Multi-tissue proteogenomic analysis for mechanistic toxicology studies in non-model species.

New approach methodologies (NAM), including omics and in vitro approaches, are contributing to the implementation of 3R (reduction, refinement and replacement) strategies in regulatory science and risk assessment. In this study, we present an integrative transcriptomics and proteomics analysis workflow for the validation and revision of complex fish genomes and demonstrate how proteogenomics expression matrices can be used to support multi-level omics data integration in non-model species in vivo and in vitro. Using Atlantic salmon as an example, we constructed proteogenomic databases from publicly available transcriptomic data and in-house generated RNA-Seq and LC-MS/MS data. Our analysis identified ∼80,000 peptides, providing direct evidence of translation for over 40,000 RefSeq structures. The data also highlighted 183 co-located peptide groups that supported a single transcript each, and in each case, either corrected a previous annotation, supported Ensembl annotations not present in RefSeq, or identified novel previously unannotated genes. Proteogenomics data-derived expression matrices revealed distinct profiles for the different tissue types analyzed. Focusing on proteins involved in defense against xenobiotics, we detected distinct expression patterns across different salmon tissues and observed homology in the expression of chemical defense proteins between in vivo and in vitro liver systems. Our study demonstrates the potential of proteogenomic analyses in extending our understanding of complex fish genomes and provides an advanced bioinformatic toolkit to support the further development of NAMs and their application in regulatory science and (eco)toxicological studies of non-model species.

Use of (Q)SAR genotoxicity predictions and fuzzy multicriteria decision-making for priority ranking of ethoxyquin transformation products.

Ethoxyquin (EQ; 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed for pets and food-producing animals, including farmed fish such as Atlantic salmon. In Europe, the authorization for use of EQ as a feed additive was suspended, due to knowledge gaps concerning the presence and toxicity of EQ transformation products (TPs). Recent analytical studies focusing on the detection of EQ TPs in farmed Atlantic salmon feed and fillets reported the detection of a total of 27 EQ TPs, comprising both known and previously not described EQ TPs. We devised and applied an in silico workflow to rank these EQ TPs according to their genotoxic potential and their occurrence data in Atlantic salmon feed and fillet. Ames genotoxicity predictions were obtained applying a suite of five (quantitative) structure-activity relationship ((Q)SAR) tools, namely VEGA, TEST, LAZAR, Derek Nexus and Sarah Nexus. (Q)SAR Ames genotoxicity predictions were aggregated using fuzzy analytic hierarchy process (fAHP) multicriteria decision-making (MCDM). A priority ranking of EQ TPs was performed based on combining both fAHP ranked (Q)SAR predictions and analytical occurrence data. The applied workflow prioritized four newly identified EQ TPs for further investigation of genotoxicity. The fAHP-based prioritization strategy described here, can easily be applied to other toxicity endpoints and groups of chemicals for priority ranking of compounds of most concern for subsequent experimental and mechanistic toxicology analyses.

Levels of omega 3 fatty acids, vitamin D, dioxins and dioxin-like PCBs in oily fish; a new perspective on the reporting of nutrient and contaminant data for risk-benefit assessments of oily seafood.

Oily seafood is an important food source which contains several key nutrients beneficial for human health. On the other hand, oily seafood also contains persistent organic pollutants (POPs), including the dioxin-like compounds (DLCs) polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like-polychlorinated biphenyls (dl-PCBs), potentially detrimental to human health. For a comprehensive comparison of the beneficial and potentially adverse health effects of seafood consumption, risk-benefit analyses are necessary. Risk-benefit analyses require reliable quantitative data and sound knowledge of uncertainties and potential biases. Our dataset comprised more than 4000 analyses of DLCs and more than 1000 analyses each of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and vitamin D in the three most important Norwegian commercial oily seafood species: Atlantic herring (Clupea harengus), Atlantic mackerel (Scomber scombrus) and farmed Atlantic salmon (Salmo salar). The levels of several DLC congeners were below the limit of quantification (LOQ), making estimation of true levels challenging. We demonstrate that the use of upper bound substitution of censored data will overestimate, while lower bound substitution will underestimate the actual levels of DLCs. Therefore, we implement an alternative robust statistical method by combining Maximum Likelihood Estimation, Regression on Order Statistics and Kaplan-Meier analyses, which is better suited for providing estimations of levels of these contaminants in seafood. Moreover, we illustrate the impact of the toxic equivalency factor (TEF) system on estimation of the sums of DLCs by comparing the TEF system to an alternative system of relative effect potency (REP) factors (Consensus Toxicity Factors). The levels of nutrients and contaminants were related to adequate intake (AI) and tolerable weekly intake (TWI), respectively. We used AI and the TWI values established by the European Food Safety Authority (EFSA). The benefit and the risk were further viewed in the context of the Norwegian average intake of oily fish, and the Norwegian governmental official dietary recommendations of oily fish. Our results showed that both benefit and risk are met at the levels found of nutrients and DLCs in oily seafood. The comprehensive quantitative data presented here will be a key for future risk-benefit assessment of oily fish consumption. Together, our results underline that a refined formalized integrative risk-benefit assessment of oily fish in the diet is warranted, and that the data and methodology presented in this study are highly relevant for future integrated and multidisciplinary assessment of both risks and benefits of seafood consumption for human health.

