Chromosomal genotoxicity of nitrobenzene and benzonitrile
Chromosomal genotoxicity of nitrobenzene and benzonitrile
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DOI：
10.1007/s00204-003-0508-1
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发表时间：
2004-01-01
影响因子：
6.1
通讯作者：
Bolt, HM
中科院分区：
文献类型：
--
作者：
Bonacker, D;Stoiber, T;Bolt, HM
为了研究硝基苯和苯甲腈的染色体遗传毒性，我们研究了这些供试化合物在V79细胞中诱导微核（MN），以及对微管形成和稳定性以及对运动蛋白功能的影响。在用高达1 mM硝基苯或1 mM苯甲腈处理18 h后，在V79细胞培养物中未观察到中性红摄取方面的细胞毒性。随后，在MN诱导的实验中使用高达100 μ M的浓度范围。与溶剂（DMSO）对照相比，两种供试化合物均显示出弱但肯定的阳性试验结果。硝基苯和苯甲腈的最小效应浓度低至0.01 μ M，无效应浓度在0.001和0.005 μ M之间。在0.1 μ M和更高浓度下发现明显增强的MN率。硝基苯和苯甲腈均主要诱导动粒（CREST）阳性微核，因此将染色体效应表征为非整倍体效应。在无细胞测定中，在不添加DMSO的情况下，在1 mM硝基苯下观察到对微管蛋白组装的轻微影响。较高浓度（5 mM）导致继发性效应。在1%DMSO存在下，硝基苯对微管蛋白组装没有可检测的影响，直到在水中的溶解度极限约为15 mM。对于苄腈，在DMSO存在下，在37 ℃下，在2 mM的无作用浓度以上观察到抑制微管蛋白组装的明显剂量反应，IC 50为13 mM，蛋白质变性开始于高于约20 mM的水平。硝基苯和苯甲腈对微管蛋白结合形成微管的作用的性质通过电子显微镜证实。通过5 mM硝基苯或13 mM苯甲腈加1%DMSO的处理使微管结构保持完整，而在不存在DMSO的情况下，5 mM硝基苯导致不规则的簇形成。实验表明，硝基苯和苯甲腈，在毫摩尔浓度范围内，可能会导致干扰微管蛋白组装在无细胞系统。使用微管滑动试验评估了微管蛋白-驱动蛋白运动蛋白系统的功能。硝基苯以浓度依赖性方式影响滑行速度，从约7.5 μ M开始，在30 μ M时达到完全抑制运动，而苯甲腈高达200 μ M不影响驱动蛋白驱动的滑行速度。微核试验数据证明了硝基苯和苯甲腈遗传毒性的染色体终点。这两种化合物的非整倍体效应发生在非常低的浓度下，最低效应浓度为0.1 μ M。这表明了与细胞纺锤体相互作用的相关性。
In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 muM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 muM, and no-effect-concentrations were between 0.001 and 0.005 muM. Clearly enhanced MN rates were found at 0.1 muM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37degreesC was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 muM and reaching complete inhibition of motility at 30 muM, whereas benzonitrile up to 200 muM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 muM. This points to the relevance of interactions with the cellular spindle apparatus.