Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements

Donald H Atha,Jennifer A Hollingsworth,Bryant C Nelson,Rashi Iyer,Andrea Steinbrück,Varsha Dua,Allison M Dennis,Amber Nagy

doi:10.1186/s12951-017-0312-3

Abstract

BackgroundWhen 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.ResultsThe 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.ConclusionsThis 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.

Highlights

When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action
In the current study we compare 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 deoxyribonucleic acid (DNA) stand breaks
We compared the responses of cultured normal human bronchial epithelial (NHBE) cells to two types of semiconductor quantum dots (QDs) that were chosen based on their known difference in cytotoxicity: cadmium selenide (CdSe) QDs, which are known to produce significant toxic effects in cultured mammalian cells and indium phosphide (InP) QDs, which are reported to induce minimal toxicity to mammalian cells [10, 11, 17,18,19,20,21,22]

Summary

Introduction

When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. Gene expression analysis affords the opportunity to evaluate these mechanisms of toxicity, through the monitoring of regulatory genes that are affected Cellular processes such as the induction of inflammatory cytokines, autophagy, necrosis, and apoptosis have been shown to be affected by physical properties of ENMS, like size and charge, as well as chemical properties, including the core composition and surface functionalization [12,13,14,15,16]. In this respect, it is critical to know how gene expression data can be correlated with common cytotoxicity assays, to know what genes will be useful to monitor as potential indicators of toxicity and to characterize the sensitivity and reproducibility of the measurements. Considering these previous studies, CdSe and InP QDs functionalized with negatively charged mercaptoundecanoic acid (MUA) were chosen as well-characterized test materials to compare the results of the cytotoxicity assays and to determine if certain transcriptional changes related to DNA damage and repair and mitochondrial function can be used as predictive toxicological indicators in conjunction with prototypical cytotoxicity assays

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Results

Discussion

Conclusion