Kikkawa Rie, Fujikawa Masaaki, Yamamoto Toshinori, Hamada Yoshimasa, Yamada Hiroshi, Horii Ikuo
Worldwide Safety Sciences, Pfizer Global Research and Development, Nagoya Laboratories, Pfizer Inc., 5-2 Taketoyo, Aichi 470-2393, Japan.
J Toxicol Sci. 2006 Feb;31(1):23-34. doi: 10.2131/jts.31.23.
For the establishment of a high throughput screening system using primary cell cultures, investigation of elucidated toxicities to assess the correlation between in vitro and in vivo hepatotoxicity is necessary in the safety evaluation of the compound. In the previous study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hepatocytes culture screening system, we conducted a single-dose in vivo study with relatively high dose of hepatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochemistry. We also carried out a proteomics analysis for estimating the reliable and sensitive biomarkers. Histopathologically, compound-specific hepatotoxicity was detected at 24 hr after administration in all compounds except amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative stress-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealed several protein biomarkers related to oxidative stress and mitochondrial metabolism-regulation, which had been previously detected by proteomics analysis in in vitro screening system. Oxidative stress-related proteins were considered as useful biomarkers of hepatotoxicity; since they were detected by immunohistochemistry and proteomics analysis prior to appearance of compound-specific histopathological changes detected by light microscopy. Considering the relevance of in vitro system to in vivo system from the aspect of new biomarkers related to the toxicogenomics/toxicoproteomics, in vitro primary cell culture system would be sufficient to detect hepatotoxicity in the early stage of drug discovery.
为了建立使用原代细胞培养的高通量筛选系统，在化合物的安全性评估中，研究已阐明的毒性以评估体外和体内肝毒性之间的相关性是必要的。在先前的研究中，我们报道了大鼠原代培养肝细胞在建立高通量筛选系统中的可用性。为了确认大鼠原代肝细胞培养筛选系统的可靠性，我们使用4种参考化合物（对乙酰氨基酚、胺碘酮、四环素、四氯化碳）对大鼠进行了相对高剂量肝毒性药物的单剂量体内研究，并通过免疫组织化学研究了组织病理学变化和氧化应激相关蛋白的表达。我们还进行了蛋白质组学分析以估计可靠且敏感的生物标志物。在组织病理学上，除已知会诱导磷脂沉着症的胺碘酮外，所有化合物在给药后24小时均检测到化合物特异性肝毒性。在免疫组织化学方面，所有处理组在给药后6小时内氧化应激相关蛋白均增加。蛋白质组学分析揭示了几种与氧化应激和线粒体代谢调节相关的蛋白质生物标志物，这些标志物先前已在体外筛选系统的蛋白质组学分析中检测到。氧化应激相关蛋白被认为是肝毒性的有用生物标志物；因为它们在光学显微镜检测到化合物特异性组织病理学变化出现之前就通过免疫组织化学和蛋白质组学分析被检测到了。从与毒理基因组学/毒理蛋白质组学相关的新生物标志物的角度考虑体外系统与体内系统的相关性，体外原代细胞培养系统足以在药物发现的早期阶段检测肝毒性。
