Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes.

Mechanisms of chloroform (CHCl3) and carbon tetrachloride (CCl4) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl3 and CCl4 was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl3 (5 mM) and CCl4 (2.5 mM) used and with the longest duration of treatment (20 hr). CCl4 was approximately 16 times more toxic than CHCl3 to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25 microM) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl3 and CCl4 toxicity. The toxicity of CHCl3 and CCl4 could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25 microM), alpha-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl3 and CCl4 are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl3 and CCl4; and free radicals may be important mediators of the toxicity of these two halomethanes.


Introduction
Chloroform (CHC13) and carbon tetrachloride (CC14) are formed as by-products of sewage and drinking water chlorination and have been identified in public drinking water (1). Both compounds are toxic to mammalian liver in vivo and to isolated liver cells, with CC14 being more toxic than CHC13 (2). CC14 is hepatocarcinogenic in both rats and mice (3), whereas CHC13 has been shown to be hepatocarcinogenic only in the B6C3F1 strain of mouse when administered by corn oil gavage (4). Both compounds may be functioning as liver tumor promoters (5).
The metabolism of CC14 and CHC13 may provide clues to their relative cytotoxic potency and their capability as tumor promoters. It is becoming evident that free radicals generated during xenobiotic metabolism play a role in cellular toxicity (6), abnormal growth control (7), and tumor promotion (8) CHC13 has been extensively studied in both rats and mice. The first step in CC14 metabolism is a one-electron reduction and homolytic cleavage catalyzed by cytochrome P450 of the mixed function oxidase system (MFOS) to yield the trichloromethyl radical (9) [Eq. (1)].
CHCL3-[CCl3O0H]---COCl2 + HCl (3) Phosgene can covalently bind to macromolecules if not detoxified by conjugation with glutathione (GSH), or can be further metabolized to carbon dioxide (CO2) by reaction with water (11). It has also been suggested that free radicals are generated during CHC13 metabolism and that they play a role in CHC13 toxicity (12,13). Most studies, however, have failed to detect enhanced levels of free radicals or of lipid peroxidation, although no study has used the B6C3F1 strain of mouse.
To provide evidence that the hepatotoxicity of CHC13 and CC14 in B6C3F1 mice might be caused by free radical production during the metabolism of these compounds, the present study was undertaken. We examined whether the modifiers of hepatocyte xenobiotic metabolism, SKF-525A and diethyl maleate (DEM), and the antioxidants, ot-tocopherol acetate (vitamin E), N,N'-diphenyl-p-phenylenediamine (DPPD), and superoxide dismutase (SOD), could alter the toxicity of CHC13 and CC14 to isolated B6C3F1 mouse hepatocytes. SKF-525A is an inhibitor of the MFOS, DEM decreases intracellular GSH levels, Vitamin E and DPPD are freeradical scavengers, and SOD dismutates superoxide radicals to hydrogen peroxide.

Materials and Methods
Animals Six-month-old male B6C3F1 mice were used in this study. They were bred at the Medical College of Ohio from C3H/He sires and C57BI/6 dams (Charles River Laboratories, Inc., Wilmington, MA). Mice were kept in plastic cages (five per cage) containing corncob bedding and given water and certified Purina Lab Chow ad libitum.

Hepatocyte Isolation and Culture
Mouse hepatocytes were isolated by two-stage in situ perfusion (14). Cell viability of the isolated cells was 90%-95%, as determined by exclusion of trypan blue. Hepatocytes were cultured in L-15 medium supplemented with glucose (1 mg/mL), dexamethasone (1 ,uM), fetal bovine serum (10%), and gentamicin (50 ,ug/mL). The hepatocytes were plated at a density of 0.5 x 106 cells per 60 mm dish and incubated in a humidified 100% air incubator at 37°C.

Cytotoxicity Assay
After initial plating and a 4-hr attachment period, the cultured cells were washed once and re-fed with 5 mL of fresh medium. The cultures were then treated with the test compounds. All compounds, except SOD, were dissolved in DMSO. The final concentration of DMSO in all cultures, except for untreated cultures, was 0.4%. After 2, 4, and 20 hr of treatment, aliquots of the medium were removed, filtered through Nitrex nylon mesh to remove cells, and analyzed for lactate dehydrogenase activity (LDH) on a Beckman Multistat Analyzer (Beckman Instrument Corp., Palo Alto, CA). Total LDH per culture was also determined from 0.01% Triton X-100 lysates of untreated cultures, mean of three dishes.    LDH in unused, fresh culture medium was also determined to indicate initial LDG levels in the cultures. Cytotoxicity in a culture was expressed as the "percent total LDH released" by using the formula (15)(16)(17): %LDH LDH in medium of treated culture -LDH in unused medium mean total LDH -LDH in untreated medium Values for the percent total LDH released were determined in three replicate cultures for each treatment and sampling time, and the means ± S.D. were determined. Different treatments were compared statistically using Student's t-test.

