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

Abstract

The cytochrome P4501A (CYP1A) enzymes play important roles in the metabolic activation and detoxification of numerous environmental carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, we tested the hypothesis that hepatic CYP1A2 differentially regulates mouse hepatic and pulmonary CYP1A1 expression and suppresses transcriptional activation of human CYP1A1 (hCYP1A1) promoter in response to 3-methylcholanthrene (MC) in vivo. Administration of wild-type (WT) (C57BL/6J) or Cyp1a2-null mice with a single dose of MC (100 mol/kg i.p.) caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein content, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not Cyp1a2-null, animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for up to 8 days in both genotypes, with Cyp1a2-null mice displaying a greater extent of CYP1A1 expression. It is noteworthy that MC caused significant augmentation of human CYP1A1 promoter activation in transgenic mice expressing the hCYP1A1 and the reporter luciferase gene on a Cyp1a2-null background, compared with transgenic mice on the WT background. In contrast, the mouse endogenous hepatic, but not pulmonary, persistent CYP1A1 expression was repressed by MC in the hCYP1A1-Cyp1a2-null mice. Liquid chromatography-mass spectrometry experiments showed that CYP1A2 catalyzed the formation of 1-hydroxy-3-MC and/or 2-hydroxy-3-MC, a metabolite that may contribute to the regulation of CYP1A1 expression. In conclusion, the results suggest that CYP1A2 plays a pivotal role in the regulation of hepatic and pulmonary CYP1A1 by PAHs, a phenomenon that potentially has important implications for PAH-mediated carcinogenesis.

Prenatal administration of the cytochrome P4501A inducer, Β-naphthoflavone (BNF), attenuates hyperoxic lung injury in newborn mice: Implications for bronchopulmonary dysplasia (BPD) in premature infants

“…The separated proteins on the gels were transferred to polyvinylidene difluoride membranes, followed by Western blotting using monoclonal antibodies to CYP1A1, which cross-reacts with CYP1A2 (Moorthy et al, 2000; Couroucli et al, 2000; Jiang et al, 2004; 2010). For loading controls, the membranes were stripped and incubated with antibodies against β-actin, followed by electrochemical detection of bands.…”

“…The relative expression levels of the target gene were calculated according to the equation, 2 −ΔcT , where ΔcT = Ct target gene − Ct 18S gene . (Jiang et al, 2004; 2010). NQO1 mRNA levels were determined similarly, except that NQO1-specific primers and probes were used.…”

“…BNF levels in lung and liver tissues of mice prenatally exposed to BNF were determined by LC/MS/MS (Jiang et al, 2010). Briefly, mouse tissues were weighed to 0.1 mg and placed in polypropylene microvials.…”

Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo

“…Mice were anesthetized with isoflurane/oxygen and placed on the imaging station. Ventral and dorsal images were collected for 1 second using the IVIS imaging system 100 (Xenogen Corporation) [30], and luciferase expression was determined in vivo by bioluminescent imaging (Xenogen Corporation (Alameda, CA) [30–32]. Photons emitted from the liver region were quantified using Series Livingimage software [30–32].…”

“…Gene-specific primers in the presence of TaqMan reverse transcription reagents and RT reaction mix (Applied Biosystems) were used to reverse transcribe RNA, and TaqMan Universal PCR Master Mix and Assays-on-Demand Gene Expression probes (Applied Biosystems) were used for PCR amplification, as reported earlier [18,31,32]. The relative mRNA levels for P4501A1 were normalized to their 18S content, and relative expression levels of CYP1A1 and 1A2 genes were calculated as reported earlier [18,31,32]. …”