Transcriptome profile analysis reveals specific signatures of pollutants in Atlantic eels.

Identifying specific effects of contaminants in a multi-stress field context remain a challenge in ecotoxicology. In this context, "omics" technologies, by allowing the simultaneous measurement of numerous biological endpoints, could help unravel the in situ toxicity of contaminants. In this study, wild Atlantic eels were sampled in 8 sites presenting a broad contamination gradient in France and Canada. The global hepatic transcriptome of animals was determined by RNA-Seq. In parallel, the contamination level of fish to 8 metals and 25 organic pollutants was determined. Factor analysis for multiple testing was used to identify genes that are most likely to be related to a single factor. Among the variables analyzed, arsenic (As), cadmium (Cd), lindane (γ-HCH) and the hepato-somatic index (HSI) were found to be the main factors affecting eel's transcriptome. Genes associated with As exposure were involved in the mechanisms that have been described during As vasculotoxicity in mammals. Genes correlated with Cd were involved in cell cycle and energy metabolism. For γ-HCH, genes were involved in lipolysis and cell growth. Genes associated with HSI were involved in protein, lipid and iron metabolisms. Our study proposes specific gene signatures of pollutants and their impacts in fish exposed to multi-stress conditions.

Effect of low-dose cadmium exposure on DNA methylation in the endangered European eel.

There is increasing evidence that epigenetics can play a key role in the etiology of diseases engendered by chronic pollutant exposure. Although epigenetics has received significant attention in the field of biomedicine during the last years, epigenetics research is surprisingly very limited in ecotoxicology. The aim of the present study was to investigate the possible effects of low-dose cadmium exposure on the DNA methylation profile in a critically endangered fish species, the European eel. Eels were exposed to environmentally realistic concentrations of cadmium (0.4 and 4 µg·L(-1)) during 45 days. The global CpG methylation status of eel liver was determined by means of a homemade ELISA assay. We then used a methylation-sensitive arbitrarily primed PCR method to identify genes that are differentially methylated between control and Cd-exposed eels. Our results show that cadmium exposure is associated with DNA hypermethylation and with a decrease in total RNA synthesis. Among hypermethylated sequences identified, several fragments presented high homologies with genes encoding for proteins involved in intracellular trafficking, lipid biosynthesis, and phosphatidic acid signaling pathway. In addition, few fragments presented high homologies with retrotransposon-like sequences. Our study illustrates how DNA methylation can be involved in the chronic stress response to Cd in fish.

Environmental modulators of diluted bitumen effects in juvenile pink salmon (Oncorhynchus gorbuscha).

Recent and potential expansions in the transportation of diluted bitumen (dilbit) through marine terminals in coastal regions of British Columbia require the examination of potential risks to estuarine species such as Pacific salmon. The estuarine habitat of out-migrated pink salmon (Oncorhynchus gorbuscha) exhibits dynamic temperature and salinity regimes, possibly modifying dilbit exposure, bioavailability and/or its effects. To examine dilbit toxicity and its modification by environmental stressors, juvenile pinks were subchronically exposed for 3 months to the water-accommodated fraction (WAF) of Cold Lake Blend dilbit (winter) in seawater at three salinities (7, 14, and 28‰ [temperature 12.5 °C]) and three temperatures (8.5, 12.5, and 16.5 °C [salinity of 28‰]). Temperature and salinity alone did not affect any measured endpoints in control fish. Dilbit exposure induced higher mortality at high (16.5 °C) and low temperatures (8.5 °C) as well as at higher salinity (28‰) in fish exposed to the highest dilution of WAF [total polycyclic aromatic compounds (TPAC) = 128.9 µg/L]. A concentration-dependent reduction of growth was evident in fish exposed to the medium (TPAC = 97.3 µg/L) and high dilution of WAF at higher temperatures (12.5 and 16.5 °C) and high salinity (28‰). At 28‰, swimming performance (Uburst) was decreased in fish exposed to the highest concentration of dilbit at all 3 temperatures. Gill Na+-K+-ATPase activity, white muscle lactate, glycogen, and triglyceride concentrations were altered by dilbit exposure and modified by temperature and salinity. In addition, gene expression associated with phase I biotransformation, energy metabolism, mitochondrial activity, and inflammation showed significant upregulation with exposure and temperature stress. Dilbit exposure at PAC concentrations in the ppb range, affected pink salmon at the molecular, biochemical, and whole organism level; effects that were exacerbated by environmental temperature and salinity.

