J F Sinclair

Ethanol-mediated increase in cytochrome P-450 in cultured hepatocytes.

Abstract Cellular levels of cytochrome P-450 were increased on exposure of cultured chick embryo hepatocytes to ethanol. This increase correlated with increases in the enzymatic activities of both benzphetamine demethylase [associated with the species of P-450 induced by propylisoproplyacetamide (PIA) or phenobarbital] and ethoxyresorufin deethylase [associated with the P-448 species induced by β-naphthoflavone (β-NF)]. The increased enzymatic activity relative to P-450 more closely resembled that caused by PIA than by β-NF. Glucuronidation of phenol red was also increased by ethanol; the dose response resembled that for increases in cytochrome P-450. PIA induced the glucuronidation of phenol red, whereas β-NF did not. Therefore, ethanol exposure was found to increase cytochrome P-450 and glucuronyltransferase in these cells in a pattern similar to increases observed with PIA.

Alcohol-mediated increases in acetaminophen hepatotoxicity: role of CYP2E and CYP3A.

This commentary focuses on the roles of CYP3A and CYP2E in alcohol-mediated increases in acetaminophen hepatotoxicity. CYP2E has been considered to be the main form of P450 responsible for such toxicity in animals and humans. However, CYP3A, which is also induced by alcohol, has been shown to have a greater affinity for acetaminophen than CYP2E. Previous experiments implicating CYP2E in alcohol-mediated increases in acetaminophen hepatotoxicity have used inhibitors of this form of P450 that are now proving to be non-specific. Triacetyloleandomycin (TAO) is a potent inhibitor of CYP3A that maintains specificity in vitro over a large concentration range. In rats treated with ethanol or the combination of ethanol and isopentanol, the major higher chain alcohol in alcoholic beverages, TAO protects animals from increases in acetaminophen hepatotoxicity, suggesting a major role of CYP3A. CYP2E may not have a major role due to the rapid loss of induced levels in the absence of continued exposure to ethanol. Knockout mice, which are being used to define the role of particular proteins in biological responses, have been developed for CYP2E1 and CYP1A2 but not CYP3A. Cyp2e1(-/-) and Cyp1a2(-/-) mice are more resistant to acetaminophen hepatotoxicity than wild-type strains, even though the amounts of the other forms of P450s are unaltered in the liver. These findings suggest that the relative amounts of P450s and not just kinetic characteristics determine their role in acetaminophen hepatotoxicity. The clinical implications of the findings that CYP3A can have a major role in acetaminophen-mediated hepatotoxicity are discussed.

Increases in cytochrome P-450 in cultured hepatocytes mediated by 3- and 4-carbon alcohols

The amount of cytochrome P-450 was increased to different extents after treatment of cultured chick embryo hepatocytes with n-propanol, isopropanol, n-butanol, or isobutanol. These increases were associated with increases in benzphetamine demethylase activity, a cytochrome P-450-catalyzed oxidation, and glucuronidation of phenol red, catalyzed by UDP-glucuronyl transferase. The responses were similar to those obtained with ethanol or propylisopropylacetamide, which the phenobarbital-like inducers. Pretreatment of cells with cycloheximide prevented the increases in both cytochrome P-450 and glucuronidation of phenol red, indicating that protein synthesis was required for these responses.

Improved retention of heme with increased resolution of microsomal proteins in polyacrylamide gel electrophoresis

Abstract Polyacrylamide gel electrophoresis, using lithium dodecyl sulfate instead of the sodium salt, was used to analyze rat hepatic microsomal hemoproteins. Good resolution of hepatic microsomal proteins was obtained with retention of approximately half of the total microsomal heme on the proteins with molecular weights of 45,000 to 50,000. Both the protein resolution and heme retention are better than with electrophoresis procedures previously described. Treatment of rats with chemicals that either increase or decrease microsomal cytochrome P-450 produced proportional changes in the heme associated with the proteins of 45,000 to 50,000 molecular weights on the gel.

Toxicity of paracetamol in cultured chick hepatocytes treated with methotrexate

Cultured chick hepatocytes were used to investigate the hepatotoxicity of methotrexate alone and in combination with paracetamol. Treatment with methotrexate alone at concentrations as high as 1 mg/ml resulted in no toxicity in cultured chick hepatocytes, as indicated by no detachment of cells and no effect on protein synthesis or on release of the intracellular enzyme lactate dehydrogenase. However, treatment with methotrexate alone resulted in a 30% decrease in reduced glutathione levels. Combined treatment with methotrexate and paracetamol was toxic, but only in cells preinduced for cytochrome P450 1A by treatment with beta-naphthoflavone. Under these conditions, methotrexate lowered the threshold concentration of paracetamol at which toxicity was observed. This methotrexate-mediated increase in paracetamol toxicity was associated with decreased formation of the glucuronide, sulfate and thiol metabolites of paracetamol and with increased covalent binding of radiolabeled paracetamol to macromolecules. In cells pretreated with beta-naphthoflavone, additional treatment with either methotrexate or buthionine sulfoximine, an inhibitor of glutathione synthesis, together with paracetamol, was associated with decreased restoration of glutathione levels. These results suggest that methotrexate increased paracetamol toxicity by decreasing the amount of glutathione available for conjugation with reactive metabolites of paracetamol.

Metabolism of the liver tumor promoter ethinyl estradiol by primary cultures of rat hepatocytes

Previously, we reported that relatively high micromolar concentrations of the liver tumor promoter 17 alpha-ethinyl estradiol (EE2) stimulated DNA synthesis and enhanced the DNA synthetic response to epidermal growth factor (EGF) in primary cultures of female rat hepatocytes [J.D. Yager, B.D Roebuck, T.L. Paluszcyk, and V.A. Memoli, Carcinogenesis 7, 2007-2014 (1986); Y.E. Shi and J.D. Yager, Cancer Res. 49, 3574-3580 (1989)]. In this study, our goal was to examine the metabolism of EE2 in cultured hepatocytes. After 4, 24, and 48 hr of culture, hepatocytes maintained their ability to convert up to 95% of a 4 nM concentration of [3H]EE2 to polar conjugates within 4 hr. EE2 at 2 microM was also 95% metabolized within 4 hr. HPLC analysis of the metabolites confirmed the rapid disappearance of [3H]EE2 and the formation of polar conjugates as detected by organic extraction. HPLC separation of hydrolyzed conjugates indicated that the major aglycone was the parent compound, EE2. In general, the metabolites differed both qualitatively and quantitatively from those reported in vivo in the rat. The rapid metabolism of EE2 by hepatocytes in culture may, at least in part, explain the high concentrations of EE2 required to stimulate DNA synthesis in cultured hepatocytes and to potentiate the response to EGF.