Quantitative proteomics analysis of zebrafish exposed to sub-lethal dosages of β-methyl-amino-L-alanine (BMAA)

The non-protein amino acid β-methylamino-L-alanine (BMAA) is a neurotoxin present in microalgae and shown to accumulate in the food web. BMAA has been linked to the complex neurodegenerative disorder of Guam and to increased incidents sporadic ALS. Two main neurotoxic routes are suggested; an excitotoxic by acting as an agonist towards glutamate receptors and a metabolic by misincorporating into cellular proteins. We have used zebrafish, an increasingly used model for neurodegenerative diseases, to further identify signaling components involved in BMAA-induced toxicity. Zebrafish embryos were exposed to sub-lethal dosages of BMAA and a label-free proteomics analysis was conducted on larvae 4 days post fertilization. The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms. Search towards a reviewed database containing 2968 entries identified 480 proteins. Only 17 of these were regulated 2-fold or more in the exposed larvae. Seven of these proteins could be associated to glutamate receptor signaling and recycling. The remaining nine have all been linked to disturbance in protein homeostasis, reactive oxygen species (ROS) development or neuronal cell death. We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.publishedVersio

Quantitative transcriptomics, and lipidomics in evaluating ovarian developmental effects in Atlantic cod (Gadus morhua) caged at a capped marine waste disposal site

In the present study, a previously capped waste disposal site at Kollevåg (Norway) was selected to study the effects of contaminant leakage on biomarkers associated with Atlantic cod (Gadus morhua) reproductive endocrinology and development. Immature cod were caged for 6 weeks at 3 locations, selected to achieve a spatial gradient of contamination, and compared to a reference station. Quantitative transcriptomic, and lipidomic analysis was used to evaluate the effects of the potential complex contaminant mixture on ovarian developmental and endocrine physiology. The number of expressed transcripts, with 0.75 log2-fold differential expression or more, varied among stations and paralleled the severity of contamination. Particularly, significant bioaccumulation of ∑PCB-7, ∑DDTs and ∑PBDEs were observed at station 1, compared to the other station, including the reference station. Respectively 1416, 698 and 719 differentially expressed genes (DEGs), were observed at stations 1, 2 and 3, compared to the reference station, with transcripts belonging to steroid hormone synthesis pathway being significantly upregulation. Transcription factors such as esr2 and ahr2 were increased at all three stations, with highest fold-change at Station 1. MetaCore pathway maps identified affected pathways that are involved in ovarian physiology, where some unique pathways were significantly affected at each station. For the lipidomics, sphingolipid metabolism was particularly affected at station 1, and these effects paralleled the high contaminant burden at this station. Overall, our findings showed a novel and direct association between contaminant burden and ovarian toxicological and endocrine physiological responses in cod caged at the capped Kollevåg waste disposal site.publishedVersio

Modulation of neuro-dopamine homeostasis in juvenile female Atlantic cod (Gadus morhua) exposed to polycyclic aromatic hydrocarbons and perfluoroalkyl substances

The dopaminergic effect of PAH and PFAS mixtures, prepared according to environmentally relevant concentrations, has been studied in juvenile female Atlantic cod (Gadus morhua). Benzo[a]pyrene, dibenzothiophene, fluorene, naphthalene, phenanthrene, and pyrene were used to prepare a PAH mixture, while PFNA, PFOA, PFOS, and PFTrA were used to prepare a PFAS mixture. Cod were injected intraperitoneally twice, with either a low (1×) or high (20×) dose of each compound mixture or their combinations. After 2 weeks of exposure, levels of plasma 17β-estradiol (E2) were significantly elevated in high PAH/high PFAS treated group. Brain dopamine/metabolite ratios (DOPAC/dopamine and HVA+DOPAC/dopamine) changed with E2 plasma levels, except for high PAH/low PFAS and low PAH/high PFAS treated groups. On the transcript levels, th mRNA inversely correlated with dopamine/metabolite ratios and gnrh2 mRNA levels. Respective decreases and increases of drd1 and drd2a after exposure to the high PAH dose were observed. Specifically, high PFAS exposure decreased both drds, leading to high plasma E2 concentrations. Other studied end points suggest that these compounds, at different doses and combinations, have different toxicity threshold and modes of action. These effects indicate potential alterations in the feedback signaling processes within the dopaminergic pathway by these contaminant mixtures.acceptedVersio