Effects on Trout Alevins of Chronic Exposures to Chemically Dispersed Access Western Blend and Cold Lake Blend Diluted Bitumens.

The present study assessed the chronic toxicity of 2 chemically enhanced water accommodated fractions (CEWAFs) of diluted bitumens (dilbits), Access Western Blend (AWB) and Cold Lake Blend (CLB), to rainbow trout alevins. Chemical dispersion was used to overcome the resistance to dispersion of dilbits and to generate test solutions that contained more and smaller oil droplets for increased partitioning of petroleum hydrocarbons into water. Test solutions were characterized by fluorescence spectroscopy, a rapid and inexpensive analytical tool to compare toxicity endpoints measured by fluorescence (total petroleum hydrocarbons measured by fluorescence [TPH-F]). Cumulative mortality and the prevalence and severity of malformations increased following exposure of alevins to dispersed dilbits. Toxicity curves overlapped for AWB and CLB when expressed as TPH-F and 22- to 24-d median lethal and effect concentrations ranged from 0.36 to 1.5 mg/L. Gene expression in alevins was also altered following exposure to dispersed dilbit, with relative cytochrome P450-1A mRNA levels increasing up to 170-fold for AWB and up to 240-fold for CLB. Access Western Blend and CLB caused similar toxicity to rainbow trout alevins as light to medium conventional crude oils, and rainbow trout alevins were more sensitive than yellow perch, Japanese medaka, and fathead minnow embryos exposed to dispersed AWB and CLB. The present study is the first to assess the embryotoxicity of dilbits to a Canadian freshwater salmonid species. Environ Toxicol Chem 2020;39:1620-1633. © 2020 SETAC.

Impact of crude oil and the dispersant Corexit™ EC9500A on capelin (Mallotus villosus) embryo development.

Marine food webs are particularly vulnerable to oil spills if keystone species are impacted. To quantify lethal and sublethal toxicity in a key Holarctic forage fish, capelin embryos were exposed to Hibernia crude oil water accommodated fraction (WAF) produced at an oil-to-water ratio of 1:9 (v:v) and chemically-enhanced WAF (CEWAF) produced with the dispersant Corexit™ EC9500A at a dispersant-to-oil ratio of 1:10 (CEWAF H) or 1:50 (CEWAF L). Corexit alone yielded similar embryotoxicity to CEWAF. 10% CEWAF H, with total polycyclic aromatic hydrocarbons of 99.2 µg/L, decreased embryo survival following 10 h of exposure, while continual exposed to 1% CEWAF L decreased hatching and heart rates. Concentrations down to 0.1% CEWAF L increased in a dose-dependent manner the transcript level of cytochrome P4501a1 (cyp1a1) in hatched larvae. These data indicate that embryo-larval survival of capelin is likely at risk if an oil spill coincides in space and time with spawning.

Comparative toxicity of two diluted bitumens to developing yellow perch (Perca flavescens).

Increasing demand for diluted bitumen (dilbit) has led to the development of the oil sands industry and the expansion of transcontinental pipelines. Dilbit is an unresolved complex mixture with variable diluent and bitumen composition. Thus, it is important to understand the effects of the two most transported dilbits in Canada, Access Western Blend (AWB) and Cold Lake Blend (CLB) on a North America native and freshwater fish species, the yellow perch (Perca flavescens). Fertilized embryos were exposed to both dilbits for 16 days, from <24 h post-fertilization until hatch. The treatment regime was a static daily renewal of water accommodated fractions (WAF) and chemically-enhanced water accommodated fractions (CEWAF) at concentrations ranging from 0.01 to 21.3 µg/L of total polycyclic aromatic compounds (TPACs). Hatched embryos were assessed for malformations and changes in the expression of genes associated with phase I and II detoxification and oxidative stress. The prevalence of developmental malformations increased significantly at the highest concentrations of all treatments, with WAF treatments yielding a higher prevalence than CEWAF. The EC50s for AWB and CLB WAF and CEWAF solutions ranged from 9.8 to 24 µg/L TPACs, with the CEWAF of AWB being the least toxic. Relative mRNA levels of cyp1a showed induction by up to 18-fold in WAF and 50-fold in CEWAF treatments at similar concentrations of measured dilbit in solution. Complementary DNA methylation analysis was assessed and fish embryos exposed to AWB CEWAF and CLB WAF showed decreased DNA methylation profiles with increasing exposure to dilbit, suggesting that global gene expression is increasing in these treatments. With recent approvals of pipelines in North America, these data will support site-specific risk assessments and monitoring of Canadian ecosystems should a pipeline spill occur.

