The project consisted of three work packages (WPs). Briefly, in WPs1&2 the behavioural consequences of developmental exposure to MeHg were to be tested first in wild-type zebrafish (WP1) and in fish lines that were mutated in two Gst genes associated with increased susceptibility to MeHg neurotoxicity in humans (WP2). In WP3, levels of MeHg retention and oxidative-stress levels were to be measured in fish following exposure, as well as effects of MeHg on gene expression. All main project aims were achieved fully or partially.
First, we established a ‘low-dose’ range, i.e. exposure levels that do not cause overt toxicity, by conducting preliminary exposures using a wide range of MeHg concentrations (5 nM-1 µM). The exposure doses were adjusted until a dose range was achieved where there was no effect on embryonic and larval survival or on swimming capacity (5-30 nM MeHg). We then characterised the behavioural effects of low-dose MeHg developmental exposure in wild-type zebrafish larvae and adults (WP1). In 6 days-old larvae we found that MeHg exposure caused significant increases in locomotor response to light/dark transitions, that increased in a dose dependent manner, indicating an elevated startle response. In adults, a significant and dose dependent effect was found on response to the novel tank dive test, which is an assay for anxiety-related behaviour triggered by introduction to a novel environment. We found that the lower dose exposure led to an increase in the dive response, indicating elevated anxiety, while higher doses also caused reduction in locomotor activity which may indicate motor impairment. Together, these behavioural changes suggest that developmental exposure to very low-doses of MeHg alters normal response to anxiety-promoting situations from early age into adulthood, while slightly higher dosages also impair locomotor activity. Interestingly, the locomotor effect was only observed in adult fish, several months after exposure termination, suggesting a delayed effect.
For WP2 we applied the CRISPR-Cas9 method to generate two new zebrafish lines with mutations in the genes Gstt1a and Gstp1. The effects of low-dose MeHg developmental exposure were tested in Gstt1a larvae, but not in adults due to time constraints. Results from the testing are still being analysed.
In WP3, we conducted comparative gene expression analysis using qPCR in wild-type (non-mutant) exposed and non-exposed fish to identify molecular pathways related to MeHg-induced neurotoxicity. We measured the expressions of a selected sample of genes related to the glutathione and oxidative stress pathways, the hypothalamus-pituitary-interrenal (HPI) axis and dopamine function. We found that MeHg exposure caused changes in the expressions of several HPI-axis genes either in larval or adult stages, as well as altered expressions of the dopamine receptor d2 and dopamine transporter, that may be related to the behavioural alterations observed.
Achievement of the project aims was accompanied by the establishment of new behavioural testing procedures and generation of new mutant zebrafish lines using the cutting-edge CRISPR-Cas9 method. The research results are still in preparation for publication, however preliminary data has already been presented at scientific conferences and seminar series in the UK and abroad.
