, and Ha-ras 6-24 h after treatment. In male rat liver from our cell proliferation studies, only a slight increase in myc expression was seen at the end of week 1 of treatment. However, beginning at week 3 and increasing at week 6, up to a 15-fold increase over control values was observed in the expression of myc in the treated animals. The only other notable increase in expression observed in any animals from the cell proliferation study was a threefold increase in myc at week 6 in treated female rats. The absence of an increase in Ha-ras expression in the male mouse liver suggests that the unique pattern of Ha-ras mutations previously reported in furan-induced mouse liver tumors is not due to increased mutational susceptibility related to overexpression of this gene. The lack of sustained expression of myc, fos, and Ha-ras in rapidly proliferating liver suggests that continuous expression of these genes is not necessary to maintain increased rates of cell replication. The large increase in myc expression in male but not female rats suggests an adaptive change that may be related to the sex-specific incidence of furan-induced hepatocellular carcinomas in rats.

Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation.

Quantitative knowledge of gene expression can provide valuable information for understanding the action of chemicals that alter cell proliferation and cancer. Accurate quantification of mRNA levels requires the normalization of the gene of interest to a gene with transcriptional levels that do not vary through the cell cycle or with a particular treatment. Changes in expression were examined in proliferating or non-proliferating rat liver for three constitutively expressed 'housekeeping' genes commonly used to normalize mRNA levels from Northern blots. In addition, a direct method of quantifying poly(A)+ mRNA by hybridization with a radiolabelled polythymidylate--poly(T)--probe was compared with traditional methods. Hepatocyte cytolethality and a subsequent wave of hepatocyte proliferation were induced in male Fischer-344 rats by treatment with a single gavage dose of carbon tetrachloride. Induced cell proliferation peaked at 48 h after treatment. Expression of the housekeeping genes actin, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and albumin, as well as the proto-oncogene H-ras, was determined by Northern blot analysis at times from 0.5 h to 4 days after treatment. Time-dependent changes were observed in the expression of these genes relative to the levels observed in the untreated control animals. Actin expression peaked at 3.4-fold over control and GAPDH expression was increased by 1.9-fold over control. Albumin mRNA levels varied the least, 1.4-fold over control, indicating that this gene is more appropriate than actin or GAPDH for normalization of proto-oncogene expression under experimental conditions that induce cell proliferation in rat liver. The direct quantification of poly(A)+ mRNA using a poly(T) probe was not influenced by the induction of cell proliferation. This method may be useful when the expression of housekeeping genes is affected by treatment.

Tissue-specific genotoxic effects of acrylamide and acrylonitrile.

Acrylamide (AA) has been reported to induce dominant lethal mutations in male rat germ cells and tumors in a variety of organs, including the scrotum, thyroid and mammary glands, but not the liver of rats. The structurally similar vinyl monomer acrylonitrile (ACN) does not induce dominant lethal mutations but does induce tumors of the brain, Zymbal gland, forestomach and mammary gland, but not the liver of rats. Several in vitro and/or in vivo unscheduled DNA synthesis (UDS) assays were employed to examine the potential tissue-specific genotoxic activity of these compounds. Neither AA nor ACN induced DNA repair in either the in vitro or in vivo hepatocyte DNA repair assays. Glycidamide (GA), a mutagenic metabolite of AA, induced DNA repair in the in vitro hepatocyte DNA repair assay. Cyanoethylene oxide (CEO), a mutagenic metabolite of ACN, did not yield a DNA repair response in the in vitro hepatocyte DNA repair assay, but was highly toxic and could not be tested at doses equivalent to GA. AA, but not ACN, produced a DNA repair response in the in vivo spermatocyte DNA repair assay. AA produced a slight response in the in vitro human mammary epithelial cell (HMEC) DNA repair assay in normal cells derived from discarded surgical samples from five different women. GA produced a strong UDS response in all cases in the same assay. CEO, but not its parent compound ACN, produced a response in the HMEC DNA repair assay. These results show a highly tissue-specific pattern of genotoxic activity for AA and ACN that correlates, to the extent that it has been examined, with the tissue-specific pattern of carcinogenic and dominant lethal activity. The induction of DNA repair by GA and CEO confirms the genotoxic potential of these metabolites. While the observation of genotoxic activity of AA in the HMEC DNA repair assay suggests that mammary cells might be a target for carcinogenic activity of this compound in humans, other factors such as pharmacokinetics and epidemiology must be evaluated to establish that effect.