Trans-species 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, the dominant froms 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 model. 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 respective promoters. We used trans-species gene transfer in cellulo as a new approach for determining the basis for qualitative differences among species in liver expression of different forms of CYP3A. When we transfected into cultured rat hepatocytes vectors containing 5'-flanking DNA from CYP3A23, CYP3A4, or CYP3A6 genes, we found that CAT activity was induced on treatment with dexamethasone or pregnenolone-16 alpha-carbonitrile only if consensus II sequences were included. Rifampicin treatment had no effect. When the same constructions containing consensus II were transfected into rabbit hepatocytes, increased activity was observed 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 gene dictates the pattern of CYP3A inducibility. The application of this new model system will provide a unique technique for identifying mechanisms of induction and advancing the development of appropriate toxicological models for human safety assessment.
Barwick
J L
JL
University of Colorado Health Sciences Center, Denver 80262, USA.
Quattrochi
L C
LC
Mills
A S
AS
Potenza
C
C
Tukey
R H
RH
Guzelian
P S
PS
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008297:Male; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology; D013045:Species Specificity; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection / Q000379:methods*
D005938:Glucocorticoids; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011285:Pregnenolone Carbonitrile; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C100638:cytochrome P-450 CYP3A6 (rabbit); C510163:CYP3A4 protein, human; D015500:Chloramphenicol O-Acetyltransferase; D012293:Rifampin