Role of CYP2A13 in the bioactivation and lung tumorigenicity of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: in vivo studies using a CYP2A13-humanized mouse model

Abstract

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is abundant in tobacco smoke, is a potent lung procarcinogen. The present study was aimed to prove that transgenic expression of human cytochrome P450 2A13 (CYP2A13), known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, will enhance NNK bioactivation and NNK-induced tumorigenesis in the mouse lung. Kinetic parameters of NNK bioactivation in vitro and incidence of NNK-induced lung tumors in vivo were determined for wild-type, Cyp2a5-null and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. As expected, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in Michaelis constant (K m) values for the formation of 4-oxo-4-(3-pyridyl)-butanal, representing the reactive intermediate that can lead to the formation of O(6)-methylguanine (O(6)-mG) DNA adducts; however, the gain of CYP2A13 at a fraction of the level of mouse lung CYP2A5 led to recovery of the activity in the lung, but not in the liver. The levels of O(6)-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of CYP2A13-humanized mice than in Cyp2a5-null mice. Moreover, incidences of lung tumorigenesis were significantly greater in CYP2A13-humanized mice than in Cyp2a5-null mice, and the magnitude of the differences in incidence was greater at low (30mg/kg) than at high (200mg/kg) NNK doses. These results indicate that CYP2A13 is a low K m enzyme in catalyzing NNK bioactivation in vivo and support the notion that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans.

Fig. 1.

Systemic levels of NNK and NNAL at various time points after i.p. administration of NNK. A/J-WT, A/J-N5-Cyp2a5-null and A/J-N5-CYP2A13-humanized mice (2-month-old, female) were injected with NNK at either 30mg/kg (A) or 200mg/kg (B), i.p., and plasma levels of NNK and NNAL were determined. Data represent means ± SD (n = 4). No significant difference was found among the three strains, at any time point, for either NNK or NNAL (Student’s t-test).

Fig. 2.

Body weights of mice during NNK tumorigenesis study. A/J-WT, A/J-N5-Cyp2a5-null and A/J-N5-CYP2A13-humanized female mice were treated with single injection (i.p.) of NNK at the indicated dose or with saline at the beginning of the ninth week of age. Body weights were recorded at weekly intervals for a batch of 10 mice per group. The results shown are mean values, with standard deviation (data not shown) smaller than 10% of the mean. (A) Comparisons of weekly body weights among the treatment groups for each strain. *P < 0.05, compared with the saline group (one-way analysis of variance followed by Dunnett’s test). (B) Comparisons of weekly body weights among the three different strains for the 200 mg/kg NNK dose (P > 0.05; one-way analysis of variance followed by Tukey’s test).

Fig. 3.

Dose-response of NNK-induced lung tumorigenesis. (A) Tumor multiplicity data from Table III are plotted to show dose-dependent increase. Values represent means ± standard error (n = 22). *P < 0.05, compared with A/J-WT and A/J-N5-CYP2A13-humanized mice (one-way analysis of variance for each dose followed by Tukey’s test). (B) Extent of increase (fold) in tumor multiplicity (mean values) in A/J-N5-CYP2A13-humanized mice, compared with A/J-Cyp2a5-null mice, at various NNK doses, showing greater contribution of CYP2A13 to lung tumorigenesis at the two lower doses tested.