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High-throughput screening platform for engineered nanoparticle-mediated genotoxicity using CometChip technology.

ABSTRACT: The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO>Ag>Fe2O3>CeO2>SiO2 in TK6 cells at 4 h and Ag>Fe2O3>ZnO>CeO2>SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies.

SUBMITTER: Watson C

PROVIDER: S-EPMC3971959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genotoxicity is a critical component of a comprehensive toxicological profile. The Tox21 Program used five quantitative high-throughput screening (qHTS) assays measuring some aspect of DNA damage/repair to provide information on the genotoxic potential of over 10?000 compounds. Included were assays detecting activation of p53, increases in the DNA repair protein ATAD5, phosphorylation of H2AX, and enhanced cytotoxicity in DT40 cells deficient in DNA-repair proteins REV3 or KU70/RAD54. Each assay measures a distinct component of the DNA damage response signaling network; >70% of active compounds were detected in only one of the five assays. When qHTS results were compared with results from three standard genotoxicity assays (bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus), a maximum of 40% of known, direct-acting genotoxicants were active in one or more of the qHTS genotoxicity assays, indicating low sensitivity. This suggests that these qHTS assays cannot in their current form be used to replace traditional genotoxicity assays. However, despite the low sensitivity, ranking chemicals by potency of response in the qHTS assays revealed an enrichment for genotoxicants up to 12-fold compared with random selection, when allowing a 1% false positive rate. This finding indicates these qHTS assays can be used to prioritize chemicals for further investigation, allowing resources to focus on compounds most likely to induce genotoxic effects. To refine this prioritization process, models for predicting the genotoxicity potential of chemicals that were active in Tox21 genotoxicity assays were constructed using all Tox21 assay data, yielding a prediction accuracy up to 0.83. Data from qHTS assays related to stress-response pathway signaling (including genotoxicity) were the most informative for model construction. By using the results from qHTS genotoxicity assays, predictions from models based on qHTS data, and predictions from commercial bacterial mutagenicity QSAR models, we prioritized Tox21 chemicals for genotoxicity characterization.

Project description:Higher-throughput, mode-of-action-based assays provide a valuable approach to expedite chemical evaluation for human health risk assessment. In this study, we combined the high-throughput alkaline DNA damage-sensing CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq®, as an integrated genotoxicity testing approach. We used metabolically competent differentiated human HepaRG™ cell cultures to enable the identification of chemicals that require bioactivation to cause genotoxicity. We studied 12 chemicals (nine DDI, three non-DDI) in increasing concentrations to measure and classify chemicals based on their ability to damage DNA. The CometChip® classified 10/12 test chemicals correctly, missing a positive DDI call for aflatoxin B1 and propyl gallate. The poor detection of aflatoxin B1 adducts is consistent with the insensitivity of the standard alkaline comet assay to bulky lesions (a shortcoming that can be overcome by trapping repair intermediates). The TGx-DDI biomarker accurately classified 10/12 agents. TGx-DDI correctly identified aflatoxin B1 as DDI, demonstrating efficacy for combined used of these complementary methodologies. Zidovudine, a known DDI chemical, was misclassified as it inhibits transcription, which prevents measurable changes in gene expression. Eugenol, a non-DDI chemical known to render misleading positive results at high concentrations, was classified as DDI at the highest concentration tested. When combined, the CometChip® assay and the TGx-DDI biomarker were 100% accurate in identifying chemicals that induce DNA damage. Quantitative benchmark concentration (BMC) modeling was applied to evaluate chemical potencies for both assays. The BMCs for the CometChip® assay and the TGx-DDI biomarker were highly concordant (within 4-fold) and resulted in identical potency rankings. These results demonstrate that these two assays can be integrated for efficient identification and potency ranking of DNA damaging agents in HepaRG™ cell cultures.

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High-throughput screening platform for engineered nanoparticle-mediated genotoxicity using CometChip technology.

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