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RESEARCH PRODUCT

Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies.

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A quantitative RT-PCR assay has been developed that is able to measure the mRNA content of the major human CYPs (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). The technique is highly specific, reproducible, rapid, and sensitive enough to quantitate low and high abundant mRNAs. The PCR primers were selected to specifically match each CYP mRNA, to have a very close annealing temperature, and to render PCR products of similar sizes. The PCR conditions were designed to allow the simultaneous measurement of the various human liver CYPs in a single run. To achieve precise and reproducible quantitation of each cytochrome mRNA, a external standard (luciferase mRNA) is added to the probes to monitor the efficiency of the RT step. The degree of amplification is estimated using appropriate cDNA standards and quantitation of the amplified products by fluorescent measurement. This assay can be used to quantify the most relevant CYPs in human liver and cultured human hepatocytes. CYPs 3A4 and 2E1 were the most abundant mRNAs in human liver (2.5 and 1.7 x 10(8) molecules/microgram of total RNA respectively), whereas 1A1 and 2D6 were the least abundant isoforms (1.2 and 2.1 x 10(6) molecules/microgram of total RNA). A similar pattern was also found in short-term cultured human hepatocytes. This technique is also suitable for assessing CYP mRNA induction by xenobiotics. Cells exposed to 3-methylcholanthrene showed a characteristic increased expression of CYP1A2 and 1A1 mRNAs. Upon incubation with phenobarbital and rifampin (rifampicin), human hepatocytes increased CYP 2B6, 3A4, and 3A5 among others.