Persistent induction of cytochrome P450 (CYP)1A enzymes by 3-methylcholanthrene in vivo in mice is mediated by sustained transcriptional activation of the corresponding promoters

Abstract

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens. Cytochrome P450 (CYP)1A enzymes play key roles in the metabolic activation of PAHs to carcinogenic metabolites. We previously showed persistent induction of CYP1A enzymes by 3-methylcholanthrene (MC) in vivo in rodents. In this study, we tested the hypothesis that MC elicits persistent induction of CYP1A1 and 1A2 in vivo by mechanisms entailing sustained transcriptional activation of the corresponding promoters. Adult male wild type (WT) (Cd-1) mice, transgenic mice expressing the human CYP1A1 promoter or the mouse CYP1A2 promoter were treated with the vehicle corn oil (CO) or the carcinogenic PAH, 3-methylcholanthrene (MC), once daily for 4 days, and luciferase reporter gene expression was determined at 1, 8. 15, and 22 days after MC withdrawal by bioluminescent imaging. Pulmonary and hepatic endogenous expression of CYP1A1 and 1A2 was also determined at the enzymatic, protein, and mRNA levels. The major findings were that MC elicited marked enhancement in the luciferase expression in the CYP1A1-luc as well CYP1A2-luc transgenic mice that was sustained for up to 22 days, the magnitude of induction being more pronounced in the CYP1A1-luc mice. MC also caused persistent induction of endogenous CYP1A1 and 1A2 expression in the WT, CYP1A1-luc, and 1A2-luc mice for up to 22 days. In conclusion, our results support the hypothesis that MC elicits sustained CYP1A1 and 1A2 expression by sustained transcriptional activation of the corresponding promoters. Thus, these novel transgenic models should be very useful for further understanding of the molecular mechanisms of persistent CYP1A induction, in relation to PAH-mediated carcinogenesis.

Persistent Induction of Cytochrome P4501A1 in Human Hepatoma Cells by 3-Methylcholanthrene: Evidence for Sustained Transcriptional Activation of the CYP1A1 Promoter

“…The dose response of EROD (CYP1A1) induction by MC suggested that the kinetics of induction correlated with the cellular concentration of the ligand. Our results showing marked induction of EROD activities and apoproteins for up to 96 h strongly suggested that MC caused persistent CYP1A1 induction in human hepatoma cells for up to 96 h. This finding was in agreement with our previous findings showing long-term induction of CYP1A1 enzymes by MC in rodent models (Moorthy et al, 1993;Moorthy, 2000;Kondraganti et al, 2002;Jiang et al, 2009).…”

Disruption of the Gene forCYP1A2, which Is Expressed Primarily in Liver, Leads to Differential Regulation of Hepatic and Pulmonary Mouse CYP1A1 Expression and Augmented Human CYP1A1 Transcriptional Activation in Response to 3-Methylcholanthrene In Vivo

“…We also reported that MC elicits sustained CYP1A1 and CYP1A2 induction in vivo via transcriptional activation of the corresponding promoters (Jiang et al, 2009). Furthermore, we showed that MC elicits persistent induction of CYP1A1 in HepG2 cells in vitro by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter (Fazili et al, 2010).…”

“…The mice were back-crossed with C57BL/6J mice for 12 generations to generate theoretically a Ͼ99.2% C57BL/6J background. Transgenic mice expressing the human 13.2-kb CYP1A1 promoter on a CD-1 background were generated via pronuclear microinjection by using Crl:CD-1 strain oocytes and were obtained from Xenogen Corporation (Alameda, CA) (Zhang et al, 2004;Jiang et al, 2009). The hCYP1A1-luc mice were backcrossed with Cyp1a2-null on the C57BL/6J background for several generations, such that two types of transgenic mice were generated: hCYP1A1-luc-WT (B6:CD-1) and hCYP1A1-luc-Cyp1a2-null (B6:CD-1).…”

“…Mice were anesthetized with isoflurane/oxygen and placed on the imaging station. Ventral and dorsal images were collected for 1 s by using the IVIS imaging system 100 (Xenogen Corporation) (Zhang et al, 2004;Jiang et al, 2009), and luciferase expression was determined in vivo by bioluminescent imaging (Xenogen Corporation) (Zhang et al, 2004). The animals were acclimatized for 7 days before the start of the experiment and randomized into three groups of six animals each.…”

“…Ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities in microsomes were assayed essentially as described previously (Moorthy, 2000;Kondraganti et al, 2002;Jiang et al, 2009). Each assay was conducted in duplicate using individual liver or lung microsomes from at least three individual mice for each group.…”