Application of quantitative transcriptomics in evaluating the ex vivo effects of per- and polyfluoroalkyl substances on Atlantic cod (Gadus morhua) ovarian physiology

Because of their global consumption and persistence, per- and polyfluoroalkyl substances (PFASs), are ubiquitously distributed in the environment, as well as in wildlife and humans. In the present study, we have employed an ex vivo organ culture technique, based on the floating agarose method, of Atlantic cod ovarian tissue to investigate the effects of three different concentrations of PFOS, PFOA (1, 5 and 25 μM) and PFNA (0.5, 5 and 50 μM), used singly and in also in combination (1×, 20× and 100×). In the 1× exposure mixture, concentrations were decided based on their proportional levels (in molar equivalents) relative to PFOS, which is the most abundant PFAS in cod liver from a 2013 screening project. To investigate the detailed underlying mechanisms and biological processes, transcriptome sequencing was performed on exposed ovarian tissue. The number of differentially expressed genes (DEGs) having at least 0.75 log2-fold change was elevated in high, compared to low and medium concentration exposures. The highest PFNA, PFOA and PFOS concentrations, and the highest (100×) mixture exposure, showed 40, 68, 1295, and 802 DEGs, respectively. The latter two exposure groups shared a maximum of 438 DEGs. In addition, they both shared the majority of functionally enriched pathways belonging to biological processes such as cellular signaling, cell adhesion, lipid metabolism, immunological responses, cancer, reproduction and metabolism. Shortlisted DEGs that were specifically annotated to reproduction associated gene ontology (GO) terms were observed only in the highest PFOS and mixture exposure groups. These transcripts contributed to ovarian key events such as steroidogenesis (star, cyp19a1a), oocyte growth (amh), maturation (igfbp5b, tgfβ2, tgfβ3), and ovulation (pgr, mmp2). Contrary to other PFAS congeners, the highest PFOS concentration showed almost similar transcript expression patterns compared to the highest mixture exposure group. This indicates that PFOS is the active component of the mixture that significantly altered the normal functioning of female gonads, and possibly leading to serious reproductive consequences in teleosts

Application of quantitative transcriptomics in evaluating the ex vivo effects of per- and polyfluoroalkyl substances on Atlantic cod (Gadus morhua) ovarian physiology

Because of their global consumption and persistence, per- and polyfluoroalkyl substances (PFASs), are ubiquitously distributed in the environment, as well as in wildlife and humans. In the present study, we have employed an ex vivo organ culture technique, based on the floating agarose method, of Atlantic cod ovarian tissue to investigate the effects of three different concentrations of PFOS, PFOA (1, 5 and 25 μM) and PFNA (0.5, 5 and 50 μM), used singly and in also in combination (1×, 20× and 100×). In the 1× exposure mixture, concentrations were decided based on their proportional levels (in molar equivalents) relative to PFOS, which is the most abundant PFAS in cod liver from a 2013 screening project. To investigate the detailed underlying mechanisms and biological processes, transcriptome sequencing was performed on exposed ovarian tissue. The number of differentially expressed genes (DEGs) having at least 0.75 log2-fold change was elevated in high, compared to low and medium concentration exposures. The highest PFNA, PFOA and PFOS concentrations, and the highest (100×) mixture exposure, showed 40, 68, 1295, and 802 DEGs, respectively. The latter two exposure groups shared a maximum of 438 DEGs. In addition, they both shared the majority of functionally enriched pathways belonging to biological processes such as cellular signaling, cell adhesion, lipid metabolism, immunological responses, cancer, reproduction and metabolism. Shortlisted DEGs that were specifically annotated to reproduction associated gene ontology (GO) terms were observed only in the highest PFOS and mixture exposure groups. These transcripts contributed to ovarian key events such as steroidogenesis (star, cyp19a1a), oocyte growth (amh), maturation (igfbp5b, tgfβ2, tgfβ3), and ovulation (pgr, mmp2). Contrary to other PFAS congeners, the highest PFOS concentration showed almost similar transcript expression patterns compared to the highest mixture exposure group. This indicates that PFOS is the active component of the mixture that significantly altered the normal functioning of female gonads, and possibly leading to serious reproductive consequences in teleosts

Quantitative transcriptomics, and lipidomics in evaluating ovarian developmental effects in Atlantic cod (Gadus morhua) caged at a capped marine waste disposal site