Results
The toxicity of CHC13 to isolated B6C3F1 mouse hepatocytes was dependent on dose and treatment duration (Fig. 1). Little increase in LDH leakage was evident in cultures treated with less than 1.0 mM CHC13. CC14 toxicity was also dependent on dose and treatment duration (Fig. 2). As with CHC13, little difference in LDH release was evident between the 2-hr and 4-hr sampling times, and LDH release was greatest after 20 hr of treatment. CC14 was approximately 16 times more toxic than CHC13 to the hepatocytes. Percent total LDH activities of 50% occurred with approximately 0.25 mM CC14 and 4.0 mM CHC13 at the 20-hr sampling time ( Figs. 1 and 2).
The addition of SKF-525A (25 ,uM) to CHC13or CC14treated cultures could be shown to significantly reduce LDH release below that of cultures treated only with CHC13 or CC14 (Tables 1 and 2). This effect was evident only at the 2-hr and 4-hr sampling times. At 20 hr, SKF-525A was cytotoxic by itself and either increased or had no effect on LDH release in CHC13-and CC14-treated cultures. Adding DEM (0.25 mM) to CHC13or CC14treated cultures significantly increased LDH release above CHC13or CCl4-only treated cultures (Tables 1   and 2). This effect was evident at 2, 4, and 20 hr. DEM was not toxic by itself.
The antioxidants, DPPD (25 ,uM), vitamin E (0.1 mM), and SOD (100 U/mL), when added to CHC13and CC14-treated cultures, significantly reduced hepatocyte toxicity below that of cultures treated with CHC13 or CC14 alone (Tables 3 and 4). With the high dose of CHC13 (5 mM), vitamin E best prevented cytotoxicity compared to SOD and DPPD (Table 3). No differences in antioxidant protection were evident with 2.5 mM CHC13. DPPD provided the greatest protection against low-dose CHC13 (1 mM) toxicity. Cytotoxicity of the low CC14 dose (1.0 mM) was best prevented by DPPD, whereas no differences between the antioxidants were apparent with the high CC14 dose (2.5 mM) ( Table 4).

Discussion
The results ofthis study indicate that CHC13 and CC14 are cytotoxic to primary cultured B6C3F1 mouse hepatocytes. Both compounds exhibited doseand timedependent effects. CC14 was approximately 16 times more toxic than CHC13. Previous studies have also indicated that CC14, is more hepatotoxic than CHC13 (2, 3).
CHC13and CCl4-induced cytotoxicity was decreased by the simultaneous treatment of SKF-525A, an inhibitor of mammalian MFOS. These results substantiate previous studies that indicated both CHC13 and CC14 are metabolized in hepatocytes by the MFOS to hepatotoxic metabolites (9,11). In the case of CC14, the initial reaction is a reduction and homolytic cleavage of CC14 to the trichloromethyl radical (KCC13). This radical may react directly with cellular macromolecules or may react with oxygen to form the trichloromethylperoxyl radical ( OOCC13), which may then attack lipids more readily than 'CC13 (10). In any event, the initiation of free radicals and attack on membrane lipids ultimately can lead to lipid peroxidation chain reactions and cell death (10).
CHC13 is thought to be first hydroxylated by a P-450 reaction to trichloromethanol (CC130H), which dehy-drochlorinates spontaneously to phosgene (COC12) (11). Phosgene is thought to covalently bind cellular macromolecules and to be the ultimate cytotoxic component (11).
However, two reports (12,13) have suggested that CHC13 is metabolized to free radicals and that it induces lipid peroxidation.
The results of the present study suggest that free radical production is an important mechanism of both CC14 and CHC13 toxicity in B6C3F1 mouse hepatocytes. The antioxidants SOD, DPPD, and vitamin E all significantly reduced the toxicity of CC14 and CHC13 to these cells. Similarly, depletion of cellular glutathione (GSH) (with DEM), increased CC14 and CHC13 toxicity to the hepatocytes. GSH functions both as an antioxidant and in the conjugation of xenobiotic metabolites (18).
Thus, the results of this initial study indicate that both CC14 and CHC13 are metabolized in B6C3F1 mouse hepatocytes by the MFOS, that cellular GSH is important in the detoxification of CC14 and CHC13 metabolites and/or induced free radicals, and that the cytotoxicity of both compounds might be partly mediated by free radical production. Previous studies have demonstrated the role of free radicals and lipid peroxidation in CC14mediated hepatotoxicity. However, evidence that CHC13-induced hepatotoxicity is mediated by free radicals has been less substantiated. The results of the present study provide additional evidence that, in mouse hepatocytes, free radical production may be an important mechanism of CHC13-induced toxicity.