Background: Bladder cancer is the seventh most common cancer in males worldwide. Recent studies have demonstrated that DNA double-strand breaks are recognized to be a serious injury leading to genome instability and cancer development. The rapid phosphorylation of histone H2AX at Serine 139, to become γH2AX, occurs in response to DNA double-strand breaks. Roles of γH2AX in carcinogenesis are still unclear, and there are few reports on the association with bladder cancer. We have previously demonstrated that γH2AX expression is induced in urinary bladders of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated rat (Toyoda et al., J Toxicol Pathol, 26: 215-221, 2013). To evaluate the value of γH2AX as a biomarker for carcinogenicity and genotoxicity to the urinary bladder, we performed an immunohistochemical analysis of γH2AX expression in chemical-treated rats.

Methods: Six-week-old male F344 rats were treated with 0.05% BBN, 1.8% 2-nitroanisole (2-NA), 2% 2,2-bis (bromomethyl)-1,3-propanediol (BMP), 0.1% phenethyl isothiocyanate (PEITC), 3% melamine, or 3% uracil in either drinking water (BBN) or diet (other chemicals) for 4 weeks. Animals were sacrificed at 4 or 6 weeks of the experiment, and immunohistochemistry for γH2AX in urinary bladders was performed.

Results: At week 4, γH2AX expression in the urothelium were significantly increased in BBN and 2-NA groups compared with the controls. γH2AX expression ratio (positive cells/1000 cells) in BBN, 2-NA and control groups were 76±21, 111±37 and 0.8±0.6, respectively. γH2AX was recognized as dot-like foci in nuclei of epithelial cells, and the positive cells were mainly located near the basal layer of the mucosa. In BMP and PEITC groups, γH2AX expression was mainly observed in umbrella cells and intermediate cells, respectively. In melamine and uracil groups, there were diffuse urothelial hyperplasia and focal expression of γH2AX especially in the basal layer. The expression ratios of γH2AX in BMP, PEITC, melamine and uracil groups were 8.3±6.6, 26±9.0, 16±11 and 11±11, respectively. Two weeks after withdrawal of administration, although the expression ratio of γH2AX was markedly reduced in all groups, it remained high in BBN (17±1.9) and 2-NA (19±3.4) groups compared with the controls (0.6±0.9).

Discussion and conclusion: BBN and 2-NA, genotoxic bladder carcinogens, induced more extensive γH2AX expression in the urothelium than other non-genotoxic (melamine and uracil) or non-carcinogenic chemicals (BMP and PEITC), and γH2AX-positive cells remained in relatively large numbers even 2 weeks after withdrawal of its administration. In addition, ratio of basal cells, intermediate cells and umbrella cells in γH2AX-positive cells showed similar patterns in BBN/2-NA and melamine/uracil groups. These data indicate that γH2AX has potential as a biomarker for early detection of genotoxic carcinogen in the urinary bladder.

Citation Format: Takeshi Toyoda, Young-Man Cho, Jun-ichi Akagi, Yasuko Mizuta, Kumiko Ogawa. Expression of γH2AX as a biomarker of genotoxic carcinogen in the urinary bladder of rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4580. doi:10.1158/1538-7445.AM2015-4580