Measurement of cytochrome P450 gene induction in human hepatocytes using quantitative real-time reverse transcriptase-polymerase chain reaction

Drug-induced changes in expression of cytochrome (CYP) P450 genes are a key cause of drug-drug interactions. Consequently, preclinical prediction of t hese changes by novel compounds is an integral component of drug developmen t. To date, in vitro models of CYP induction have used mRNA measurement, im munodetection, and substrate metabolism as reporters. Here, we describe the application of quantitative real-time reverse transcriptase polymerase cha in reaction to study CYP1A1 and CYP3A4 gene induction in 5-day-old cultures of human hepatocytes by known CYP inducers. After 5 days in culture, CYP1A 1 expression was significantly elevated (5.1- to 26-fold; P < .01) in all f our livers studied. In direct contrast, CYP3A4 mRNA levels consistently dec reased during culture (80- to 300-fold; P < .001). In three independent exp eriments, a 48-h exposure to 3-methylcholanthrene, omeprazole, and lansopra zole significantly induced CYP1A1 expression in comparison to untreated cul tures (P < .05). Rifampicin and solvent were without effect on CYP1A1 expre ssion. Under identical experimental conditions, rifampicin and lansoprazole significantly elevated CYP3A4 mRNA expression (P < .05), whereas 3-methylc holanthrene, omeprazole, and dimethyl sulfoxide were without significant ef fect. These data demonstrate the applicability of quantitative reverse tran scriptase polymerase chain reaction to the determination of gene dynamics i n human hepatocytes. This offers a highly specific alternative to quantific ation of drug effects on CYP expression using immunodetection and substrate metabolism.