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Maintenance of total cytochrome P-450 content in rat hepatocyte culture and the abundance of CYP1A2 and CYP2B1/2 mRNAs.

ABSTRACT: mRNAs encoding cytochrome P-450s CYP1A2 and CYP2B1/2 have been quantified in rat hepatocytes cultured for periods up to 72 h under several different culture conditions that maintain total cytochrome P-450 content. When hepatocytes were cultured at either 37 or 30 degrees C in Williams E media, both CYP1A2 and CYP2B1/2 mRNAs declined dramatically. However, when cultured at 30 degrees C for 24 h, the decline in these mRNAs was not as great as that observed in cells grown at 37 degrees C. The addition of dimethyl sulphoxide to cells grown at 37 degrees C did not affect the rate of disappearance of the CYP1A2 or CYP2B1/2 mRNAs. These mRNAs also declined rapidly in cells grown in 'P-450 medium' i.e. RPMI 1640 medium without cyst(e)ine but supplemented with 0.1 mM-delta-aminolaevulinic acid. However, the levels of CYP2B1/2 mRNAs were maintained when hepatocytes were cultured in Williams E medium supplemented with 0.5 mM-metyrapone. These conditions did not, however, maintain the levels of CYP1A2 mRNA.

SUBMITTER: Padgham CR

PROVIDER: S-EPMC1132884 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Project description:The role of steroids related to the adrenal androgen dehydroepiandrosterone (5-androstene-3 beta-ol-17-one; DHEA) in regulating the expression of peroxisomal and cytochrome P-450 4A (CYP4A) enzymes active in fatty acid metabolism was assessed using a primary rat hepatocyte culture system. Exposure of hepatocytes to the peroxisome proliferator, clofibric acid (10-250 microM), for 48-96 h led to substantial increases in CYP4A protein, CYP4A1, CYP4A2 and CYP4A3 mRNAs, and the mRNAs encoding both forms of peroxisomal acyl-CoA oxidase (ACOX-I and ACOX-II), as judged by Northern-blot analysis using gene-specific oligonucleotide probes. Although DHEA treatment in vivo is effective in inducing these mRNAs in rat liver, it had no effect in the cultured hepatocytes. In contrast, treatment of the cells with DHEA 3 beta-sulphate (DHEA-S; 10-250 microM) stimulated major increases in CYP4A and ACOX mRNA levels. Examination of several analogues indicated a preference for 3 beta-sulphate over 17 beta-sulphated steroids and the inactivity of a 3 alpha-hydroxy-17 beta-sulphate derivative (DHEA-S > 5-androstene-3 beta,17 beta-diol 3-sulphate approximately 5 alpha-androstene-3 beta-ol-17-one 3-sulphate > 5-androstene-3 beta, 17 beta,17 beta-diol 17-sulphate approximately 5 beta-androstane-3 alpha-ol-17-one 3-sulphate >> 5 alpha-androstane-3 alpha, 17 beta-diol 17-sulphate). Induction of CYP4A mRNAs by either DHEA-S or clofibric acid was partially blocked by structurally diverse Ca(2+)-channel antagonists (nicardipine, nifedipine and diltiazem; 50 microM), suggesting that both the steroidal and fibrate classes of CYP4A inducers stimulate peroxisomal-proliferative responses via a Ca(2+)-dependent pathway. Retinoic acid alone slightly induced CYP4A mRNAs but did not enhance the induction by clofibrate or DHEA-S. As DHEA-S corresponds to a physiologically important major circulating androgen, these findings suggest that it may serve as an endogenous regulator of hepatic peroxisome enzyme levels. They further suggest that Ca(2+)-channel blockers may be useful pharmacological tools for the further study of the underlying cellular mechanism whereby endogenous steroids and fibrate drugs induce peroxisome proliferation, and the relationship of these events to activation of the peroxisome proliferator-activated receptor.

Project description:We have characterized the induction of acyl-CoA oxidase and cytochrome P450IVA1 RNAs in a primary hepatocyte culture system in vitro, using a sensitive and specific RNAse protection assay. Hepatocytes were cultured with a maximal inducing dose of the peroxisome proliferator clofibric acid (1 mM), or vehicle control, for 4 days, and the level of RNAs compared with the level in rats which had been treated with corn oil or clofibric acid (300 mg/kg) for 4 days. The level of acyl-CoA oxidase and P450IVA1 RNAs in 4-day-old control hepatocytes was less than 2% of that in control liver. However, the level of these RNAs in RNA from treated hepatocytes was 61% of that in liver RNA from treated rats. Hepatocytes were treated with the potent peroxisome proliferator methylclofenapate (100 microM), and the induction of RNAs determined at various times after exposure. P450IVA1 RNA was significantly induced 1 h after dosing, rising to 34-fold above control after 8 h, whereas acyl-CoA oxidase RNA was not significantly induced until 4 h, increasing to 5.2-fold above control after 8 h. A similar time course of induction was seen after treatment of hepatocytes with 100 microM-nafenopin, 100 microM-methylclofenapate, 1 mM-clofibric acid or 1 mM-mono(ethylhexyl) phthalate, suggesting that the differential time course of induction of P450IVA1 and acyl-CoA oxidase RNAs is not related to the esterification, structure or potency of the peroxisome proliferator, but is intrinsic to the process of peroxisome proliferation. Hepatocytes were treated with methylclofenapate in the presence and absence of cycloheximide. P450IVA1 RNA was significantly induced by methylclofenapate in the presence of cycloheximide, rising to 17-fold above control after 8 h. However, no induction of acyl-CoA oxidase RNA was detected in the presence of cycloheximide. Therefore we characterize the induction of acyl-CoA oxidase and P450IVA1 RNAs in primary hepatocyte culture in vitro as a faithful model of the induction response in rat liver, and suggest that induction of P450IVA1 RNA is a primary event in the process of peroxisome proliferation.

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Maintenance of total cytochrome P-450 content in rat hepatocyte culture and the abundance of CYP1A2 and CYP2B1/2 mRNAs.

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