Disruption of the gene for CYP1A2, 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 μmol/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.

Fig. 1.

Effect of MC on hepatic EROD/MROD activities and CYP1A apoprotein contents. Eight-week-old female WT or Cyp1a2-null mice were treated with a single dose of MC (100 μmol/kg) or vehicle (CO) and sacrificed 1, 8, or 15 days after dosing. A and B, EROD (A) and MROD (B) activities were estimated in the liver microsomes of these animals. Values represent mean ± S.E. (n = 3). ∗, Statistically significant differences between MC- and vehicle-treated samples at P < 0.05, as determined by two-way ANOVA and modified t tests. C, representative Western blot showing the effect of MC on CYP1A1/1A2 protein expression in liver microsomes. WT and Cyp1a2-null animals were treated with MC as described above, and CYP1A1/1A2 apoprotein expression was analyzed in microsomes (10 μg) at the indicated time points by Western blotting.

Fig. 2.

Effect of MC on pulmonary EROD activities (A) and CYP1A1 apoprotein content (B). A, eight-week-old female WT or Cyp1a2-null mice were treated with a single dose of MC as described in the legend to Fig. 1, and EROD activities were estimated in the lung microsomes of the animals. Values represent mean ± S.E. (n = 3). ∗, Statistically significant differences between MC- and vehicle-treated samples at P < 0.05, as determined by two-way ANOVA and modified t tests. B, representative Western blot showing the effect of MC on CYP1A1 protein expression in lung microsomes. WT and Cyp1a2-null animals were treated with MC as described above, and CYP1A1 apoprotein expression was analyzed in the microsomes (10 μg) at the indicated time points by Western blotting.

Fig. 3.

Representative EMSA of hepatic nuclear extracts from CO- and MC-treated mice. WT and Cyp1a2-null mice were treated with CO or MC as described in the legend to Fig. 1. Nuclear protein extracts from treated livers were subjected to EMSA, as described under Materials and Methods. The labeled nuclear proteins were separated by PAGE, and the gels were dried. The gels were exposed to autoradiography at −80°C for 24 h. The arrow indicates the interaction with the AHREs of an MC-specific nuclear protein that seems to be the MC–AHR–ARNT complex, which is competed off in the presence of 50-fold excess of cold AHRE-specific oligonucleotide (see lanes labeled Cold Probe).

Fig. 4.

LC-MS/MS analyses of MC. Parent MC in livers and lungs was analyzed by LC-MS/MS. A, UV chromatogram of MC standard (I). B, multiple reaction monitoring transition showing the primary product ion MC at m/z 253.9, which represents M⁺ + 1-15 (loss of methyl group) fragment. C, parent MC was estimated in the livers and lungs of WT and Cyp1a2-null mice exposed to MC at 1 and 15 days after treatment. Values represent mean ± S.E. (n = 3).

Fig. 5.

Chemical structures of MC (I) and the various metabolite standards (II–IX) used in the study. I, MC; II, MC-1-one; III, 11-hydroxy-3-MC; IV, 2-OH-MC; V, 1-OH-MC; 1-hydroxy-3-MC; VI, MC-11,12-dihydroepoxide; VII, 11,12-dione-3-MC; VIII, 11,12-dihydro-11,12-dihydroxy-3-MC; and IX, MC-11,12-dialdehyde.

Fig. 6.

LC-MS/MS analyses of MC metabolites. The different MC metabolite standards (II–IX) depicted in Fig. 5 were analyzed by LC-MS/MS, and the mass transitions and retention times of each metabolite are shown.

Fig. 7.

Levels of 1-OH-MC and 2-OH-MC mouse tissues exposed to MC. WT or Cyp1a2-null mice were treated with MC as described in the legend to Fig. 1, and combined levels of hepatic and pulmonary 1-OH-MC and 2-OH-MC were estimated at the 1- and 15-day time points by LC-MS/MS. Values represent mean ± S.E. (n = 3). ∗, Statistically significant differences between WT and Cyp1a2-null groups at P < 0.05, determined by Student's t test. ND, not detected.

Fig. 8.

Bioluminescent imaging of hCYP1A1-luc-WT and hCYP1A1-luc-Cyp1a2-null mice after MC treatment. A and B, adult male hCYP1A1-luc-WT (A) or hCYP1A1-luc-Cyp1a2-null (B) mice were treated with CO or MC (100 μmol/kg), once daily for 4 days, and luciferase expression was analyzed by bioluminescent imaging in real time at 1, 8, or 15 days after MC or CO withdrawal. C, quantitation of bioluminescent imaging data was conducted with IVIS imaging software. Values represent mean ± S.E. (n = 5). ∗, Statistically significant differences between MC- and CO-treated mice at P < 0.05, as determined by two-way ANOVA and modified t tests.

Fig. 9.

Effect of MC on endogenous hepatic EROD (CYP1A1), MROD, and CYP1A apoprotein contents. A and B, adult male hCYP1A1-luc-WT (A) or hCYP1A1-luc-Cyp1a2-null (B) mice were treated with CO or MC (100 μmol/kg), once daily for 4 days, and EROD (A) and MROD (B) activities were analyzed as described under Materials and Methods. Data represent mean ± S.E. of EROD or MROD activities from at least four individual animals. C, representative Western blots showing the effects of MC on endogenous hepatic CYP1A1 and CYP1A2 (left) and pulmonary CYP1A1 protein (right) expression in mice. hCYP1A1-luc-WT (left) or hCYP1A1-luc-Cyp1a2-null (right) mice were treated with CO or MC as described above, and CYP1A1/1A2 apoprotein expression was analyzed in the microsomes (20 μg) at the indicated time points by Western blotting.

Fig. 10.

Effect of MC on endogenous pulmonary EROD (CYP1A1) and CYP1A1 apoprotein content. A, adult male hCYP1A1-luc-WT or hCYP1A1-luc-Cyp1a2-null mice were treated with CO or MC (100 μmol/kg), once daily for 4 days, and EROD activities were analyzed as described under Materials and Methods. Data represent mean ± S.E. of EROD activities from at least four individual animals. B, representative Western blot showing the effect of MC on endogenous pulmonary CYP1A1 protein expression in mice. hCYP1A1-luc-WT (left) or hCYP1A1-luc-Cyp1a2-null (right) mice were treated with CO or MC as described above, and CYP1A1 apoprotein expression was analyzed in the microsomes (20 μg) at the indicated time points by Western blotting.

Fig. 11.

Possible mechanisms of the role of hepatic CYP1A2 in the regulation of hepatic and pulmonary CYP1A1 by MC. We hypothesize that MC upon entry into liver is metabolized by the liver-specific CYP1A2 to 2-OH-MC, which upon further metabolism results in the formation of a reactive metabolite such as 2-OH-MC-X, which complexes with the AHR and is transported to the nucleus. In the nucleus, the 2-OH-MC-X binds covalently to the AHREs, resulting in suppression of hepatic CYP1A1 induction. Because the lung is devoid of CYP1A2, it is possible that the 2-OH-MC generated in the liver is transported to the lung, wherein it gets activated to 2-OH-MC-X, which in turn suppresses CYP1A1 expression, eventually leading to decreased persistence of DNA adducts and attenuation of pulmonary tumorigenesis.