Identification of cytochrome P450 enzymes critical for lung tumorigenesis by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): insights from a novel Cyp2abfgs-null mouse

Abstract

Cytochrome P450 (P450) enzymes encoded by the mouse Cyp2abfgs gene cluster are preferentially expressed in the respiratory tract. Previous studies have demonstrated that pulmonary P450-mediated bioactivation is necessary for lung tumorigenesis induced by the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and that CYP2A5 mediates a noteworthy fraction, but not all, of NNK bioactivation in the lung. The aim of this study was to determine whether other P450s encoded by the Cyp2abfgs gene cluster also play significant roles in NNK lung tumorigenesis. A novel Cyp2abfgs-null mouse was generated, in which all Cyp2a, 2b, 2g, 2f and 2s genes are deleted. The Cyp2abfgs-null mouse was viable, fertile and without discernible physiological abnormalities or compensatory increases in the expression of other P450s. NNK bioactivation in vitro and NNK-induced DNA adduction and lung tumorigenesis in vivo were determined for wild-type (WT) and Cyp2abfgs-null mice; the results were compared with previous findings from Cyp2a5-null mice. The Cyp2abfgs-null mice exhibited significantly lower rates of NNK bioactivation in lung and liver microsomes, compared with either WT or Cyp2a5-null mice. The levels of lung O(6)-methyl guanine DNA adduct were also substantially reduced in Cyp2abfgs-null mice, compared with either WT or Cyp2a5-null mice. Moreover, the Cyp2abfgs-null mice were largely resistant to NNK-induced lung tumorigenesis at both low (50mg/kg) and high (200mg/kg) NNK doses, in contrast to the WT or Cyp2a5-null mice. These results indicate for the first time that, collectively, the CYP2A, 2B, 2F, 2G, and 2S enzymes are indispensable for NNK-induced lung tumorigenesis.

Fig. 1.

Generation of Cyp2abfgs-null mice. (A) Breeding steps. (B) PCR analysis of genomic DNA from WT and Cyp2s1-Cyp2f2 ^Δ/Δ mice. PCR was performed using primers for Cyp2f2, Cyp2a4/5, Cyp2b10, Cyp2g1, Cyp2s1, Cyp2s1-Cyp2f2 ^Δ and Cre. Selected bands of a 100bp DNA size marker are shown. The full length gels are shown in Supplementary Figure S2, available at Carcinogenesis Online.

Fig. 2.

Validation of the Cyp2abfgs-null mice. (A) Strategy of Southern blot analysis. DNA probes P1 and P2 are shown as solid boxes. The WT Cyp2s1 and Cyp2f2 alleles and the Cyp2s1-Cyp2f2 ^Δ allele are shown. Following BglII digestion of genomic DNA, P1 would detect a 5.2kb fragment in Cyp2f2 WT allele, whereas P2 would detect a 5.6kb fragment in Cyp2s1 WT allele; both probes would detect a 3.3kb fragment from the Cyp2s1-Cyp2f2 ^Δ allele. The positions of PCR primers (F and R) used for detection of the Cyp2s1-Cyp2f2 ^Δ allele and the residual loxP site are also shown. (B) Southern blot analysis. DNA from a WT and a Cyp2s1-Cyp2f2 ^Δ/Δ (HO) mice (10 μg each) was analyzed. The full length blots are shown in Supplementary Figure S2, available at Carcinogenesis Online. (C) Absence of CYP2F2, CYP2A4/5, CYP2B9/10/19, CYP2G1 and CYP2S1 messenger RNA expression in tissues of the Cyp2abfgs-null mice. RNA-PCR was performed using total RNA prepared from the liver, lung and OM of adult male and female (two each, 2-month-old, pooled) WT or Cyp2abfgs-null mice. PCR products were analyzed on a 1.5% agarose gel and visualized by staining with ethidium bromide. (-), no template control. The positions of selected fragments of a 100bp DNA marker are indicated. The full length gels are shown in Supplementary Figure S2, available at Carcinogenesis Online.

Fig. 3.

NNK-induced O⁶-mG adduct formation in the lung and liver. Two-month-old female WT (B6) and Cyp2abfgs-null (B6) mice were injected with NNK at a dose of 200mg/kg (intraperitoneally), and lung (A) and liver (B) levels of O⁶-mG and total guanine were determined at 1 or 4h after the injection, as described in Materials and methods. Data represent mean ± SD (n = 5–8). ^aDetection limit for lung O⁶-mG was ~1.0 pmol/μmol guanine. **P < 0.01, *P < 0.05, compared with WT mice.

Fig. 4.

Body weights of mice after saline or NNK treatment. WT (A/J-N₃) and Cyp2abfgs-null (A/J-N₃) female mice (8-week old) were treated with a single injection (intraperitoneally) of NNK at 50 or 200mg/kg, or with saline. Body weights were recorded weekly at 8–10 weeks of age, and then biweekly. The results shown are mean ± SD (n = 10–12). Data was analyzed by two-way analysis of variance with Bonferroni post hoc test. **P < 0.01.