Received for publication February 14, 2008.
Revised May 16, 2008.
Accepted for publication June 3, 2008.

Cytochrome P450 2C11 5'-flanking region and promoter mediate in vivo suppression by 3-methylcholanthrene

Abstract

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-kb, 5.6-kb and 2.4-kb of the CYP2C11 5'-flank. MC suppressed CYP2C11-luciferase activity of the 10.1-kb and 5.6-kb constructs to below 50% of vehicle levels by 24 h 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 72h. 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-kb 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.

Key words: Ah receptor, CYP gene regulation, CYP2C, cytochrome P450 regulation, polycyclic aromatic hydrocarbons, toxicology, transcriptional regulation