Periodic Reporting for period 1 - GSTHgNDD (Role of GST gene variation in susceptibility to mercury (Hg)-induced neurodevelopmental disorders (NDD) in zebrafish)
Periodo di rendicontazione: 2017-06-20 al 2019-06-19
Methylmercury (MeHg), an organic form of the heavy metal mercury, is one of the most neurotoxic environmental pollutants, demonstrated to have high potency for causing developmental neurotoxicity even at low levels. However, the role of prenatal MeHg exposure in the aetiology of neurodevelopmental disorders is controversial. While many epidemiological studies identify associations with ADHD, cognitive reduction, decrease in IQ, and ASDs, other studies attest the contrary finding of no significant association between measured mercury exposure in humans and risk for developmental neurotoxicity. This observed inconsistency is hypothesized to be the result of individual genetic variation in specific genes involved in the process of detoxification. The glutathione system plays a central role in MeHg detoxification, and the glutathione-s-transferase (GST) group of genes are key genes in this system. Recent epidemiological studies revealed that specific polymorphisms and deletions in human GST genes are associated with increased mercury retention in the body at similar exposure levels, and therefore higher risk for susceptibility to toxicity.
We proposed to develop a new model for assessing the role of gene:environment interactions (GxE), specifically testing GST-related genetic predisposition for MeHg neurotoxicity, and identify early markers predictive of toxicity and behavioural impairment. We chose to use the zebrafish model, since it is a well-established model for vertebrate developmental biology and neurotoxicity studies and has important advantages over traditional rodent models for this purpose. Such advantages include robustness for conducting large experiments with strong statistical power; an embrynic development period of only 5 days after which behavioural data can be obtained; external development of transparent embryos allowing for direct exposure without maternal transfer and direct observation throughout development. Our approach was to expose wild-type and CRISPR-generated Gst mutant embryos to mercury and test whether there is genetic predisposition to increased risk for toxin-induced behavioural phenotypes. We also measured the transcriptomic effects of mercury exposure on the stress axis, the dopamine pathway and oxidative stress markers.
We proposed to develop a new model for assessing the role of gene:environment interactions (GxE), specifically testing GST-related genetic predisposition for MeHg neurotoxicity, and identify early markers predictive of toxicity and behavioural impairment. We chose to use the zebrafish model, since it is a well-established model for vertebrate developmental biology and neurotoxicity studies and has important advantages over traditional rodent models for this purpose. Such advantages include robustness for conducting large experiments with strong statistical power; an embrynic development period of only 5 days after which behavioural data can be obtained; external development of transparent embryos allowing for direct exposure without maternal transfer and direct observation throughout development. Our approach was to expose wild-type and CRISPR-generated Gst mutant embryos to mercury and test whether there is genetic predisposition to increased risk for toxin-induced behavioural phenotypes. We also measured the transcriptomic effects of mercury exposure on the stress axis, the dopamine pathway and oxidative stress markers.
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.
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.
The proposal was directly related to the EC work program for Health, demographic change and wellbeing, with respect to both 'Personalized medicine' for noncommunicable diseases and 'Early development' addressing mental health from childhood to older ages. Our results will have an impact on the ability to conduct individual and population specific toxicity risk assessment by linking markers of oxidative-stress and the endocrine stress axis to behavioural phenotypes of neurotoxicity. Our results also show the relationship between the short (larval) and long-term (adult) behavioural effects of the developmental exposure, that may lead to novel measures for early detection of potential adverse outcomes.
A great deal of progress was made in several aspects of the researcher’s professional development, including grant and fellowship writing, networking and communication, and teaching. In the duration of the fellowship 5 grant application and one early-career fellowship were submitted. Of these, one grant application was successful and provided funding for an international networking meeting that produced another grant submission and a collaboration in writing a systematic review. In addition, through local networking within QMUL, two teaching positions were secured that provided training and experience in multiple aspects of higher education teaching and mentoring.
A great deal of progress was made in several aspects of the researcher’s professional development, including grant and fellowship writing, networking and communication, and teaching. In the duration of the fellowship 5 grant application and one early-career fellowship were submitted. Of these, one grant application was successful and provided funding for an international networking meeting that produced another grant submission and a collaboration in writing a systematic review. In addition, through local networking within QMUL, two teaching positions were secured that provided training and experience in multiple aspects of higher education teaching and mentoring.