In the present study, a previously capped waste disposal site at Kollevåg (Norway) was selected to study the effects of contaminant leakage on biomarkers associated with Atlantic cod (Gadus morhua) reproductive endocrinology and development. Immature cod were caged for 6 weeks at 3 locations, selected to achieve a spatial gradient of contamination, and compared to a reference station. Quantitative transcriptomic, and lipidomic analysis was used to evaluate the effects of the potential complex contaminant mixture on ovarian developmental and endocrine physiology. The number of expressed transcripts, with 0.75 log2-fold differential expression or more, varied among stations and paralleled the severity of contamination. Particularly, significant bioaccumulation of ∑PCB-7, ∑DDTs and ∑PBDEs were observed at station 1, compared to the other station, including the reference station. Respectively 1416, 698 and 719 differentially expressed genes (DEGs), were observed at stations 1, 2 and 3, compared to the reference station, with transcripts belonging to steroid hormone synthesis pathway being significantly upregulation. Transcription factors such as esr2 and ahr2 were increased at all three stations, with highest fold-change at Station 1. MetaCore pathway maps identified affected pathways that are involved in ovarian physiology, where some unique pathways were significantly affected at each station. For the lipidomics, sphingolipid metabolism was particularly affected at station 1, and these effects paralleled the high contaminant burden at this station. Overall, our findings showed a novel and direct association between contaminant burden and ovarian toxicological and endocrine physiological responses in cod caged at the capped Kollevåg waste disposal site

Quantitative transcriptomics, and lipidomics in evaluating ovarian developmental effects in Atlantic cod (Gadus morhua) caged at a capped marine waste disposal site

In the present study, a previously capped waste disposal site at Kollevåg (Norway) was selected to study the effects of contaminant leakage on biomarkers associated with Atlantic cod (Gadus morhua) reproductive endocrinology and development. Immature cod were caged for 6 weeks at 3 locations, selected to achieve a spatial gradient of contamination, and compared to a reference station. Quantitative transcriptomic, and lipidomic analysis was used to evaluate the effects of the potential complex contaminant mixture on ovarian developmental and endocrine physiology. The number of expressed transcripts, with 0.75 log2-fold differential expression or more, varied among stations and paralleled the severity of contamination. Particularly, significant bioaccumulation of ∑PCB-7, ∑DDTs and ∑PBDEs were observed at station 1, compared to the other station, including the reference station. Respectively 1416, 698 and 719 differentially expressed genes (DEGs), were observed at stations 1, 2 and 3, compared to the reference station, with transcripts belonging to steroid hormone synthesis pathway being significantly upregulation. Transcription factors such as esr2 and ahr2 were increased at all three stations, with highest fold-change at Station 1. MetaCore pathway maps identified affected pathways that are involved in ovarian physiology, where some unique pathways were significantly affected at each station. For the lipidomics, sphingolipid metabolism was particularly affected at station 1, and these effects paralleled the high contaminant burden at this station. Overall, our findings showed a novel and direct association between contaminant burden and ovarian toxicological and endocrine physiological responses in cod caged at the capped Kollevåg waste disposal site

Toxicity and developmental effects of Arctic fuel oil types on early life stages of Atlantic cod (Gadus morhua)

Due to the heavy fuel oil (HFO) ban in Arctic maritime transport and new legislations restricting the sulphur content of fuel oils, new fuel oil types are continuously developed. However, the potential impacts of these new fuel oil types on marine ecosystems during accidental spills are largely unknown. In this study, we studied the toxicity of three marine fuel oils (two marine gas oils with low sulphur contents and a heavy fuel oil) in early life stages of cod (Gadus morhua). Embryos were exposed for 4 days to water-soluble fractions of fuel oils at concentrations ranging from 4.1 - 128.3 µg TPAH/L, followed by recovery in clean seawater until 17 days post fertilization. Exposure to all three fuel oils resulted in developmental toxicity, including severe morphological changes, deformations and cardiotoxicity. To assess underlying molecular mechanisms, we studied fuel oil-mediated activation of aryl hydrocarbon receptor (Ahr) gene battery and genes related to cardiovascular, angiogenesis and osteogenesis pathways. Overall, our results suggest comparable mechanisms of toxicity for the three fuel oils. All fuel oils caused concentration-dependant increases of cyp1a mRNA which paralleled ahrr, but not ahr1b transcript expression. On the angiogenesis and osteogenesis pathways, fuel oils produced concentration-specific transcriptional effects that were either increasing or decreasing, compared to control embryos. Based on the observed toxic responses, toxicity threshold values were estimated for individual endpoints to assess the most sensitive molecular and physiological effects, suggesting that unresolved petrogenic components may be significant contributors to the observed toxicity

