Biosystems and Biomaterials Division, National Institute of Standards and Technology, Bld. 227, Rm. A247, MS 8313, 100 Bureau Drive, Gaithersburg, MD, 20899, USA.
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA.
J Nanobiotechnology. 2017 Nov 9;15(1):79. doi: 10.1186/s12951-017-0312-3.
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
背景：在评估工程纳米材料（ENMS）的毒性时，使用基于不同作用机制的多种生物测定法非常重要。在这方面，我们评估了使用基因表达和常见细胞毒性测量的方法，使用两种具有已知毒性差异的纳米颗粒作为测试材料，即 5nm 巯基十一酸（MUA）封端的 InP 和 CdSe 量子点（QDs）。我们使用四种常见的细胞毒性测定法，即用于测定活性氧（ROS）的二氯荧光素测定法、用于测定膜活力的乳酸脱氢酶（LDH）测定法、用于测定线粒体功能的线粒体脱氢酶测定法和用于测定 DNA 链断裂的彗星测定法，在 0.5 至 160μg/mL 的浓度范围内测试了这些 QDs 对培养的正常人类支气管上皮（NHBE）细胞的影响。
结果：在 80μg/mL 下暴露 24 小时时，细胞毒性测定法显示出相似的趋势，与暴露于 InP QDs 相比，暴露于 CdSe QDs 时 ROS 增加了三倍，而 ROS 水平无明显变化；与暴露于 InP QDs 相比，LDH 坏死测定法中 NHBE 细胞的 LDH 增加了两倍，而暴露于 CdSe QDs 时减少了 50%；与暴露于 InP 时的最小增加相比，暴露于 CdSe QDs 时的线粒体功能测定法降低了 60%，而暴露于 CdSe QDs 时则出现了明显的 DNA 链断裂，而暴露于 InP 时则没有明显的 DNA 链断裂。暴露于 80μg/mL 浓度下 6 小时的细胞的高通量实时聚合酶链反应（qRT-PCR）数据与细胞毒性测定法一致，表明 CdSe 和 InP QDs 对 DNA 损伤、DNA 修复和线粒体功能基因调控反应有明显差异。观察到 BRCA2、CYP1A1、CYP1B1、CDK1、SFN 和 VEGFA 基因的上调，这表明它们可能可作为毒性的生物标志物。
结论：本研究可作为比较传统细胞毒性测定法和基因表达测定法的模型，并确定用于评估 ENMs 生物相容性的候选生物标志物。
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