Technical University of Denmark, National Food Institute, Mørkhøj Bygade, 19-2860 Søborg, Denmark.
Mutat Res. 2010 Jul 19;700(1-2):18-25. doi: 10.1016/j.mrgentox.2010.04.021. Epub 2010 Apr 28.
The natural clay mineral montmorillonite (Cloisite) Na+) and an organo-modified montmorillonite (Cloisite 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-microm pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141microg/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p<0.01 and p<0.001) and (p<0.05 and p<0.01), respectively. The unfiltered samples were tested up to concentrations of 170microg/ml and the filtered samples up to 216microg/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57microg/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite) 30B.
天然粘土矿物蒙脱石（Cloisite）Na+和有机改性蒙脱石（Cloisite 30B）被研究为潜在的遗传毒性物质，以原状悬浮液和通过 0.2 微米孔径过滤器过滤的悬浮液的形式存在，以去除纳米级以上的颗粒。在沙门氏菌/微粒体试验中，使用测试菌株 TA98 和 TA100，浓度高达 141μg/ml 的原状粘土的过滤和未过滤水悬浮液均未诱导突变。在碱性彗星试验中，经过 24 小时暴露后，Caco-2 细胞中未检测到过滤和未过滤 Cloisite）Na+悬浮液在培养基中引起的 DNA 链断裂。然而，Cloisite 30B 的过滤和未过滤样品均以浓度依赖的方式诱导 DNA 链断裂，并且两个最高测试浓度与对照样品（p<0.01 和 p<0.001）和（p<0.05 和 p<0.01）相比，产生了统计学上显著不同的结果。在过滤之前，未过滤的样品测试浓度高达 170μg/ml，过滤的样品测试浓度高达 216μg/ml。在与彗星试验相同的浓度范围内进行测试时，在无细胞测试系统（DCFH-DA 测定）中，没有一种粘土产生 ROS。电感耦合等离子体质谱法（ICP-MS）用于使用铝作为粘土特征示踪元素检测过滤样品中的粘土颗粒。结果表明，过滤样品中不存在粘土颗粒，这一点通过动态光散射测量得到了独立证实。通过 HPLC-Q-TOF/MS 对过滤样品中的游离季铵改性剂进行了检测和鉴定，并发现混合物中总浓度为 1.57μg/ml 的季铵类似物。这些发现表明，有机改性蒙脱石的遗传毒性是由有机改性剂引起的。检测到的有机改性剂混合物被合成，彗星试验结果表明，在过滤的 Cloisite）30B 悬浮液中，等摩尔浓度的有机改性剂的合成有机改性剂的遗传毒性效力处于相同数量级，因此至少可以部分解释 Cloisite）30B 的遗传毒性作用。
