Division of Pathology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, 210-9501, Japan.
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
J Appl Toxicol. 2019 Jun;39(6):868-876. doi: 10.1002/jat.3775. Epub 2019 Jan 31.
Phosphorylated histone H2AX (γ-H2AX) has been demonstrated as a DNA damage marker both in vitro and in vivo. We previously reported the effects of genotoxic carcinogens in the urinary bladder of rats by immunohistochemical analysis of γ-H2AX using samples from 28-day repeated-dose tests. To evaluate the application of γ-H2AX as a biomarker of carcinogenicity in the bladder, we examined species differences in γ-H2AX formation in the urinary bladder of mice. Six-week-old male B6C3F mice were treated orally with 12 chemicals for 4 weeks. Immunohistochemical analysis demonstrated that N-butyl-N-(4-hydroxybutyl)nitrosamine, p-cresidine and 2-acetylaminofluorene (2-AAF), classified as genotoxic bladder carcinogens, induced significant increases in γ-H2AX levels in the bladder urothelium. In contrast, genotoxic (2-nitroanisole, glycidol, N-nitrosodiethylamine and acrylamide) and non-genotoxic (dimethylarsinic acid and melamine) non-bladder carcinogens did not upregulate γ-H2AX. Importantly, 2-nitroanisole, a potent genotoxic bladder carcinogen in rats, significantly increased the proportion of γ-H2AX-positive cells in rats only, reflecting differences in carcinogenicity in the urinary bladder between rats and mice. Significant upregulation of γ-H2AX was also induced by uracil, a non-genotoxic bladder carcinogen that may be associated with cell proliferation, as demonstrated by increased Ki67 expression. 2-AAF caused γ-H2AX formation mainly in the superficial layer, together with reduced and disorganized expression of uroplakin III, unlike in rats, suggesting the mouse-specific cytotoxicity of 2-AAF in umbrella cells. These results suggest γ-H2AX is a useful biomarker reflecting species differences in carcinogenicity in the urinary bladder.
磷酸化组蛋白 H2AX（γ-H2AX）已被证明是体外和体内的 DNA 损伤标志物。我们之前通过对 28 天重复剂量试验样本进行γ-H2AX 的免疫组织化学分析，报告了遗传毒性致癌剂在大鼠膀胱中的作用。为了评估 γ-H2AX 作为膀胱致癌性生物标志物的应用，我们检查了小鼠膀胱中 γ-H2AX 形成的种属差异。用 12 种化学物质对 6 周龄雄性 B6C3F 小鼠进行口服处理，持续 4 周。免疫组织化学分析表明，N-丁基-N-（4-羟丁基）亚硝胺、对甲酚和 2-乙酰氨基芴（2-AAF）被归类为遗传毒性膀胱致癌剂，可显著增加膀胱尿路上皮中 γ-H2AX 的水平。相比之下，遗传毒性（2-硝基苯甲醚、缩水甘油、N-亚硝二乙胺和丙烯酰胺）和非遗传毒性（二甲砷酸和三聚氰胺）非膀胱致癌剂不会上调 γ-H2AX。重要的是，2-硝基苯甲醚是一种在大鼠中具有很强遗传毒性的膀胱致癌剂，仅在大鼠中显著增加了 γ-H2AX 阳性细胞的比例，反映了大鼠和小鼠膀胱致癌性的差异。非遗传毒性膀胱致癌剂尿嘧啶也显著上调了 γ-H2AX，这可能与细胞增殖有关，如 Ki67 表达增加所证明的那样。2-AAF 主要在浅层引起 γ-H2AX 的形成，同时 uroplakin III 的表达减少和紊乱，与大鼠不同，提示 2-AAF 在伞细胞中具有小鼠特异性的细胞毒性。这些结果表明，γ-H2AX 是一种反映膀胱致癌性种属差异的有用生物标志物。
