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.

Effects of cadmium exposure on the gill proteome of Cottus gobio: modulatory effects of prior thermal acclimation.

Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. The aim of the present study was to investigate whether acclimation to elevated temperature affects the response of the European bullhead (Cottus gobio) to subsequent cadmium (Cd) exposure by using enzymatic and proteomic approaches. Fish acclimated to 15 (standard temperature), 18 or 21 °C for 28 days were exposed to 1mg Cd/L for 4 days at the respective acclimation temperature. First, exposure to Cd significantly decreased the activity of the lactate dehydrogenase (LDH) in gills of fish acclimated to 15 or 18 °C. However, an acclimation to 21 °C suppressed the inhibitory effect of Cd. Second, using a proteomic analysis by 2D-DIGE, we observed that thermal acclimation was the first parameter affecting the protein expression profile in gills of C. gobio, while subsequent Cd exposure seemed to attenuate this temperature effect. Moreover, our results showed opposite effects of these two environmental stressors at protein expression level. From the 52 protein spots displaying significant interaction effects of temperature and Cd exposure, a total of 28 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet algorithms of Scaffold software. The identified differentially expressed proteins can be categorized into diverse functional classes, related to protein turnover, folding and chaperoning, metabolic process, ion transport, cell signaling and cytoskeleton. Within a same functional class, we further reported that several proteins displayed reverse responses following sequential exposure to heat and Cd. This work provides insights into the molecular pathways potentially involved in heat acclimation process and the interactive effects of temperature and Cd stress in ectothermic vertebrates.

Proteomics to assess the role of phenotypic plasticity in aquatic organisms exposed to pollution and global warming.

Nowadays, the unprecedented rates of anthropogenic changes in ecosystems suggest that organisms have to migrate to new distributional ranges or to adapt commensurately quickly to new conditions to avoid becoming extinct. Pollution and global warming are two of the most important threats aquatic organisms will have to face in the near future. If genetic changes in a population in response to natural selection are extensively studied, the role of acclimation through phenotypic plasticity (the property of a given genotype to produce different phenotypes in response to particular environmental conditions) in a species to deal with new environmental conditions remains largely unknown. Proteomics is the extensive study of the protein complement of a genome. It is dynamic and depends on the specific tissue, developmental stage, and environmental conditions. As the final product of gene expression, it is subjected to several regulatory steps from gene transcription to the functional protein. Consequently, there is a discrepancy between the abundance of mRNA and the abundance of the corresponding protein. Moreover, proteomics is closer to physiology and gives a more functional knowledge of the regulation of gene expression than does transcriptomics. The study of protein-expression profiles, however, gives a better portrayal of the cellular phenotype and is considered as a key link between the genotype and the organismal phenotype. Under new environmental conditions, we can observe a shift of the protein-expression pattern defining a new cellular phenotype that can possibly improve the fitness of the organism. It is now necessary to define a proteomic norm of reaction for organisms acclimating to environmental stressors. Its link to fitness will give new insights into how organisms can evolve in a changing environment. The proteomic literature bearing on chronic exposure to pollutants and on acclimation to heat stress in aquatic organisms, as well as potential application of proteomics in evolutionary issues, are outlined. While the transcriptome responses are commonly investigated, proteomics approaches now need to be intensified, with the new perspective of integrating the cellular phenotype with the organismal phenotype and with the mechanisms of the regulation of gene expression, such as epigenetics.

Evidence that elevated water temperature affects the reproductive physiology of the European bullhead Cottus gobio.

Climate change is predicted to increase the average water temperature and alter the ecology and physiology of several organisms including fish species. To examine the effects of increased water temperature on freshwater fish reproduction, adult European bullhead Cottus gobio of both genders were maintained under three temperature regimes (T1: 6-10, T2: 10-14 and T3: 14-18°C) and assessed for gonad development (gonadosomatic index-GSI and gonad histology), sex steroids (testosterone-T, 17ß-estradiol-E2 and 11-ketotestosterone-11-KT) and vitellogenin (alkali-labile phosphoprotein phosphorus-ALP) dynamics in December, January, February and March. The results indicate that a 8°C rise in water temperature (T3) deeply disrupted the gonadal maturation in both genders. This observation was associated with the absence of GSI peak from January to March, and low levels of plasma sex steroids compared with T1-exposed fish. Nevertheless, exposure to an increasing temperature of 4°C (T2) appeared to accelerate oogenesis with an early peak value in GSI and level of plasma T recorded in January relative to T1-exposed females. In males, the low GSI, reduced level of plasma 11-KT and the absence of GSI increase from January to March support the deleterious effects of increasing water temperature on spermatogenesis. The findings of the present study suggest that exposure to elevated temperatures within the context of climate warming might affect the reproductive success of C. gobio. Specifically, a 4°C rise in water temperature affects gametogenesis by advancing the spawning, and a complete reproductive failure is observed at an elevated temperature of 8°C.