Contaminant accumulation and biological responses in Atlantic cod (Gadus morhua) caged at a capped waste disposal site in Kollevåg, Western Norway

The aim of this study was to assess whether fish in Kollevåg, a sheltered bay on the western coast of Norway, previously utilized as a waste disposal site, could be affected by environmental contaminants leaking from the waste. Farmed, juvenile Atlantic cod (Gadus morhua) were caged for six weeks at three different locations in Kollevåg bay and at one reference location. Sediments and cod samples (bile and liver) were analyzed for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), per-and polyfluoroalkyl substances (PFASs) and polycyclic aromatic hydrocarbon (PAH) metabolites, revealing a contamination gradient at the four stations. Furthermore, hepatosomatic index (HSI) and Fulton's condition factor (CF) were significantly lower in cod caged closest to the disposal site. Levels and activities of biomarker proteins, such as vitellogenin (Vtg), metallothionein (Mt), and biotransformation and oxidative stress enzymes, including cytochrome P450 1a and 3a (Cyp1a, Cyp3a), glutathione s-transferase (Gst) and catalase (Cat), were quantified in blood plasma and liver tissue. Hepatic Cat and Gst activities were significantly reduced in cod caged at the innermost stations in Kollevåg, indicating modulation of oxidative stress responses. However, these results contrasted with reduced hepatic lipid peroxidation. Significant increases in transcript levels were observed for genes involved in lipid metabolism (fasn and acly) in cod liver, while transcript levels of ovarian steroidogenic enzyme genes such as p450scc, cyp19, 3β-hsd and 20β-hsd showed significant station-dependent increases. Cyp1a and Vtg protein levels were however not significantly altered in cod caged in Kollevåg. Plasma levels of estradiol (E2) and testosterone (T) were determined by enzyme immunoassay (EIA) and showed elevated E2 levels, but only at the innermost station. We conclude that the bay of Kollevåg did not fullfill adequate environmental condition based on environmental quality standards (EQSs) for chemicals in coastal waters. Following a six weeks caging period, environmental contaminants accumulated in cod tissues and effects were observed on biomarker responses, especially those involved in reproductive processes in cod ovary

Contaminant accumulation and biological responses in Atlantic cod (Gadus morhua) caged at a capped waste disposal site in Kollevåg, Western Norway

The aim of this study was to assess whether fish in Kollevåg, a sheltered bay on the western coast of Norway, previously utilized as a waste disposal site, could be affected by environmental contaminants leaking from the waste. Farmed, juvenile Atlantic cod (Gadus morhua) were caged for six weeks at three different locations in Kollevåg bay and at one reference location. Sediments and cod samples (bile and liver) were analyzed for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), per-and polyfluoroalkyl substances (PFASs) and polycyclic aromatic hydrocarbon (PAH) metabolites, revealing a contamination gradient at the four stations. Furthermore, hepatosomatic index (HSI) and Fulton's condition factor (CF) were significantly lower in cod caged closest to the disposal site. Levels and activities of biomarker proteins, such as vitellogenin (Vtg), metallothionein (Mt), and biotransformation and oxidative stress enzymes, including cytochrome P450 1a and 3a (Cyp1a, Cyp3a), glutathione s-transferase (Gst) and catalase (Cat), were quantified in blood plasma and liver tissue. Hepatic Cat and Gst activities were significantly reduced in cod caged at the innermost stations in Kollevåg, indicating modulation of oxidative stress responses. However, these results contrasted with reduced hepatic lipid peroxidation. Significant increases in transcript levels were observed for genes involved in lipid metabolism (fasn and acly) in cod liver, while transcript levels of ovarian steroidogenic enzyme genes such as p450scc, cyp19, 3β-hsd and 20β-hsd showed significant station-dependent increases. Cyp1a and Vtg protein levels were however not significantly altered in cod caged in Kollevåg. Plasma levels of estradiol (E2) and testosterone (T) were determined by enzyme immunoassay (EIA) and showed elevated E2 levels, but only at the innermost station. We conclude that the bay of Kollevåg did not fullfill adequate environmental condition based on environmental quality standards (EQSs) for chemicals in coastal waters. Following a six weeks caging period, environmental contaminants accumulated in cod tissues and effects were observed on biomarker responses, especially those involved in reproductive processes in cod ovary