Gene transcription profiling in wild and laboratory-exposed eels: Effect of captivity and in situ chronic exposure to pollution.

Aquatic ecosystems are subjected to a variety of man-induced stressors but also vary spatially and temporally due to variation in natural factors. In such complex environments, it remains difficult to detect, dissociate and evaluate the effects of contaminants in wild organisms. In this context, the aim of this study was to test whether the hepatic transcriptome profile of fish may be used to detect in situ exposure to a particular contaminant. Transcriptomic profiles from laboratory-exposed and wild eels sampled along a contamination gradient were compared. During laboratory experiments, fish were exposed during 45days to different pollutants (Hg, PCBs, OCPs or Cd) or natural factors (temperature, salinity or low food supply) at levels close to those found in the sampling sites. A strong difference was observed between the transcriptomic profiles obtained from wild and laboratory-exposed animals (whatever the sites or experimental conditions), suggesting a general stress induced by captivity in the laboratory. Among the biological functions that were up-regulated in laboratory eels in comparison to wild eels, histone modification was the most represented. This finding suggests that laboratory conditions could affect the epigenome of fish and thus modulate the transcriptional responses developed by fish in response to pollutant exposure. Among experimental conditions, only the transcription profiles of laboratory animals exposed to cold temperature were correlated with those obtained from wild fish, and more significantly with fish from contaminated sites. Common regulated genes were mainly involved in cell differentiation and liver development, suggesting that stem/progenitor liver cells could be involved in the adaptive response developed by fish chronically exposed to pollutant mixtures.

Detecting the exposure to Cd and PCBs by means of a non-invasive transcriptomic approach in laboratory and wild contaminated European eels (Anguilla anguilla).

Detecting and separating specific effects of contaminants in a multi-stress field context remain a major challenge in ecotoxicology. In this context, the aim of this study was to assess the usefulness of a non-invasive transcriptomic method, by means of a complementary DNA (cDNA) microarray comprising 1000 candidate genes, on caudal fin clips. Fin gene transcription patterns of European eels (Anguilla anguilla) exposed in the laboratory to cadmium (Cd) or a polychloro-biphenyl (PCBs) mixture but also of wild eels from three sampling sites with differing contamination levels were compared to test whether fin clips may be used to detect and discriminate the exposure to these contaminants. Also, transcriptomic profiles from the liver and caudal fin of eels experimentally exposed to Cd were compared to assess the detection sensitivity of the fin transcriptomic response. A similar number of genes were differentially transcribed in the fin and liver in response to Cd exposure, highlighting the detection sensitivity of fin clips. Moreover, distinct fin transcription profiles were observed in response to Cd or PCB exposure. Finally, the transcription profiles of eels from the most contaminated site clustered with those from laboratory-exposed fish. This study thus highlights the applicability and usefulness of performing gene transcription assays on non-invasive tissue sampling in order to detect the in situ exposure to Cd and PCBs in fish.

Gonadal transcriptome analysis of wild contaminated female European eels during artificial gonad maturation.

Since the early 1980s, the population of European eels (Anguilla anguilla) has dramatically declined. Nowadays, the European eel is listed on the red list of threatened species (IUCN Red List) and is considered as critically endangered of extinction. Pollution is one of the putative causes for the collapse of this species. Among their possible effects, contaminants gradually accumulated in eels during their somatic growth phase (yellow eel stage) would be remobilized during their reproductive migration leading to potential toxic events in gonads. The aim of this study was to investigate the effects of organic and inorganic contaminants on the gonad development of wild female silver eels. Female silver eels from two sites with differing contamination levels were artificially matured. Transcriptomic analyses by means of a 1000 candidate gene cDNA microarray were performed on gonads after 11weeks of maturation to get insight into the mechanisms of toxicity of contaminants. The transcription levels of several genes, that were associated to the gonadosomatic index (GSI), were involved in mitotic cell division but also in gametogenesis. Genes associated to contaminants were mainly involved in the mechanisms of protection against oxidative stress, in DNA repair, in the purinergic signaling pathway and in steroidogenesis, suggesting an impairment of gonad development in eels from the polluted site. This was in agreement with the fact that eels from the reference site showed a higher gonad growth in comparison to contaminated fish.