TRANSSPECIES GENE-TRANSFER FOR ANALYSIS OF GLUCOCORTICOID-INDUCIBLE TRANSCRIPTIONAL ACTIVATION OF TRANSIENTLY EXPRESSED HUMAN CYP3A4 AND RABBIT CYP3A6 IN PRIMARY CULTURES OF ADULT-RAT AND RABBIT HEPATOCYTES

Interindividual variation in the spontaneous and in the glucocorticoid - or rifampicin-inducible expression of the CYP3A cytochromes P450, th e dominant forms of this supergene family that catalyze the oxidation of numerous drugs and environmental chemicals in human liver, remains largely unexplained, due in part to the lack of a validated animal mod el. We analyzed the 5'-flanking sequences of CYP3A genes from the rat (CYP3A23, CYP3A2), rabbit (CYP3A6), and human (CYP3A4, CYP3A5, CYP3A7) and found variable regions separated by three areas (consensus I, II, and III) of sequence homology immediately upstream of their respectiv e promoters. We used trans-species gene transfer in cellule as a new a pproach for determining the basis for qualitative differences among sp ecies in liver expression of different forms of CYP3A. When we transfe cted into cultured rat hepatocytes vectors containing 5'-flanking DNA from CYP3A23, CYP3A4, or CYP3A6 genes, we found that GAT activity was induced on treatment with dexamethasone or pregnenolone-16 alpha-carbo nitrile only if consensus II sequences were included. Rifampicin treat ment had no effect. When the same constructions containing consensus I I were transfected into rabbit hepatocytes, increased activity was obs erved on treatment of the cells with dexamethasone or with rifampicin but not with pregnenolone-16 alpha-carbonitrile. These results suggest that the host cellular environment rather than the structure of the g ene dictates the pattern of CYP3A inducibility. The application of thi s new model system will provide a unique technique for identifying mec hanisms of induction and advancing the development of appropriate toxi cological models for human safety assessment.