Pharmacology & Toxicology 1992, 70, 453458.

Evidence for Cytochrome P450 2El-Mediated Toxicity of N-Nitrosodimethylamine in Cultured Perivenous Hepatocytes from Ethanol Treated Rats Irene Anundi and Kai 0. Lindros* Biomedical Research Center, ALKO Ltd., Box 350, SF-00101 Helsinki, Finland (Received October 22, 1991; Accepted February 20, 1992) Abstract: The involvement of cytochrome P450 in the liver toxicity of the potent carcinogen, N-nitrosodimethylamine (NDMA) was investigated in hepatocytes isolated from the periportal or perivenous region by digitonin-collagenase perfusion. Exposure of hepatocytes in culture to NDMA (0.5 or 5 mM) for up to 18 hrs caused little damage, but after 42 hr loss of cell viability became evident, and the extent of cell death was higher in perivenous cells than in periportal cells. Pretreatment of rats with ethanol caused a dramatically enhanced cell damage in perivenous cells (80%) compared to periportal cells (45%). This ethanol pretreatment caused a several-fold induction of cytochrome P450 2E1, as determined both with Western blot and as NDMA demethylase activity, and the effect was observed almost exclusively in perivenous cells. Isoniazid, an inhibitor of cytochrome P450 2E1, completely protected against NDMA toxicity. Glutathione dependent cytoprotective mechanisms and lipid peroxidation did not appear to be critical in NDMA toxicity, as evidence by lack of potentiation of toxicity by buthionine sulfoximine, an inhibitor of glutathione synthesis, and by the absence of increased lipid peroxidation. Instead, the higher expression of cytochrome P450 2E1 in the perivenous cells seems to be the main determinant for the regiospecific toxicity of NDMA, and, consequently, probably also for the associated genotoxicity.

N-Nitrosodimethylamine (NDMA) is a potent carcinogen belonging to the nitrosamines. Its hepatotoxic and genotoxic effects are dependent on metabolic activation (Lai et al. 1979; Lai & Arcos 1980) but the detailed mechanisms of this process are not known. The key step is believed to be a hydroxylation of the a-carbon atom to an unstable intermediate, a-hydroxymethylnitrosamine, which decomposes to formaldehyde and an electrophilic alkylating agent (Lai & Arcos 1980; Preussmann & Stewart 1984). The activation reaction of NDMA is catalyzed by at least two cytochrome P450 forms of microsomal NDMA demethylase (NDMAd), a high and a low Km form (Hong & Yang 1985; Lai & Arcos 1980; Thomas et al. 1987), but probably only the low Km form is of importance for the cytotoxic and genotoxic effects (Lai & Arcos 1980; Thomas et al. 1987). Compounds such as acetone and ethanol induce NDMAd in rats in vivo (Peng et al. 1982) and in experiments with reconstituted systems where the catalytic activity of several different cytochrome P450 isozymes was compared, the P450 2E1 form was found to be the isozyme with the highest demethylase activity (Levin et al. 1986). Thus, although indirect evidence suggests a role for cytochrome P450 2E1 in the toxicity of NDMA, the relationship has not been clearly established. Zonation of various enzyme systems and metabolic processes in the liver acinus is well-known, but the functional importance and mechanisms underlying this heterogeneity are still poorly understood. Although many cytochrome P450 isozymes seem to have a higher expression in the perivenous region (Gooding et al. 1987; Buhler et

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To whom correspondence should be addressed.

al. 1992) this heterogeneity appears to be particularly pronounced for cytochrome P450 2E1 (Ingelman-Sundberg et al. 1988; Tsutsumi et al. 1989). Furthermore, induction of P450 2E1 by chronic ethanol-treatment seems to occur predominantly in the pv region (Ingelman-Sundberg et al. 1988) and the resultant enhanced expression of cytochrome P450 2E1 in these cells was recently shown to be associated with an increased vulnerability to carbon tetrachloride in vitro toxicity (Lindros et al. 1990). The aim of the present study was to directly investigate how NDMA cytotoxicity is related to changes in P450 2E1 levels. Because NDMA has been shown to cause centrilobular necrosis in vivo and P450 2E1 is thought to be involved in its metabolism, NDMA toxicity was studied in hepatocytes with different levels of P450 2E1, i.e. periportal and perivenous hepatocytes, isolated by the digitonin-collagenase perfusion technique (Lindros & Penttila 1985; Quistorff 1985). Similar to several other hepatotoxic carcinogens (e.g. aflatoxin B1) (Hayes et al. 1986), the toxic effect of NDMA was delayed. Furthermore, perivenous cells were indeed found to be more sensitive towards toxic effects of NDMA during prolonged exposure of cells in primary culture. Importantly, chronic administration of ethanol to rats potentiated cell death selectively in perivenous hepatocytes, suggesting a central role of cytochrome P450 2E1 in NDMA toxicity.