Low dose exposure to HBCD, CB-153 or TCDD induces histopathological and hormonal effects and changes in brain protein and gene expression in juvenile female BALB/c mice.

Developmental health risks of chronical exposure to low doses of foodborne persistent organic pollutants (POP) are recognized but still largely uncharacterized. Juvenile female BALB/c mice exposed to either HBCD, CB-153 or TCDD at doses relevant to human dietary exposures (49.5 µg, 1.35 µg and 0.90 ng kg-1 bw-1 day-1, respectively) for 28 days displayed histopathological changes in liver (HBCD, CB-153, TCDD), thymus (HBCD, CB-153) and uterus (HBCD), reduced serum oestradiol 17ß (E2) levels (HBCD), increased serum testosterone (T) levels (CB-153) and an increased T/E2 ratio (HBCD). Proteomics analysis of brain provided molecular support for the HBCD-induced reduction in E2. Neural gene expression analysis, confirmed effects on 18 out of 30 genes previously found to be affected after exposure to higher doses to the same pollutants. Our findings indicate that exposure to POP at low doses is associated with subtle, but toxicological relevant effects on post-natal development in female mice.

Parallel in vivo and in vitro transcriptomics analysis reveals calcium and zinc signalling in the brain as sensitive targets of HBCD neurotoxicity.

Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that accumulates in humans and affects the nervous system. To elucidate the mechanisms of HBCD neurotoxicity, we used transcriptomic profiling in brains of female mice exposed through their diet to HBCD (199 mg/kg body weight per day) for 28 days and compared with those of neuronal N2A and NSC-19 cell lines exposed to 1 or 2 µM HBCD. Similar pathways and functions were affected both in vivo and in vitro, including Ca2+ and Zn2+ signalling, glutamatergic neuron activity, apoptosis, and oxidative stress. Release of cytosolic free Zn2+ by HBCD was confirmed in N2A cells. This Zn2+ release was partially quenched by the antioxidant N-acetyl cysteine indicating that, in accordance with transcriptomic analysis, free radical formation is involved in HBCD toxicity. To investigate the effects of HBCD in excitable cells, we isolated mouse hippocampal neurons and monitored Ca2+ signalling triggered by extracellular glutamate or zinc, which are co-released pre-synaptically to trigger postsynaptic signalling. In control cells application of zinc or glutamate triggered a rapid rise of intracellular [Ca2+]. Treatment of the cultures with 1 µM of HBCD was sufficient to reduce the glutamate-dependent Ca2+ signal by 50%. The effect of HBCD on zinc-dependent Ca2+ signalling was even more pronounced, resulting in the reduction of the Ca2+ signal with 86% inhibition at 1 µM HBCD. Our results show that low concentrations of HBCD affect neural signalling in mouse brain acting through dysregulation of Ca2+ and Zn2+ homeostasis.

Sensitivity and toxic mode of action of dietary organic and inorganic selenium in Atlantic salmon (Salmo salar).

