Division of Pathology, National Institute of Health Sciences.
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology.
J Toxicol Sci. 2023;48(6):323-332. doi: 10.2131/jts.48.323.
We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.
我们使用γ-H2AX 的免疫组织化学方法开发了一种具有高灵敏度和特异性的膀胱癌致癌剂早期检测方法，γ-H2AX 是一种著名的 DNA 损伤标志物。为了研究 γ-H2AX 作为肝癌早期检测生物标志物的潜在应用，我们检查了用几种不同化学物质处理 28 天的大鼠肝脏中 γ-H2AX 的形成。6 周龄雄性 F344 大鼠经口处理 28 天，用 5 种肝癌致癌物：N-亚硝基二乙胺（DEN）、邻苯二甲酸二（2-乙基己基）酯、1,4-二恶烷（DO）、3,3'-二甲基联苯胺二盐酸盐或硫代乙酰胺（TAA），或用 2 种非肝癌致癌物：4-氯邻苯二胺和 N-乙基-N-亚硝脲。在治疗期末，进行 γ-H2AX 和 Ki67 的免疫组织化学和 DNA 修复相关基因的表达分析。5 种肝癌致癌物中的 3 种（DEN、DO 和 TAA）诱导 γ-H2AX 阳性肝细胞显著增加，Rad51mRNA 表达上调，而另外 2 种肝癌致癌物和 2 种非肝癌致癌物未见变化。在 TAA 处理的大鼠中观察到 Ki67 表达显著增加，Brip1、Xrcc5 和 Lig4 上调，表明直接 DNA 损伤和细胞复制应激引起的继发性 DNA 损伤都可能与 γ-H2AX 的形成有关。这些结果表明，γ-H2AX 免疫染色法对肝癌致癌物的早期检测具有潜在价值，但需要检查更多化学物质的影响，以及是否应将 γ-H2AX 免疫染色法与其他标志物结合使用以提高敏感性。使用福尔马林固定石蜡包埋标本的 γ-H2AX 免疫染色可以很容易地纳入现有的 28 天重复剂量毒性研究中，并且预计该方法将进一步改进。
