REGULATION OF HEPATIC P450S BY LOVASTATIN AND OXYSTEROLS

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Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, is widely used in the treatment of hypercholesterolemia, a major risk factor for the development of coronary artery disease. Recent evidence from this laboratory has demonstrated that lovastatin is also a novel inducer of cytochromes P450 in cultured rat hepatocytes, inducing P450 2B2 and 4A1 mRNAs when administered alone, and potentiating the inductions of 2B1/2 and 3A1 mRNAs in cultures cotreated with phenobarbital or clotrimazole. Also, lovastatin markedly induced P450 3A6 in cultured rabbit hepatocytes, consistent with existing human data. Induction or potentiated induction of P45Os in humans treated with lovastatin could decrease the therapeutic efficacies, or increase the toxicities, of simultaneously administered drugs. The hypothesis of this proposal is that lovastatin, by completely inhibiting cholesterol biosynthesis, prevents the synthesis and accumulation of specific oxysterols that suppress P450 expression. By extension, other chemicals that modify P450 expression, such as the imidazole antimycotic drugs, may inhibit enzymes in the sterol metabolic pathway, resulting in altered levels of regulatory oxysterols. The specific aims of this proposal are to: (1) define the effects of HMG-CoA reductase inhibitors (i.e., lovastatin, pravastatin, simvastatin and BMY-22089) on expression of rat class 2B, 3A and 4A P45Os or rabbit P450 3A6 at the mRNA (by Northern blot analysis), protein (by Western blot analysis) and enzymatic activity levels in primary cultured hepatocytes, (2) define the effects of lovastatin on expression of rat class 2B, 3A and 4A P45Os or rabbit P450 3A6 at the mRNA, protein and enzymatic activity levels in vivo, (3) determine the general mechanisms for the effects of lovastatin on P450 expression (as defined in aims 1 and 2) by examining, as appropriate, effects of treatments on rates of transcription (by nuclear run on analysis), mRNA stability (by measuring mRNA levels after inhibition of transcription), protein stability (by pulse-chase experiments) or enzyme activity, (4) define the effects of oxysterol treatments (i.e., mevalonate, 25- hydroxycholesterol, low density lipoproteins and squalistatin 1) on expression of rat class 2B, 3A and 4A P45Os or rabbit P450 3A6 at the mRNA, protein and enzymatic activity levels in cultured hepatocytes and (5) examine the effects of lovastatin and imidazole antimycotic drugs on incorporation of [14C)labeled acetate or mevalonate into oxysterols in cultured rat or rabbit hepatocytes by high performance liquid chromatography, and correlate changes in specific oxysterols to changes in P450 expression. The significance of this work is that it will provide information that could impact on the safety and efficacy of lovastatin in humans, will allow testing of a new hypothesis for the cellular basis of hepatic P450 induction, and may identify new targets for the development of more effective and/or less toxic hypocholesterolemic drugs.