Analysis of mixture toxicity for dual combinations of heavy metals and herbicides using toxicogenomic approach

单一化学细胞毒性试验表明，重金属和银纳米颗粒毒性较高，而除草剂的毒性相对较低。相反，双重组合的细胞毒性显示，银纳米颗粒和重金属的组合往往比银纳米颗粒或重金属单独的毒性大得多，不包括银纳米颗粒和铬的组合(AgNPs-Cr)。然而，除草剂与重金属联合作用的毒性与单独使用除草剂的毒性相似。根据细胞毒性结果，使用5μM的银纳米颗粒、2.6μM的镉、7μM的砷和1.1μM的铬进行基因芯片分析，以进一步阐明混合毒性的机制。AgNPs-CD(70%细胞存活率)、AgNPs-AS(100%细胞存活率)和AgNPs-Cr(140%细胞存活率)处理分别改变了107个、500个和196个基因的表达水平。在DNA复制分叉的重要基因MCM2、MCM5、MCM6和GINS2上，两种组合(AgNPs-Cr和AgNPs-Cd)与单独使用银纳米颗粒相比差异有统计学意义。此外，在AgNPs-AS联合作用下，FABP1基因下调了41倍。FABP在脂类向细胞内特定隔室的运输中起着重要作用。综上所述，毒物基因组学方法可以解释AgNPs与重金属的协同毒性，这清楚地表明了DNA微阵列分析用于混合物毒性评价的可能性。

The single chemical cytotoxicity showed that heavy metals and silver nanoparticles had high toxicity, respectively, but herbicides had relatively low toxicity. In contrast, the dual combinations cytotoxicity showed that the combination of silver nanoparticles and heavy metals tended to be much more toxic than silver nanoparticles or heavy metals alone, excluding the combination of silver nanoparticles and chromium (AgNPs-Cr). However, the toxicity of the combination of herbicides and heavy metals was similar to that of herbicides exposure alone. From cytotoxicity results, 5 μM of silver nanoparticles, 2.6 μM of cadmium, 7μM of arsenic and 1.1 μM of chromium were used for the DNA microarray analyses to further elucidate the mechanisms of mixture toxicity. The treatments with AgNPs-Cd (70% cytoviability), AgNPs-As (100% cytoviability) and AgNPs-Cr (140% cytoviability) altered the expression levels of 107 genes, 500 genes and 196 genes, respectively. There was a significant difference between 2 combinations (AgNPs-Cr and AgNPs-Cd) and silver nanoparticles alone on DNA replication genes such as MCM2, MCM5, MCM6, and GINS2 that are important for DNA replication fork. Furthermore, FABP1 gene was down regulated 41-fold by AgNPs-As combination. FABPs play an important role in the transport of lipids to specific compartments in the cell. In conclusion, toxicogenomic approach could elucidate synergistic toxicity by AgNPs and heavy metals, which clearly demonstrate the possible use of DNA microarray analysis for evaluation of mixture toxicity.