Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single‐cell gel electrophoresis and cytokinesis‐block micronucleus assay

Abstract

To protect human and ecosystem health, it is necessary to develop sensitive assays and to identify responsive cells and species (and their life stages). In this study, the relative genotoxicity of two inorganic arsenicals: trivalent sodium arsenite (As(3+)) and pentavalent sodium arsenate (As(5+)), was evaluated in two cell lines of phylogenetically different origin, using alkaline single-cell gel electrophoresis (i.e., the Comet assay) and the cytokinesis-block micronucleus (MN) assay. The cell lines were the rainbow trout gonad-2 (RTG-2) and Chinese hamster ovary-K1 (CHO-K1) lines. Following optimization and validation of both assays using reference chemicals (i.e., 1-100 microM hydrogen peroxide for the Comet assay and 1-10 mM ethylmethane sulfonate for the MN assay), cells were exposed to 1-10 microM of both arsenicals to determine the relative extent of genetic damage. The unexposed controls showed similar (background) levels of damage in both cell lines and for both assays. Treatment with the arsenicals induced concentration-dependent increases in genetic damage in the two cell lines. Arsenite was more potent than arsenate in inducing DNA strand breaks in the Comet assay; at the highest concentration (10 microM) arsenite produced similar levels of DNA damage in CHO-K1 and RTG-2 cells, while 10 microM arsenate was significantly more genotoxic in RTG-2 cells. MN induction was consistently higher in RTG-2 cells than in CHO-K1 cells, with 10 microM arsenite inducing an approximate 10-fold increase in both cell lines. MN induction also was positively correlated with DNA strand breaks for both arsenicals. Overall, the study demonstrated that the fish cells are more sensitive than the mammalian cells at environmentally realistic concentrations of both arsenicals, with arsenite being more toxic.

Intracellular Arsenic Speciation and Quantification in Human Urothelial and Hepatic Cells

“…We detected significant genotoxic effects of all tested trivalent arsenic compounds (As(III), MMA(III), and DMA(III)) and the pentavalent arsenate already after 30 min of exposure. These results are in compliance with the findings of previous studies using several mammalian cell types (Schwertdtle et al, 2003;Raisuddin & Jha, 2004;Dopp et al, 2005). This emphasises how urgently the knowledge of the tissuedependent arsenic biotransformation ( Fig.…”

Comet Assay measurements: a perspective

“…Attempts have been made to make this assay widely acceptable, by correlating the results with other well-established assays (e.g. micronucleus assay) whilst determining the genotoxicity (Raisuddin and Jha 2004). Before it can be accepted as a regulatory tool, this assay has to be harmonised in terms of its methodology and interpretations and should be demonstrated to be reliable, accurate and transferable between laboratories.…”

Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro

“…Cytotoxicity was determined using the neutral red retention (NRR) assay. While the comet and MN assays are well-established methods for evaluating the potential genotoxic effects in ecotoxicological studies (Dixon et al 2002;Jha 2004Jha , 2008Raisuddin and Jha 2004;Reeves et al 2008), the NRR assay has been recommended for regulatory use and in particular phototoxic evaluation of chemicals by OECD and NIH (Spielmann et al 1998). …”

“…microtubules) and the DNA (Dopp et al 1997). Elucidation of such mechanisms is important given that origin and properties of the cells could influence the toxic response (Raisuddin and Jha 2004). For example, different cell types (e.g.…”

“…This cell line has also been found to be more sensitive than standard mammalian cells (i.e. Chinese hamster ovary or CHO cell line) for different genotoxic endpoints (Raisuddin and Jha 2004). Prior to their genotoxic and cytotoxic evaluation, the mean size of the ENPs was characterised along with their effects on cellular adhesion and surface distribution using electron microscopy (i.e.…”