Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran.
Physiology Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Kerman, Iran.
Toxicol In Vitro. 2020 Jun;65:104796. doi: 10.1016/j.tiv.2020.104796. Epub 2020 Feb 15.
For in vitro studies, non-toxic doses of nanomaterials are routinely selected by quantification of live cells after exposing to different concentrations of nanoparticles but considering only morphological changes or viability of cells is not sufficient to conclude that these nanomaterials are non-cytotoxic. Here we investigated if secondary toxicity is active in the cells exposed to non-toxic doses of graphene oxide (GO) and graphene quantum dots (GQDs). Non-cytotoxic dose of 15 μg mL of GO (100 nm) and GQDs (50 nm) was selected according to MTT and Hoechst 33342/PI double staining assays. In order to investigate the secondary toxicity, the expression of miR-21, miR-29a and three genes at both mRNA and protein level were evaluated in MCF-7, HUVEC, KMBC/71 cells 4 and 24 h post exposure. Mitochondrial membrane potential (MMP) was assessed by Rhodamine 123 staining. According to our results, there was no significant decrease in viability of cells after exposure to the non-cytotoxic dose of GO and GQDs, but we observed significant alterations in the expression level of miR-21, miR-29a, Bax, Bcl2 and PTEN genes after treatment in all three cells. In addition to molecular changes, we observed alteration in mitochondrial activity at cellular level. However, we also observed that GO influenced the basal level of genes and MMP more compare to GQDs. Considering that all these genes are involved in breast tumor development and metastasis, the observed changes in miRNA expression and protein synthesis may alter cell fate and susceptibility and cause deviation in the desired outcome of GO and GQDs application in medical research.
对于体外研究，通常通过暴露于不同浓度的纳米颗粒后对活细胞进行定量来选择无毒剂量的纳米材料，但仅考虑细胞的形态变化或活力不足以得出这些纳米材料没有细胞毒性的结论。在这里，我们研究了暴露于无毒剂量的氧化石墨烯（GO）和石墨烯量子点（GQDs）的细胞中是否存在继发性毒性。根据 MTT 和 Hoechst 33342/PI 双重染色试验，选择了非细胞毒性剂量的 15μg/mL GO（100nm）和 GQDs（50nm）。为了研究继发性毒性，在 MCF-7、HUVEC、KMBC/71 细胞中，在暴露后 4 和 24 小时评估了 miR-21、miR-29a 和三个基因的 mRNA 和蛋白水平的表达。通过 Rhodamine 123 染色评估线粒体膜电位（MMP）。根据我们的结果，暴露于 GO 和 GQDs 的无毒剂量后，细胞活力没有明显下降，但我们观察到所有三种细胞中 miR-21、miR-29a、Bax、Bcl2 和 PTEN 基因的表达水平都发生了显著改变。除了分子变化外，我们还观察到细胞水平线粒体活性的改变。然而，我们还观察到 GO 比 GQDs 更能影响基因和 MMP 的基础水平。考虑到所有这些基因都参与乳腺癌的发展和转移，miRNA 表达和蛋白质合成的观察到的变化可能会改变细胞命运和易感性，并导致 GO 和 GQDs 在医学研究中的应用偏离预期结果。
