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Cytochrome P450 2C11 5'-Flanking Region and Promoter Mediate in Vivo Suppression by 3-Methylcholanthrene

Department of Pharmacology and Toxicology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada

Aromatic hydrocarbons such as 3-methylcholanthrene (MC) elicit toxic and adaptive responses through the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons act via an unknown mechanism to suppress the transcription of CYP2C11, a growth hormone-regulated gene encoding the male-specific rat hepatic cytochrome P450 2C11. We hypothesize that suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene's promoter and 5'-flank. Using hydrodynamics-based injections to deliver plasmid DNA to the liver of live rats, we studied the MC responsiveness of luciferase constructs containing 10.1, 5.6, and 2.4 kilobases (kb) of the CYP2C11 5'-flank. MC suppressed CYP2C11-luciferase activity of the 10.1- and 5.6-kb constructs to less than 50% of vehicle levels by 24 and 72 h. Luciferase activity of the 2.4-kb CYP2C11 construct was decreased to 63% of vehicle levels 24 h after MC treatment, but no suppression was detected by 72 h. Negative regulatory element(s) responsible for CYP2C11 reporter suppression by MC exist in the proximal 2.4 kb of the 5'-flank; however, additional cis-acting elements located between –5.6 and –2.4 kb mediate persistent reporter suppression. As a positive control for AHR activation, MC dramatically induced the luciferase activity of a Cyp1a1-driven luciferase plasmid under AHR control. Modulation of reporter gene activity by MC was accompanied by induction of endogenous CYP1A1 and suppression of endogenous CYP2C11 mRNA/protein. This is the first demonstration of aromatic hydrocarbon-mediated suppression of a CYP2C11-luciferase construct, and this finding suggests that the 5'-flanking region and promoter mediate down-regulation of this gene in the intact rat.