Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements.
Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements.
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DOI：
10.1186/s12951-017-0312-3
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发表时间：
2017-11-09
影响因子：
10.2
通讯作者：
Nelson BC
中科院分区：
文献类型：
--
作者：
Atha DH;Nagy A;Steinbrück A;Dennis AM;Hollingsworth JA;Dua V;Iyer R;Nelson BC
在评估工程纳米材料（ENMS）的毒性时，重要的是要根据不同的作用机制使用多种生物测定法。在这方面，我们评估了基因表达的使用和常见的细胞毒性测量，使用作为测试材料，两种选择的纳米颗粒具有已知的毒性差异，5 nm巯基十一烷酸（MUA）-封端的InP和CdSe量子点（QD）。我们使用四种常见的细胞毒性试验测试了浓度范围为0.5至160 µg/mL的这些QD对培养的正常人支气管上皮（NHBE）细胞的影响：用于活性氧（ROS）的二氯荧光素试验，用于膜活力（LDH）的乳酸脱氢酶试验，用于线粒体功能的线粒体脱氢酶试验和用于DNA链断裂的彗星试验。当暴露于80 μg/mL的纳米颗粒24小时时，细胞毒性测定显示出类似的趋势，与暴露于InP QD后ROS水平的不显著变化相比，暴露于CdSe QD时ROS增加了三倍，与暴露于InP QD时50%的降低相比，暴露于CdSe QD时NHBE细胞中LDH坏死测定增加了两倍，与InP情况下的最小增加相比，暴露于CdSe QD后线粒体功能测定降低60%，并且与InP情况下无显著DNA链断裂相比，暴露于CdSe QD后显著DNA链断裂。在80 µg/mL浓度下暴露6 h的细胞的高通量定量实时聚合酶链反应（qRT-PCR）数据与细胞毒性试验一致，表明对CdSe和InP QD的DNA损伤、DNA修复和线粒体功能基因调控反应存在重大差异。BRCA 2，CYP 1A 1，CYP 1B 1，CDK 1，SFN和VEGFA基因被观察到上调，特别是从增加的CdSe暴露，并建议其可能的效用作为生物标志物的毒性。这项研究可以作为一个模型，比较传统的细胞毒性试验和基因表达的测量，并确定候选生物标志物评估ENM的生物相容性。本文的在线版本（10.1186/s12951-017-0312-3）包含补充材料，可供授权用户使用。
When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. This study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs. The online version of this article (10.1186/s12951-017-0312-3) contains supplementary material, which is available to authorized users.
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