Krishna G, Kropko M L, Theiss J C
Department of Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105.
Mutat Res. 1989 Jan;222(1):63-9. doi: 10.1016/0165-1218(89)90036-0.
The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.
已对细胞松弛素B（CYB）阻断双核细胞试验进行了探索，以使用两种已知的诱变致癌物分析V79中国仓鼠肺细胞中的微核和细胞周期动力学。为了确定获得用于微核分析的最大数量双核细胞的最佳时间，将指数生长细胞的重复培养物用3微克/毫升CYB处理不同持续时间（8 - 48小时）。在存在CYB的情况下，双核细胞在16小时出现峰值，表明这是双核V79细胞微核分析的最佳时间。为了评估V79细胞中微核诱导能力，在重复培养物中测试了两种诱变致癌物，丝裂霉素C（0.125 - 1.0微克/毫升）和环磷酰胺（2 - 12微克/毫升）。丝裂霉素C是一种直接作用的烷化剂，在测试的最高浓度（1.0微克/毫升）下，与对照相比微核频率增加了约18倍，并且这种增加呈剂量相关方式（r = 0.92）。测试的丝裂霉素C浓度也导致了明显的剂量相关细胞周期延迟，因此表明对V79细胞具有细胞毒性。环磷酰胺是一种间接作用的烷化剂，需要存在S9混合物，在测试的最高浓度（12微克/毫升）下，与对照相比微核频率增加了约17倍，具有明显的剂量反应（r = 0.99）。不同浓度的环磷酰胺也以剂量相关方式引起细胞毒性。因此，本研究证明了胞质分裂阻断法在V79细胞中作为分析微核诱导和细胞毒性的可能筛选方法的有用性。由于这种方法比进行结构染色体分析所需的劳动力少得多，该试验作为致断裂活性的初步筛选以及研究致断裂性潜在机制的工具都具有巨大潜力。
