Roscher E, Wiebel F J
Institut für Toxikologie, Gesellschaft für Strahlen- und Umweltforschung, München, Neuherberg, FRG.
Mutagenesis. 1988 May;3(3):269-76. doi: 10.1093/mutage/3.3.269.
The present studies were aimed at evaluating the suitability of the differentiated Reuber hepatoma cells H4IIEC3/G- for monitoring permanent damage to the DNA caused by hepatotrophic chemicals. First we determined the profile of xenobiotic metabolizing enzymes. The cells expressed various cytochrome P-450-dependent monooxygenases, UDP-glucuronosyl-, phenol sulpho- and glutathione S-transferase, cytochrome c (P-450) reductase and carboxylesterases. We then established the conditions for genotoxicity testing in H4IIEC/G- cells. Induction of resistance against 6-thioguanine and appearance of micronuclei served as indicators for mutagenicity and clastogenicity, respectively. 6-Thioguanine-resistant H4IIEC3/G- cells were phenotypically stable for at least 30 cell cycles; recovery of 6-thioguanine-resistant cells was not significantly affected by the number of cells seeded for mutant selection up to at least 10(6) cells/100-mm dish; expression time of chemically induced mutants was 12-15 days; a period of 24 h after treatment appeared to be sufficient to allow for the formation of micronuclei. Finally we tested the genotoxic effects of promutagens which are typically activated or inactivated in liver. Aflatoxin B1, N-nitrosodiethylamine and cyclophosphamide were genotoxic to H4IIEC3/G- cells at concentrations of 10-30 nM, 2-20 mM and 1 mM, respectively. N-Nitrosodimethylamine and benzo[a]pyrene were not or only weakly cytotoxic and genotoxic to the cells, but this appears most likely to be due to protective mechanisms rather than to lack of metabolic activation. The results indicate that differentiated hepatoma cells such as H4IIEC3/G- offer a means of studying the potential of chemicals for inducing permanent DNA damage in liver cells.
本研究旨在评估分化的鲁伯肝癌细胞H4IIEC3/G-用于监测肝营养化学物质对DNA造成的永久性损伤的适用性。首先，我们测定了异生物质代谢酶的谱。这些细胞表达了各种细胞色素P-450依赖性单加氧酶、UDP-葡糖醛酸基转移酶、酚磺基转移酶和谷胱甘肽S-转移酶、细胞色素c（P-450）还原酶和羧酸酯酶。然后，我们确定了H4IIEC/G-细胞遗传毒性测试的条件。对6-硫鸟嘌呤抗性的诱导和微核的出现分别作为致突变性和染色体断裂剂的指标。对6-硫鸟嘌呤抗性的H4IIEC3/G-细胞在至少30个细胞周期内表型稳定；对于突变体选择，接种细胞的数量高达至少10(6)个细胞/100-mm培养皿时，6-硫鸟嘌呤抗性细胞的恢复不受显著影响；化学诱导突变体的表达时间为12-15天；处理后24小时的时间段似乎足以形成微核。最后，我们测试了通常在肝脏中被激活或失活的前诱变剂的遗传毒性作用。黄曲霉毒素B1、N-亚硝基二乙胺和环磷酰胺分别在10-30 nM、2-20 mM和1 mM的浓度下对H4IIEC3/G-细胞具有遗传毒性。N-亚硝基二甲胺和苯并[a]芘对细胞无或仅有微弱的细胞毒性和遗传毒性，但这似乎最有可能是由于保护机制而非缺乏代谢激活。结果表明，分化的肝癌细胞如H4IIEC3/G-提供了一种研究化学物质诱导肝细胞永久性DNA损伤潜力的方法。
