David, Rhiannon
(2009).
Xenobiotic metabolism and markers of genotoxicity in regulatory non-vertebrate ecotoxicological test species.
University of Birmingham.
Ph.D.
Abstract
Genotoxicants are well known and important environmental contaminants. However, current ecotoxicological testing strategies generally focus on apical and reproductive endpoints, with little consideration given to subtle sub-lethal effects such as genotoxicity. Moreover, even less information is available on the responses of lower animals and plants to such substances. In this study, the sub-lethal responses of the water flea Daphnia magna, and the unicellular alga Chlamydomonas reinhardtii were investigated following exposure to known genotoxic substances. These studies were aimed at providing fundamental information on the responses of these two species thus addressing their relevance as potential alternatives to the use of vertebrates (e.g. fish) for the ecotoxicological testing of the effects of such compounds. Methods were developed to allow the Comet assay to be applied to algal and daphnid cells. Statistically significant increases in DNA strand breaks were detected following exposure of algal cells to selected direct- and indirect-acting genotoxicants. An apparent relatively low sensitivity was not elevated by using a wall-free mutant. In contrast, the DNA damage responses in D. magna did not achieve statistical significance. Methods were also developed to allow the xenobiotic biotransformation capability (via 7-ethoxyresorufin-O-deethylase or EROD activity) of both organisms to be measured. C. reinhardtii and D. magna possessed low, but measurable, basal EROD activity (0.03 pmol/minute/106 algal cells and 7 fmol/minute/daphnid). In C. reinhardtii, a 48h exposure to β-naphthoflavone (0.2 and 1nM) did not significantly induce activity, and in D. magna, EROD activity was not significantly affected by the presence of dimethylsulfoxide (up to 0.1%) or methanol (up to 0.05%). In addition to the analysis of effects at the sub-cellular level, gene expression analysis using D. magna oligonucleotide microarrays was also undertaken to assess the effect of these gentoxicants, on DNA repair, at the transcriptomic level. The microarrays revealed unique expression profiles for adults and neonates and significantly higher expression of some DNA repair genes in adults. Following exposure to a mixture of sodium dichromate and benzo[a]pyrene, a greater number of DNA repair genes showed up-regulation in adults compared to neonates. The majority of modulated genes implicated sodium dichromate as the primary stressor (e.g. glutathione-S-transferases, peroxiredoxins, ferritins), and potential reproductive and population level effects were identified. In all this study has highlighted the potential use of C. reinhardtii and D. magna in genotoxicity testing, using the methods developed herein. These studies revealed that both species are able to activate and respond to genotoxicants, although there were some clear differences in terms of sensitivity to the compounds applied. Furthermore, novel gene expression profiling in D. magna also offers a potential complementary measurement of genotoxicant exposure, via the assessment of DNA repair capacity. This methodology could be applied in addition to standard mutation and DNA damage assays to provide a battery approach to non-vertebrate
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