A critique of methods to measure cytotoxicity in mammalian cell genotoxicity assays

O’Donovan, Mike

doi:10.1093/mutage/ges045

Cited by 33 publications

(28 citation statements)

References 25 publications

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“…However, the proliferative capacity of the utilized cells remained at acceptable levels at all concentrations of nanoparticles, which has been shown to be a more sensitive index of cytotoxicity in genotoxicity testing (Figure 3F). 65 These data are in agreement with the numerous reports of deleterious effects of nanosilver not only in mammalian cells but also in aquatic organisms. 66−68 The use of silver ENPs may have several benefits; however, understanding the risks are essential to ensure product safety and eliminate public and environmental health concerns.…”

Section: Resultssupporting

confidence: 92%

High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

Watson¹,

Cohen³

et al. 2014

ACS Nano

145

125

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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.

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Section: Resultssupporting

confidence: 92%

High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

Watson¹,

Cohen³

et al. 2014

ACS Nano

145

125

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show abstract

“…Importantly, monitoring cytotoxicity is essential in genotoxicity assessments as high levels of toxicity can confound genotoxicity results. In both the ZnO NP and AgNP exposures, we observed a decline in cellular viability and proliferation of less than 30%, which is an acceptable level of toxicity when using the comet assay (73). …”

Section: Introductionmentioning

confidence: 92%

Emerging metrology for high-throughput nanomaterial genotoxicology

Nelson

Wright

Ibuki

et al. 2016

MUTAGE

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The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated ‘Fluorimetric Detection of Alkaline DNA Unwinding’ (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided.

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“…Increasing the specificity of currently used in vitro genotoxicity tests is a major challenge that can be addressed either through the optimization of conventional tests, or by the inclusion of novel short‐term assays [Kirkland, ]. According to several authors, the specificity of in vitro genotoxic assays could be greatly improved by a careful choice of the cell line used [Pfuhler et al, ; Fowler et al, ] and by more accurate toxicity measurements [Fellows et al, ,2012b; Clarke et al, ; O'Donovan, ]. The use of in vitro assays would also significantly reduce the number of animals required for genotoxicity assessment.…”

Section: Introductionmentioning

confidence: 99%

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

Khoury

Zalko

Audebert

2013

Environ and Mol Mutagen

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In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96-well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In-Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90-100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high-throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics.

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A critique of methods to measure cytotoxicity in mammalian cell genotoxicity assays

Cited by 33 publications

References 25 publications

High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

Emerging metrology for high-throughput nanomaterial genotoxicology

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

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