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

Temperature 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 (CdCl(2) at nominal concentrations of 0.01 and 1mg/L) for 4 days at either 15°C or 21°C. First, exposure to 1mg 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.

Ecotoxicoproteomics in gills of the sentinel fish species, Cottus gobio, exposed to perfluorooctane sulfonate (PFOS).

The environmental persistence, bioaccumulative tendency and potential toxicity of perfluorooctane sulfonate (PFOS) have generated great concern. This study aimed at evaluating the toxicity of short-term PFOS exposure in gills of the European bullhead Cottus gobio, a candidate sentinel species, by monitoring the response of some enzymes (citrate synthase CS, cytochrome c oxidase CCO, and lactate dehydrogenase LDH), and by undertaking a proteomic analysis using 2D-DIGE. First, a 96-h exposure to 1mg PFOS/L significantly altered the activity of mitochondrial CS and CCO. Second, 2D-DIGE gels were used to compare gills from the control fish group with tissues from fish exposed for 96h to either 0.1 or 1mg PFOS/L. From the 27 protein spots displaying significant changes in abundance following PFOS exposure, a total of 20 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet of Scaffold software. The differentially expressed proteins that were identified are involved in the general stress response, ubiquitin-proteasome system, energy metabolism, and actin cytoskeleton, which provide clues on the cellular pathways and components mainly affected by PFOS. Moreover, our results showed that most proteins were differentially expressed at the low but not at the high PFOS concentration. This work provides insights into the biochemical and molecular events in PFOS-induced toxicity in gill tissue, and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of PFOS toxicity in fish.

Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio.

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.

A differential proteomic approach to assess the effects of chemotherapeutics and production management strategy on giant tiger shrimp Penaeus monodon.

The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg(-1)) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.

The genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute exposure to the androgen, 17beta-trenbolone.

We investigated the genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute (4 days) exposure to 0.1 or 1.0microg/L of 17beta-trenbolone (TB), the active metabolite of an anabolic androgenic steroid used as a growth promoter in cattle and a contaminant of concern in aquatic systems. Our objectives were to investigate the gene expression profile induced by TB, define biomarkers of exposure to TB, and increase our understanding of the mechanisms of adverse effects of TB on fish reproduction. In female gonad tissue, microarray analysis using a 22K oligonucleotide microarray (EcoArray Inc., Gainesville, FL) showed 99 significantly upregulated genes and 741 significantly downregulated genes in response to 1microg TB/L. In particular, hydroxysteroid (17beta) dehydrogenase 12a (hsd17b12a), zona pellucida glycoprotein 2.2 (zp2.2), and protein inhibitor of activated STAT, 2 (pias2) were all downregulated in gonad. Q-PCR measurements in a larger sample set were consistent with the microarray results in the direction and magnitude of these changes in gene expression. However, several novel potential biomarkers were verified by Q-PCR in the same samples, but could not be validated in independent samples. In liver, Q-PCR measurements showed a significant decrease in vitellogenin 1 (vtg1) mRNA expression. In brain, cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b, previously known as aromatase B) transcript levels were significantly reduced following TB exposure. Our study provides a candidate gene involved in mediating the action of TB, hsd17b12a, and two potential biomarkers sensitive to acute TB exposure, hepatic vtg1 and brain cyp19a1b.

Oxidative stress, protein carbonylation and heat shock proteins in the black tiger shrimp, Penaeus monodon, following exposure to endosulfan and deltamethrin.

The impact of commonly used pesticides, endosulfan and deltamethrin, on the molecular stress level in black tiger shrimp Penaeus monodon, was assessed using classical oxidative stress biomarkers, protein carbonylation profiles, and levels of heat shock proteins. Results showed that 4 days exposure to 0.1 µg L(-1) deltamethrin significantly (p<0.05) increased lipid peroxidation (LPO) level in gills (64.3 ± 3.2 compared to 34.2 ± 5.3 nmol MDA equiv.g(-1) tissue at day 0). However, no pesticide treatment had significant effect on the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Carbonylated protein profiles were determined on gills following 2,4-dinitrophenylhydrazine derivatization and 2D-PAGE along with Western blotting. Immunoblotting with dinitrophenol-specific antibody revealed 17 protein spots carbonylated in response to 4 days exposure to 0.1 µg L(-1) deltamethrin while 24 protein spots specifically oxidized at day 0 were no longer detected after deltamethrin treatment. On the other hand, endosulfan exposure at 0.1 and 1 µg L(-1) induced up to 2.1-fold increase of HSP90 level in muscle. This approach is providing new insights into the molecular impacts of deltamethrin and endosulfan on an economically important crustacean. While deltamethrin has shown a pro-oxidant effect in gills, endosulfan exposure rather induced proteotoxic effects in muscles. This argues that LPO level, protein carbonylation specificities, and HSP90 levels may be potential discriminating biomarkers to assess the chemical stress level in farm shrimp.