Direct and rapid transcript analysis assay for CYP mRNA expression and inducibility in human primary hepatocytes.

Abstract

Induction of cytochrome P450 (CYP) enzymes is commonly analyzed in cultured human primary hepatocytes (HPHs) by measuring CYP1A2, CYP2B6 and CYP3A4/3A5 activities after exposure to test and reference compounds. Because chemicals can both inhibit and induce CYP enzymes, this traditional approach fails to distinguish such simultaneous effects. Regulatory authorities have therefore suggested that measurement of CYP expression levels should complement activity measurements. We aimed to compare a hybridization and bead-based assay termed transcript analysis with the aid of affinity capture (TRAC) with the routinely used quantitative real-time PCR (qRT-PCR) assay and to study its suitability for CYP induction studies on mRNA level. HPHs from three donors were treated with vehicle, reference substances omeprazole, phenobarbital and rifampicin and six test compounds on 48-well plates. The mRNA expression of ten CYP isoforms important for drug metabolism was determined by TRAC and qRT-PCR methods in order to validate the novel TRAC method. The fold-increases of CYP mRNA levels showed a good correlation between the assays. With TRAC, the marker CYP mRNAs for induction could be easily detected from about 10 000 hepatocytes per sample, with a coefficient of variation below 10% between triplicates. Time spent for TRAC analysis was significantly shorter. Thus, TRAC is a sensitive and reproducible high-throughput assay, which enables accurate and direct detection of multiple mRNA targets simultaneously from large number of samples without enzymatic reactions inherent to qRT-PCR. It is a valuable method to study CYP induction and expandable to other genes relevant for drug metabolism and toxicity.