DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation

DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors

Combined effects of heat stress and xenobiotic exposure in the European bullhead Cottus gobio. Clues provided by enzymatic and proteomic approaches

Combined exposure to elevated temperature and pollutants is an environmentally relevant scenario that is expected to become more widespread in the future with global warming. Accordingly, the general objective of the present study was to investigate the interactive effects of temperature stress and xenobiotic exposure in the European bullhead, Cottus gobio, a candidate sentinel species. Before studying the combined stresses, we have firstly characterized the toxicity of two ubiquitous contaminants in aquatic environments, i.e., cadmium (Cd) and perfluorooctane sulfonate (PFOS), in C. gobio. Exposure to high pollutants concentrations significantly altered the activity of several enzymes involved in energy metabolism, namely, citrate synthase, cytochrome c oxidase and lactate dehydrogenase, either in liver and/or in gills. Additionally, a proteomic approach was used to further our understanding of toxicity of the selected pollutants in C. gobio. The results showed that most of the identified differentially expressed proteins are associated with central aspects of the evolutionarily conserved cellular stress response and are part of the stress proteome. Secondly, as temperature and Cd are known to induce oxidative stress in aquatic organisms, their combined effects on the oxidative stress status of bullhead were assessed. Overall, we have observed that elevated temperature and Cd exposure independently influenced the antioxidant defence system with clear tissue-specific and stress-specific antioxidant responses. Further, the 20S proteasome activity was increased in response to heat stress in liver and gills. Finally, we have evaluated the effects of temperature and Cd exposure in sequential combination, i.e., fish acclimated to enhanced temperature and subsequently exposed to Cd. A combination of enzyme activity assays and protein expression patterns clearly indicated that increases in water temperature consistent with climate change predictions may modulate the ability of C. gobio to tolerate subsequent exposure to another stressor, such as Cd. Furthermore, the proteomic analysis brings clues to the potential molecular pathways involved in the acclimation process and by which a heat acclimation could interfere with an additional stress to Cd thereby highlighting shared mechanisms of action. As a corollary, the present study demonstrated that an integrative approach using enzymatic and proteomic analyses is worthwhile to better characterize the interactive effects of multiple stressors, such as temperature and metal pollution on fish species, and suggests that additional studies to link the cellular phenotype as observed here to changes at higher levels of biological organization could provide essential information to inform on organisms’ health in their natural environments.L'augmentation de la température moyenne globale et les différents polluants présents dans l’environnement peuvent occasionner des effets néfastes aux organismes aquatiques. Toutefois, l’importance des effets combinés d’une hausse de la température et d’une exposition à un/des xénobiotique(s) reste encore inconnue à ce jour. Dans le présent travail, nous avons premièrement caractérisé la toxicité de deux polluants omniprésents dans les milieux aquatiques, le cadmium (Cd) et le perfluorooctane sulfonate (PFOS), chez le chabot, préalablement aux stress combinés. Des modifications de l'activité de plusieurs enzymes impliquées dans le métabolisme énergétique (la citrate synthétase, la cytochrome c oxydase et la lactate déshydrogénase) ont été observées dans le foie et/ou les branchies après une exposition à de fortes concentrations en polluants. Une analyse protéomique a également été réalisée afin d’approfondir la compréhension des effets toxiques du Cd et du PFOS chez le chabot. Ainsi, nous avons mis en évidence la présence de variations dans l’expression de diverses protéines impliquées dans des voies très diverses et faisant partie du protéome de stress. Deuxièmement, les capacités antioxydantes du chabot ont été étudiées suite à une exposition combinée à la température et au Cd. Les résultats montrent que les différents systèmes antioxydants enzymatiques sont soit altérés par l’augmentation de la température, soit par le Cd. L’observation des résultats souligne également l’existence d’une réponse variable selon le tissu considéré. De plus, une augmentation de l’activité du protéasome 20S a été mesurée dans le foie et les branchies après un stress thermique. Enfin, nous avons tenté de déterminer si une acclimatation à la chaleur chez le chabot interfère avec une exposition subséquente au Cd. La mesure de l’activité de la lactate déshydrogénase et l’analyse des profils d’expression protéiques ont clairement démontré qu’une acclimatation à une augmentation de la température, conforme aux prévisions du réchauffement climatique, peut modifier la capacité des organismes à faire face à une autre perturbation environnementale comme une exposition à un xénobiotique. Par ailleurs, l'analyse protéomique apporte des indices sur les mécanismes moléculaires possibles mis en place pendant une acclimatation thermique et pouvant interférer avec les effets du Cd. En résumé, ce travail démontre qu'une approche intégrative utilisant des analyses enzymatiques et protéomiques, fournit une meilleure compréhension de l’interaction entre une hausse de la température et une exposition à un polluant chez un organisme aquatique.(DOCSC03) -- FUNDP, 201

Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium

International audienceTemperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl2 at nominal concentrations of 0.01 and 1 mg/L) for 4 days at either 15 °C or 21 °C. First, exposure to 1 mg Cd/L induced a high mortality rate (67%) in fish held at 21 °C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21 °C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15 °C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues