Kimura Masayuki, Mizukami Sayaka, Watanabe Yousuke, Onda Nobuhiko, Yoshida Toshinori, Shibutani Makoto
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan; Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan.
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan.
Exp Toxicol Pathol. 2016 Aug;68(7):399-408. doi: 10.1016/j.etp.2016.06.002. Epub 2016 Jul 9.
The present study aimed to determine the onset time of hepatocarcinogen/hepatocarcinogenic tumor promoter-specific cell proliferation, apoptosis and aberrant cell cycle regulation after post-initiation treatment. Six-week-old rats were treated with the genotoxic hepatocarcinogen, carbadox (CRB), the marginally hepatocarcinogenic leucomalachite green (LMG), the tumor promoter, β-naphthoflavone (BNF) or the non-carcinogenic hepatotoxicant, acetaminophen, for 2, 4 or 6 weeks during the post-initiation phase using a medium-term liver bioassay. Cell proliferation activity, expression of G2 to M phase- and spindle checkpoint-related molecules, and apoptosis were immunohistochemically analyzed at week 2 and 4, and tumor promotion activity was assessed at week 6. At week 2, hepatocarcinogen/tumor promoter-specific aberrant cell cycle regulation was not observed. At week 4, BNF and LMG increased cell proliferation together with hepatotoxicity, while CRB did not. Additionally, BNF and CRB reduced the number of cells expressing phosphorylated-histone H3 in both ubiquitin D (UBD)(+) cells and Ki-67(+) proliferating cells, suggesting development of spindle checkpoint dysfunction, regardless of cell proliferation activity. At week 6, examined hepatocarcinogens/tumor promoters increased preneoplastic hepatic foci expressing glutathione S-transferase placental form. These results suggest that some hepatocarcinogens/tumor promoters increase their toxicity after post-initiation treatment, causing regenerative cell proliferation. In contrast, some genotoxic hepatocarcinogens may disrupt the spindle checkpoint without facilitating cell proliferation at the early stage of tumor promotion. This suggests that facilitation of cell proliferation and disruption of spindle checkpoint function are induced by different mechanisms during hepatocarcinogenesis. Four weeks of post-initiation treatment may be sufficient to induce hepatocarcinogen/tumor promoter-specific cellular responses.
本研究旨在确定启动后处理后肝癌致癌物/肝癌致癌肿瘤促进剂特异性细胞增殖、凋亡及异常细胞周期调控的起始时间。使用中期肝脏生物测定法，对六周龄大鼠在启动后阶段用遗传毒性肝癌致癌物卡巴多司（CRB）、边缘性肝癌致癌剂孔雀石绿（LMG）、肿瘤促进剂β-萘黄酮（BNF）或非致癌肝毒物对乙酰氨基酚进行2、4或6周的处理。在第2周和第4周对细胞增殖活性、G2至M期及纺锤体检查点相关分子的表达和凋亡进行免疫组织化学分析，并在第6周评估肿瘤促进活性。在第2周，未观察到肝癌致癌物/肿瘤促进剂特异性异常细胞周期调控。在第4周，BNF和LMG在导致肝毒性的同时增加了细胞增殖，而CRB则没有。此外，BNF和CRB在泛素D（UBD）（+）细胞和Ki-67（+）增殖细胞中均减少了表达磷酸化组蛋白H3的细胞数量，提示纺锤体检查点功能障碍的发生，而与细胞增殖活性无关。在第6周，所检测的肝癌致癌物/肿瘤促进剂增加了表达谷胱甘肽S-转移酶胎盘型的癌前肝灶。这些结果表明，一些肝癌致癌物/肿瘤促进剂在启动后处理后毒性增加，导致再生细胞增殖。相反，一些遗传毒性肝癌致癌物可能在肿瘤促进早期不促进细胞增殖的情况下破坏纺锤体检查点。这表明在肝癌发生过程中，细胞增殖的促进和纺锤体检查点功能的破坏是由不同机制诱导的。启动后4周的处理可能足以诱导肝癌致癌物/肿瘤促进剂特异性细胞反应。
