Regulation of Constitutive Mouse Hepatic Cytochromes P450 and Growth Hormone Signaling Components by 3-Methylcholanthrene

Abstract

ABSTRACT:3-Methylcholanthrene (MC) activates the aryl hydrocarbon receptor and increases expression of cytochrome P450 (P450) enzymes such as CYP1A1. MC also decreases expression of CYP2C11, the major hepatic P450 in male rats that is regulated by pulsatile growth hormone (GH) secretion via a pathway partially dependent on signal transducer and activator of transcription 5b (STAT5b). If disruption of this GH signaling pathway is important for MC's ability to suppress CYP2C11 transcription, we hypothesize that MC suppresses other male-specific genes (e.g., mouse Cyp2d9) regulated by pulsatile GH with STAT5b dependence. We examined the time course of MC's effects on hepatic P450s and GH signaling components in male C57BL/6 mice. P450 content, heme content, and NADPH P450 oxidoreductase activity were induced 2.3-, 1.8-, and 1.3-fold, respectively, by MC. MC dramatically induced CYP1A1 mRNA, protein, and catalytic activity. MC caused a 42% decrease in CYP2D9 protein, a 28% decrease in CYP2D9 mRNA, and a 27% decrease in testosterone 16␣-hydroxylation activity. MC caused a pronounced decrease in CYP3A protein; however, there was no apparent change in testosterone 6␤-hydroxylation activity, and changes in mRNA levels for CYP3A forms were relatively small. Expression of GH receptor and major urinary protein 2, a gene regulated by GH with STAT5b dependence, was decreased by MC at the mRNA level. These results show that MC suppresses mouse Cyp2d9, a pulsatile GH-and STAT5b-dependent male-specific gene, via a pretranslational mechanism that may involve disrupted GH signaling. Mouse CYP3A protein levels are dramatically decreased by MC via a mechanism that is not yet understood.

Aryl hydrocarbon receptor-dependence of dioxin’s effects on constitutive mouse hepatic cytochromes P450 and growth hormone signaling components

“…Similar to the suppression of rat CYP2C11 by aromatic hydrocarbons, we showed that MC treatment of male mice caused downregulation of hepatic Cyp2d9 at the mRNA, protein and catalytic activity levels (Lee et al 2006), and this was accompanied by decreased mRNA levels for the GH receptor (GHR) and major urinary protein 2 (MUP2), a GH-regulated and STAT5b-dependent transcript. Others have shown that treatment of mice with MC leads to an AHR-dependent decrease in hepatic levels of mRNA encoding GHR, Janus kinase 2 (JAK2) and two distinct STAT5b targets, MUP2 and cytokine-inducible Src homology 2 domain-containing protein (CIS) (Nukaya et al 2004).…”

“…We showed previously that MC treatment caused a dramatic decrease in mouse hepatic CYP3A11 content, particularly at the protein level (Lee et al 2006). TCDD caused a 76% decrease in hepatic CYP3A11 mRNA levels in wild-type mice and this suppression response was not observed in Ahr −/− mice (Fig.…”

“…Our previous work showed that mouse hepatic POR catalytic activity was increased by up to 33% following MC treatment (Lee et al 2006). POR mRNA levels were increased by 55% following TCDD treatment in wild-type mice and this small magnitude induction response was not see in Ahr −/− mice (Fig.…”

“…The reverse transcription step was carried out as described previously (Lee et al 2006). Hepatic levels of mRNAs encoding CYP2D9, CYP3A11, NADPH-cytochrome P450 oxidoreductase (POR), serum amyloid protein P (SAP), suppressor of cytokine signaling 3 (SOCS3) and GHR, normalized to β-actin as the internal reference standard, were determined in triplicate for all samples using the ABI Prism 7500 Sequence Detection System.…”

“…Hepatic levels of mRNAs encoding CYP1A1, JAK2, STAT5a/b, CIS and MUP2, normalized to β-actin as the internal reference standard, were determined by measuring the intensity of PCR products on Vistra Green-stained polyacrylamide gels using the primers and experimental conditions previously described (Lee et al 2006). …”