Stabilization of cytochrome P4502E1 protein by ethanol in primary hamster hepatocyte cultures.

We report the effect of ethanol on monooxygenase activities in primary hamster hepatocyte cultures maintained on collagen-coated dishes. The addition of 50mM ethanol to cell cultures both from control and ethanol pretreated animals almost completely maintained, at least for 72hr, the P4502E1-dependent aniline hydroxylase (AnH) activity and the 2E1 immunodetectable apoprotein content at the levels of the corresponding 4-hr plated hepatocytes. On the contrary, other P450-dependent monooxygenase activities, as assayed by testosterone hydroxylases, kept decreasing falling-after 72hr of culture-to the levels of the 4-hr plated hepatocytes. In both cases, in the absence of ethanol, a rapid decline of AnH activities and 2E1 apoprotein contents were also observed, attaining undetectable levels at 72hr. The hybridizable 2E1 mRNA also rapidly declined in both cultures, but such decline was not significantly altered by the presence of 50mM ethanol in the culture medium. Furthermore, we show that P4502E1 in the liver possesses a rapid degradation phase with a half-life of about 6hr. Thus, in the hamster, P4502E1 appears regulated at post-translational level, as in rat, probably by a protein stabilization mechanism.

Induction of peroxisomal beta-oxidation and P-450 4A-dependent activities by pivalic and trichloroacetic acid in rat liver and kidney.

The influence of pivalic acid (PIV), a compound often used to make pro-drugs, and of the structurally related trichloroacetic acid (TCA), on several hepatic and renal enzymes was investigated in Sprague-Dawley rats, following a 4-day treatment period. The PIV and TCA treatments resulted in a similar and selective induction (2-3 times) of peroxisomal palmitoyl-CoA oxidase and the cytochrome P-450 4A dependent microsomal (omega)- and (omega-1)-lauric acid activities, both in liver and kidney. Western blot analysis of liver and kidney microsomes from PIV- and TCA-treated rats, using antibody to the P-450 4A1, revealed induction of members of the P-450 4A subfamily. These results suggest that PIV, like TCA, is a renal and hepatic peroxisome proliferator in rats, and further support the previously indicated close association between the peroxisomal fatty acid beta-oxidation enzymes and microsomal P-450 4A sub-family enzymes.

Synthesis and toxicity to mammalian cells of the carrot dihydroisocoumarins.

The dihydroisocoumarins (+-)-6-methoxy-8-hydroxy-3-methyl-3,4- dihydroisocoumarin (1), (+-)-6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin (2), and (+-)-6,8-dimethoxy-3-methyl-3,4-dihydroisocoumarin (3) were synthesized with high yields via metalation of o-methylbenzamides. The toxicity of these compounds and that of (-)-1 extracted from carrot cells were tested, in vitro, on Chinese hamster cells. The toxicity was determined according to the presence or absence of a hydroxyl group in the peri position of the lactonic carbonyl group and according to the stereochemistry of the dihydroisocoumarin.