Zhong B Z, Depree G J, Siegel P D
Analytical Services Branch, M/S L4218, Health Effect Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505-2888, USA.
Mutat Res. 2001 Oct 18;497(1-2):29-37. doi: 10.1016/s1383-5718(01)00246-7.
Methylenedi-p-phenyl diisocyanate (MDI) is widely used in the production of polyurethane products. Diisocyanates are reactive compounds, MDI can react under physiological conditions with various functional groups found on biological molecules resulting in conjugate formation or undergo non-enzymatic hydrolysis to form 4,4'-methylenedianiline (MDA). We have previously reported that addition of MDI directly to Chinese hamster lung fibroblasts (V79) cultures did not induce micronuclei (MN), but MDA, and the glutathione and cysteine conjugates of MDI (BisGS-MDI and BisCYS-MDI), induced a concentration-dependent increase in the frequency of MN. The conventional MN assay does not discriminate between MN produced by acentric chromosome fragments from those arising due to whole lagging chromosomes that were not incorporated into daughter nuclei at the time of cell division. The mechanism of MN induction from these potential MDI metabolites/reaction products was explored in the present study using immunofluorescent staining of kinetochore in MN of cytokinesis-blocked V79 cells. This assay discerns the presence of centromere within the MN to distinguish the MN containing centric chromosomes from those containing acentric fragments. Eighty five percent of MDA-induced MN were negative with respect to anti-kinetochore antibody binding (KC(-)). This is consistent with an interaction between MDA and DNA resulting in chromosome breakage. However, BisGS-MDI and BisCYS-MDI induced a higher percentage of MN that were positively stained by the anti-kinetochore antibody (KC(+)). These results suggest that the mechanism of MN formation induced by BisGS-MDI and BisCYS-MDI is mediated through disruption and/or by affecting the function of the mitotic spindle. This mechanism is distinctly different from the mechanism of MN induction by MDA.
亚甲基二苯基二异氰酸酯（MDI）广泛用于聚氨酯产品的生产。二异氰酸酯是反应性化合物，MDI可在生理条件下与生物分子上发现的各种官能团发生反应，导致共轭物形成，或进行非酶水解形成4,4'-亚甲基二苯胺（MDA）。我们之前报道过，将MDI直接添加到中国仓鼠肺成纤维细胞（V79）培养物中不会诱导微核（MN），但MDA以及MDI的谷胱甘肽和半胱氨酸共轭物（双谷胱甘肽-MDI和双半胱氨酸-MDI）会诱导MN频率呈浓度依赖性增加。传统的MN检测方法无法区分由无着丝粒染色体片段产生的MN和由于整个滞后染色体在细胞分裂时未并入子核而产生的MN。在本研究中，使用细胞分裂阻滞的V79细胞MN中的着丝粒免疫荧光染色，探讨了这些潜在的MDI代谢物/反应产物诱导MN的机制。该检测方法可识别MN内着丝粒的存在，以区分含有中心染色体的MN和含有无着丝粒片段的MN。85%的MDA诱导的MN与抗着丝粒抗体结合呈阴性（KC(-)）。这与MDA和DNA之间的相互作用导致染色体断裂一致。然而，双谷胱甘肽-MDI和双半胱氨酸-MDI诱导的MN中，抗着丝粒抗体阳性染色（KC(+)）的比例更高。这些结果表明，双谷胱甘肽-MDI和双半胱氨酸-MDI诱导MN形成的机制是通过破坏和/或影响有丝分裂纺锤体的功能介导的。这种机制与MDA诱导MN的机制明显不同。
