Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells

Abstract

Occupational and tobacco exposure to aromatic amines (AAs) including 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are associated with bladder cancer (BC) risk. Several epidemiological studies have also reported a possible role for structurally related heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meats with BC risk. We had screened for DNA adducts of 4-ABP, 2-NA, and several prominent HAAs formed in tobacco smoke or grilled meats including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) in the bladder DNA of BC patients, using liquid chromatography/mass spectrometry. We detected DNA adducts of 4-ABP, but not adducts of the other carcinogens. In this study, we have examined the capacity of RT4 cells, an epithelial human bladder cell line, to bioactivate AAs and HAAs to DNA damaging agents, which may contribute to BC. 4-ABP and AαC formed DNA adducts, but DNA adducts of 2-NA, PhIP, and MeIQx were not detected. 4-ABP DNA adducts were formed at tenfold higher levels than AαC adducts. Pretreatment of RT4 cells with α-naphthoflavone (1-10 µM), a specific cytochrome P450 1 (CYP1) inhibitor, decreased AαC adduct formation by 50% but did not affect the level of 4-ABP adducts. However, cell pretreatment with 8-methoxypsoralen (0.1-1 µM), a potent inhibitor of CYP2A, resulted in a 90% decrease of 4-ABP DNA adducts levels. These data signify that CYP2A and CYP1A isoforms expressed in the target urothelium bioactivate 4-ABP and AαC, respectively, and may be a critical feature of aromatic amine-induced urinary bladder carcinogenesis. The bioactivation of other tobacco and environmental AAs by bladder CYPs and their ensuing bladder DNA damage warrants further study.

Keywords: 2-Amino-9H-pyrido[2,3-b]indole; 4-Aminobiphenyl; Aromatic amines; Bladder cancer; DNA adduct; Heterocyclic aromatic amines.

Fig. 1:. AAs, HONH-AAs, HAAs, HONH-HAAs DNA adducts formation in RT4 cells.

(a) 4-ABP, 2-NA, PhIP, MeIQx, and AαC, and (b) HONH-4-ABP, HONH-2-NA, HONH-PhIP, HONH-MeIQx, and HONH-AαC. DNA adducts were measured after 24 h of carcinogen treatment by UPLC-ESI/MS³. (Three independent experiments,*P <0.05; **P <0.01, ***P < 0.005). ND: not detected.

Figure 2:. Kinetic and dose effect formation of 4-ABP and AαC DNA adducts in RT4 cells.

(a) After 3, 6, 12, and 24 h of treatment with 1 μM of 4-ABP or 10 μM AαC DNA adducts were measured by UPLC-ESI/MS³, and (b) After 24 h of treatment with 4-ABP or AαC (10 nM – 10 μM), DNA adducts were measured by UPLC-ESI/MS³. (Three independent experiments,*P <0.05; **P <0.01, ***P <0.005).

Fig. 3:. α-NF and 8-MOP inhibition of CYP1, CYP2A, and 4-ABP and AαC DNA adducts formation in RT4 cells.

(a) Following pretreatment with α-NF (1 – 10 μM), CYP1 and CYP2A activity, and (b) DNA adducts formed by 4-ABP (1 μM) and AαC (10μM) RT4 cells were measured. (c) Following pretreatment with 8-MOP (0.1 – 1 μM), CYP1 and CYP2A activity, and (d) DNA adducts formed by 4-ABP (1 μM) and AαC (10 μM) in RT4 cells were measured (Three independent experiments,*P <0.05; **P <0.01, ***P <0.005 versus Ctrl).

Fig. 4:. 4-ABP and AαC bioactivation by CYP2A6 and CYP2A13.

4-ABP and AαC (10 μM) were incubated with human recombinant CYP2A6 and CYP2A13 for 30 min at 37 °C. The formed HONH-4-ABP and HONH-AαC or the synthetic HONH-4-ABP and HONH-AαC were then incubated with CT-DNA at pH 5, for 30 min. 4-ABP and AαC DNA adducts were measured by UPLC-ESI/MS³. (Three independent experiments,*P <0.05; **P <0.01, ***P <0.005). ND: not detected.

Scheme 1:. Bioactivation of 4-ABP leading to DNA adduct formation in bladder.