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) mRNA regulation by cytokines in mouse liver. The method combines standard RT-PCR with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target template present. We show that this method provides very rapid, sensitive, and reproducible quantification of mouse Cyp 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 dexamethasone (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-1beta (500 ng/mouse), and tumor necrosis factor (TNF)-alpha (2 microgram/mouse) on Cyp mRNA expression in the mouse. Constitutive Cyp2b10 mRNA was reduced to 40% by IL-6 and 15% by IL-1beta, whereas Cyp2d9 mRNA was reduced to 70% by TNF-alpha administration. The level of Cyp1a2 mRNA was suppressed to 67% by IL-6 and 59% by TNF-alpha. Cyp3a11 and Cyp2e1 mRNAs were not affected by any of the 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.
Pan
J
J
Wyeth-Ayerst Research, Department of Drug Safety and Metabolism, Princeton, New Jersey, USA. panj@war.wyeth.com
Xiang
Q
Q
Ball
S
S
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D016207:Cytokines / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D016207:Cytokines; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System