In vitrogenotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment

Abstract

There is serious concern about the potential harmful effects of certain nanomaterials (NMs), on account of their ability to penetrate cell membranes and the increased reactivity that results from their increased surface area compared with bulk chemicals. To assess the safety of NMs, reliable tests are needed. We have investigated the possible genotoxicity of four representative NMs, derived from titanium dioxide, zinc oxide, cerium oxide and silver, in two human cell lines, A549 alveolar epithelial cells and lymphoblastoid TK6 cells. A high-throughput version of the comet assay was used to measure DNA strand beaks (SBs) as well as oxidised purines (converted to breaks with the enzyme formamidopyrimidine DNA glycosylase). In parallel, cytotoxicity was measured with the alamarBlue® assay, and the ability of NM-treated cells to survive was assessed by their colony-forming efficiency. TiO and CeO NMs were only slightly cytotoxic by the alamarBlue® test, and had no long-term effect on colony-forming efficiency. However, both induced DNA damage at non-cytotoxic concentrations; the damage decreased from 3 to 24-h exposure, except in the case of CeO-treated A549 cells. ZnO and Ag NMs affected cell survival, and induced high levels of DNA damage at cytotoxic concentrations. At lower concentrations, there was significant damage, which tended to persist over 24 h. The implication is that all four reference metal NMs tested-whether cytotoxic or not-are genotoxic. A full assessment of NM toxicity should include tests on different cell types, different times of incubation and a wide range of (especially non-cytotoxic) concentrations; a test for cell viability should be performed in parallel. Inclusion of Fpg in the comet assay allows detection of indirect genotoxic effects via oxidative stress.

Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats

“…Considering that respective findings were present only at high concentrations of >300 mg/kg BW, a potential for the induction of DNA damage via secondary rather than primary genotoxic mechanisms could be assumed for CeO 2 nanoparticles. This is further supported by in vitro testing of CeO 2 NM-212 revealing genotoxic effects at non-cytotoxic levels in different cell lines [51]. …”

Immunotoxicity, genotoxicity and epigenetic toxicity of nanomaterials: New strategies for toxicity testing?

“…DNA damage can be measured with various assays such as alkaline elution, neutral elution, DNA unwinding chromatography (Semisch et al, 2014), fluorometric detection of alkaline DNA unwinding (Moreno-Villanueva et al, 2011), or g-H2AX double strand break assay, but the most common test for detecting DNA damage after NM exposure is the comet assay (Magdolenova et al, 2014;Cowie et al, 2015;El Yamani et al, 2017).…”

“…Several cytotoxicity, immunotoxicity and genotoxicity assays have been adapted to HTS screening ). An example of the adaptation of a standard assay to HTS screening and miniaturization is the comet assay that can measure DNA breaks and specific DNA lesions (as well as, perhaps, global DNA methylation) (Watson et al, 2014;Dusinska et al, 2015;Harris et al, 2015;El Yamani et al, 2017). As well as saving time, the ability to run several hundred sample gels in one experiment means that interexperimental variation is less of an issue.…”

“…A HTS CometChip screening assay based on a microfabricated 96-well design with automated processing was also used to evaluate DNA damage caused by ZnO, Ag, Fe 2 O 3 , CeO 2 and SiO 2 NMs in human lymphoblastoid (TK6) cells in suspension, and adherent Chinese hamster ovary (H9T3) cells (Watson et al, 2014). HTS comet assay approaches, whether 12 mini-gels on a slide, 48 or 96 mini-gels on a GelBond film (Shaposhnikov et al, 2010;Gutzkow et al, 2013;Harris et al, 2015;El Yamani et al, 2017) or microfabricated 96-well CometChips (Watson et al, 2014) all show robustness, improved efficiency and reduced processing time, and in addition they reduce the risk of user bias in comparison with the standard comet assay.…”

Prevention of γ-Radiation-Induced DNA Damage in Human Lymphocytes Using a Serine-Magnesium Sulfate Mixture

“…[1][2][3] A simple, sensitive, versatile, rapid and economical method of assessing DNA damage is comet assay or single-cell gel electrophoresis; this involves embedding cells in a low-melting-point agarose (LMPA), lysis of the cells in neutral or alkaline conditions (pH >13) and electrophoresis of the suspended lysed cells. [4][5][6] A methyl-thiazole-tetrazolium (MTT) assay is a colourimetric assay for assessing cell viability and metabolic activity in which a yellow water-soluble tetrazolium dye is reduced by mitochondrial nicotinamide adenine dinucleotide phosphatedependent oxidoreductase enzymes of live, but not dead, cells to a purple formazan product that is insoluble in aqueous solutions. Under defined conditions, a MTT assay therefore reflects the number of viable cells present.…”