Materials and Methods Chemicals. Collagenase and NDMA were obtained from Sigma Chem. Co. (St. Louis, MO, U.S.A.). All other chemicals were of reagent grade and purchased from commercial sources.

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Animals. Male Wistar rats (150-180 g), given food (standard R3 rat food, Ewos AB, Sodertalje, Sweden) and water ad libitum, were used. Ethanol was continually provided to some animals for 2 weeks by incremental additions (from 3 to 12%) to the tap water as described before (Lindros et al. 1984). The mean daily ethanol intake for the whole period was 9.4 g/kg b.wt. and weight gain was normal. Isolation of hepatocytes and cell culture. Hepatocytes from the periportal or perivenous region of the liver lobule were isolated by digitonin-collagenase perfusion (Lindros & Penttila 1985; Quistorff 1985). Preparations with more than 90% of the cells initially excluding eosin were used. Cells were plated at a density of 6 x 104 cells/ cm2 on 3.5 cm 0 Nunc cell culture dishes in Krebs-Ringer buffer supplemented with foetal calf serum (5%), newborn calf serum (5%), insulin (80 ng/ml), glucagon (1.8 ng/ml), dexamethasone (0.1 pM), gentamicin (50 pg/ml), and nystatin (1 pg/ml). After 4 hr of incubation unattached cells were removed by aspirating the medium and new serum-free medium including NDMA was added. Nitrosodimethylamine was added dissolved in distilled water. The cultures were then further incubated in a humidified atmosphere of 95% air and 5% C 0 2 for 18 or 42 h at 37".

exposure to NDMA. Furthermore, toxic effects were only slightly enhanced when the concentration of NDMA was increased from 0.5 to 5 mM. After hepatocytes isolated from periportal and perivenous regions of the liver of control rats had been maintained in culture for up to 42 hr, both cell types were characterized by a normal morphology, low leakage of cytosolic LDH (20 2%) and high exclusion of eosin (about 80%) (fig. 1). Similar results were obtained using cells from ethanolpretreated rats. These rats were administered ethanol for 2 weeks by addition to the drinking water, a rather mild ethanol treatment, which was deliberately chosen so as to avoid possible unspecific effects, such as an increased initial vulnerability of cells. As previously reported (Ingelman-

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Assays. Viability was assessed by counting cells excluding eosin (0.05%) and by measuring leakage of cytosolic lactate dehydrogenase. To verify the origin of the cells, glutamine synthetase and alanine aminotransferase were assayed in periportal and perivenous cells as previously described (Lindros & Penttila 1985). The amount of P450 2E1 apoprotein in periportal and perivenous cells was analyzed by Western blot (Ingelman-Sundberg et al. 1988), using antirat P450 2E1 IgG raised in rabbits and which was a kind gift from Dr. M. Ingelman-Sundberg, Stockholm, Sweden. In some experiments microsomes were isolated from preparations of periportal and perivenous cells and rates of NDMA demethylation assayed colorimetrically as formaldehyde produced as described in (Nash 1953; Shertzer et al. 1987). Thiobarbituric acid reactive species was determined fluorometrically as in (Lindros et al. 1990). Intracellular reduced glutathione (GSH) was assayed by a fluorometric method according to Hissin & Hilf (1 976).

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The different acinar origin of the two cell populations was verified by measuring the activities of marker enzymes with known heterogeneous distribution. The mean activity ratios between periportal and perivenous preparations were 1.7 for alanine aminotransferase (periportal marker) and 0.025 for glutamine synthetase (perivenous marker). These numbers are comparable to previous reports (Ingelman-Sundberg et al. 1988; Lindros et al. 1990) and indicate a good separation of periportal and perivenous cells. In preliminary experiments with suspensions of freshly isolated periportal and perivenous hepatocytes incubated for 3 to 4 hr in the presence of 5 mM NDMA, no evidence of diminished cell viability was observed, as judged from lack of leakage of cytosolic LDH and exclusion of eosin. In subsequent experiments with cells maintained in primary culture it was observed that toxic effects were not normally apparent until after a characteristic lag period of 18 hr of

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Fig. 1. NDMA induced cell damage in periportal and perivenous cells isolated from control and ethanol-treated rats. Periportal and perivenous hepatocytes in primary culture were exposed to NDMA (0.5 and 5 mM) for 42 hr. Cell damage was assessed by leakage of cytosolic LDH (continuous lines) and exclusion of 0.05% eosin (dotted lines). Cells were isolated from control rats (A) or from rats chronically treated with ethanol (B). Values are the meansfS.D. of 3-8 different cell preparations. Statistical significance was determined with Student's t-test. *=P

Evidence for cytochrome P450 2E1-mediated toxicity of N-nitrosodimethylamine in cultured perivenous hepatocytes from ethanol treated rats.

The involvement of cytochrome P450 in the liver toxicity of the potent carcinogen, N-nitrosodimethylamine (NDMA) was investigated in hepatocytes isola...
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