Depending on its chemical form, selenium (Se) is a trace element with a narrow range between requirement and toxicity for most vertebrates. Traditional endpoints of Se toxicity include reduced growth, feed intake, and oxidative stress, while more recent finding describe disturbance in fatty acid synthesis as underlying toxic mechanism. To investigate overall metabolic mode of toxic action, with emphasis on lipid metabolism, a wide scope metabolomics pathway profiling was performed on Atlantic salmon (Salmo salar) (572±7g) that were fed organic and inorganic Se fortified diets. Atlantic salmon were fed a low natural background organic Se diet (0.35mg Se kg-1, wet weight (WW)) fortified with inorganic sodium selenite or organic selenomethionine-yeast (SeMet-yeast) at two levels (∼1-2 or 15mgkg-1, WW), in triplicate for 3 months. Apparent adverse effects were assessed by growth, feed intake, oxidative stress as production of thiobarbituric acid-reactive substances (TBARS) and levels of tocopherols, as well as an overall metabolomic pathway assessment. Fish fed 15mgkg-1 selenite, but not 15mgkg-1 SeMet-yeast, showed reduced feed intake, reduced growth, increased liver TBARS and reduced liver tocopherol. Main metabolic pathways significantly affected by 15mgkg-1 selenite, and to a lesser extent 15mgkg-1 SeMet-yeast, were lipid catabolism, endocannabinoids synthesis, and oxidant/glutathione metabolism. Disturbance in lipid metabolism was reflected by depressed levels of free fatty acids, monoacylglycerols and diacylglycerols as well as endocannabinoids. Specific for selenite was the significant reduction of metabolites in the S-Adenosylmethionine (SAM) pathway, indicating a use of methyl donors that could be allied with excess Se excretion. Dietary Se levels to respectively 1.1 and 2.1mgkg-1 selenite and SeMet-yeast did not affect any of the above mentioned parameters. Apparent toxic mechanisms at higher Se levels (15mgkg-1) included oxidative stress and altered lipid metabolism for both inorganic and organic Se, with higher toxicity for inorganic Se.

Vitamin E reduces endosulfan-induced toxic effects on morphology and behavior in early development of zebrafish (Danio rerio).

The aim of this study was to investigate if vitamin E (α-TOC) modulates the developmental toxicity of the pesticide endosulfan (ESF), using a modified zebrafish embryotoxicity test (ZET). Zebrafish (Danio rerio) embryos were exposed from 6 to 72 h post fertilization (hpf) to either ESF (0.1-50 mg/L) or α-TOC (0.01-3 mM) alone or in combination. The effects of these exposures on embryonic morphology, larval behavior and antioxidant gene expression were analyzed. Phenotypic analysis at 48 hpf showed that ESF led to a dose-dependent increase in embryonic deformities, including axis malformations, pericardial edema and reduced pigmentation. Co-exposure of ESF with α-TOC (1-3 mM) significantly (p < 0.05) reduced ESF-induced embryonic malformations. Exposure to solely α-TOC did not affect rates of survival or malformations. Behavior studies showed that ESF caused hyperactivity at 5 days post fertilization, indicating a developmental neurotoxic effect. The ESF-induced hyperactivity was ameliorated by α-TOC. Elevated ESF concentrations caused down-regulation of the antioxidant genes cuzn-sod, gpx1a and cat, suggesting that ESF promoted oxidative stress in the embryos. α-TOC did not prevent the ESF-induced dysregulation of these genes. These results demonstrate that α-TOC protects against phenotypic and behavioral effects caused by ESF but did not rescue ESF-induced aberrations in antioxidant gene expression.

Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

UNLABELLED: The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. SIGNIFICANCE: To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient.

Cross-omics gene and protein expression profiling in juvenile female mice highlights disruption of calcium and zinc signalling in the brain following dietary exposure to CB-153, BDE-47, HBCD or TCDD.

The present study assessed if eating a diet of fish, spiked with persistent organic pollutants (POPs), affects gene and protein expression in the maturing mouse brain. Juvenile female Balb/c mice (22 days of age) were exposed for 28 days to fish-based diets spiked with the dioxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) or the non dioxin-like (NDL) chemicals hexabromocyclodocecane (HBCD), 2,2'4,4'-tetrabromodiphenylether (BDE-47) or 2,2'4,4',5,5'-hexachlorobiphenyl (CB-153) at doses approximating their respective lowest observed adverse effect levels (LOAEL). It was found that all POPs elicited changes in neural gene and protein expression profiles. Bioinformatic analysis of gene expression data highlighted the importance of the aryl hydrocarbon receptor (AHR) in dioxin toxicity and revealed that zinc regulation in the brain is targeted by TCDD through the AHR. Calcium homeostasis was affected by both TCDD and the NDL chemicals. In contrast to the transcriptomic analysis, the proteomics data did not allow for a clear distinction between DL and NDL responses in the juvenile brain but indicated that proteins associated with excitotoxicity were affected in all exposure groups. Integrated interpretation of data led to the conclusion that the dietary contaminants investigated in the present study breach the blood brain barrier (BBB) and accumulate in the juvenile brain where they may induce excitotoxic insults by dysregulation of the otherwise tightly controlled homeostasis of calcium and zinc. Overall, the findings of the present study highlight the need for further assessment of the risks associated with early life exposure to foodborne POPs.

Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants (HBCD, BDE-47, PCB-153, TCDD): comparative assessment of effects in potential target tissues.

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2',4,4'-Tetrabromodiphenyl ether BDE-47; 2,2',4,4',5,5'-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 µg/kg bw/day; PCB-153 195 µg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects.