Use of a novel real-time quantitative reverse transcription-polymerase chain reaction method to study the effects of cytokines on cytochrome P450 mRNA expression in mouse liver

In this study, we developed a real-time quantitative reverse transcription- polymerase chain reaction (RT-PCR) method to study cytochrome P450 (CYP) mR NA regulation by cytokines in mouse liver. The method combines standard RT- PCR with a fluorogenic probe in which the intensity of fluorescence is prop ortional to the amount of target template present. We show that this method provides very rapid, sensitive, and reproducible quantification of mouse C yp mRNA with a wide dynamic range of starting target molecule. We validated the method by using several prototypic CYP inducers. As expected, the mRNA levels of Cyp3a11, Cyp2b10, and Cyp1a2 were induced by a single dose of de xamethasone (100 mg/kg), phenobarbital (80 mg/kg), and 3-methylcholanthrene (80 mg/kg), respectively. The method of real-time RT-PCR was then used to evaluate the effects of interleukin (IL)-6 (100 ng/mouse), IL-1 beta (500 n g/mouse), and tumor necrosis factor (TNF)-alpha (2 mu g/mouse) on Cyp mRNA expression in the mouse. Constitutive Cyp2b10 mRNA was reduced to 40% by IL -6 and 15% by IL-1 beta, whereas Cyp2d9 mRNA was reduced to 70% by TNF-alph a administration. The level of Cyp1a2 mRNA was suppressed to 67% by IL-6 an d 59% by TNF-alpha. Cyp3a11 and Cyp2e1 mRNAs were not affected by any of th e cytokines that were studied. We conclude that the real-time RT-PCR method is a powerful new tool to study CYP mRNA expression and regulation. Using this method, we are the first to report that the expression of constitutive Cyp2b10, 1a2, and 2d9 mRNAs was suppressed by proinflammatory cytokines.