Hazard Assessment of Benchmark Metal-Based Nanomaterials Through a Set of In Vitro Genotoxicity Assays.

For safety assessment of nanomaterials (NMs), in vitro genotoxicity data based on well-designed experiments is required. Metal-based NMs are amongst the most used in consumer products. In this chapter, we report results for three metal-based NMs, titanium dioxide (NM-100), cerium dioxide (NM-212) and silver (NM-302) in V79 cells, using a set of in vitro genotoxicity assays covering different endpoints: the medium-throughput comet assay and its modified version (with the enzyme formamidopyrimidine DNA glycosylase, Fpg), measuring DNA strand beaks (SBs) and oxidized purines, respectively; the micronucleus (MN) assay, assessing chromosomal damage; and the Hprt gene mutation test. The results generated by this test battery showed that all NMs displayed genotoxic potential. NM-100 induced DNA breaks, DNA oxidation damage and point mutations but not chromosome instability. NM-212 increased the level of DNA oxidation damage, point mutations and increased the MN frequency at the highest concentration tested. NM-302 was moderately cytotoxic and induced gene mutations, but not DNA or chromosome damage. In conclusion, the presented in vitro genotoxicity testing strategy allowed the identification of genotoxic effects caused by three different metal-based NMs, raising concern as to their impact on human health. The results support the use of this in vitro test battery for the genotoxicity assessment of NMs, reducing the use of more expensive, time-consuming and ethically demanding in vivo assays, in compliance with the 3 R's.

A multi-endpoint approach to the combined toxic effects of patulin and ochratoxin a in human intestinal cells.

Humans can be exposed to a complex and variable combination of mycotoxins. After ingestion, intestinal mucosa constitutes the first biological barrier that can be exposed to high concentrations of these toxins. The present study aimed to characterize the combined cytotoxicity, genotoxicity and impact on the gastrointestinal barrier integrity of patulin (PAT, 0.7 µM to 100 µM) and ochratoxin A (OTA, 1 µM to 200 µM) mixtures in Caco-2 cells. A dose-ratio deviation was verified for cytotoxicity, implying that OTA was mainly responsible for synergism when dominant in the mixture, while this pattern was changed to antagonism for the highest PAT concentrations. Genotoxicity (comet assay) results were compatible with an interactive DNA damaging effect at the highest PAT and OTA concentrations, not clearly mediated by the formation of oxidative DNA breaks. Regarding gastrointestinal barrier integrity, a potential synergism was attained at low levels of both mycotoxins, changing to antagonism at higher doses. The present results indicate that combined mycotoxins effects may arise at the intestinal level and should not be underestimated when evaluating their risk to human health.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties.

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in BBEAS-2Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities.