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.

Modelling of the feed-to-fillet transfer of ethoxyquin and one of its main metabolites, ethoxyquin dimer, to the fillet of farmed Atlantic salmon (Salmon salar L.).

Ethoxyquin (EQ) is an antioxidant supplemented to feed ingredients, mainly fish meal, which is currently under re-evaluation for use in the food production chain. EQ is partly metabolized into several metabolites of which the ethoxyquin dimer (EQDM) accumulates most in the farmed fish fillet. In this study, the feed-to-fillet transfer of dietary EQ and EQDM in Atlantic salmon fillet was investigated, and a physiologically based pharmacokinetic (PBPK-) two-compartmental model was developed, based on experimental determined EQ and EQDM uptake, metabolism, and elimination kinetics. The model was verified with an external data-set and used to simulate the long term (>1.5 years) EQ and EQDM feed-to fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth, and fillet fat deposition. The model predictions showed that initial EQDM levels in juvenile fish are the driving factor in final levels found in food-producing animals, while for EQ the levels in feed, and seasonal variations were the driving factor for food EQ levels.

Zinc uptake in fish intestinal epithelial model RTgutGC: Impact of media ion composition and methionine chelation.

Apical uptake of zinc as ionic Zn(II) or as Zn-methionine (Zn-Met) was studied in RTgutGC cell line in vitro under media compositions mirroring the gut luminal ionic concentration of freshwater (FW) and seawater (SW) acclimated salmonids. Viability of the RTgutGC cells exposed to experimental media preparations showed a time-dependent decrease in SW treated cells, with the effect being significant at 48 h (P < 0.01), but not at 12 h or 24 h. Half effective concentration of Zn exposure over 12 h (EC50, in µM) was not differentially affected by media composition (FW, 59.7 ± 12.1 or SW, 83.2 ± 7.2; mean ± SE, P = 0.43). Zinc (65Zn) influx in RTgutGC was not different between FW or SW treated cells, but increased significantly in the presence of methionine (2 mM, L-Met or DL-Met). An interaction effect was observed between Zn concentration and media ionic composition on the impact of Met on apical Zn uptake (L-met, P < 0.001; DL-met, P = 0.02). In the presence of Met, apical Zn uptake in SW medium was significantly lower compared to FW, but only at higher Zn concentrations (12 and 25 µM, P < 0.01). Further, Met facilitated Zn uptake was reduced in cells treated with an amino acid transport system blocker with the effect being more significant and stereospecific in SW ionic conditions. The findings of this study showed that (i) Zn speciation in the presence of Met improved apical Zn uptake in RTgutGC cells and Zn-Met species were possibly taken up through Met uptake system. (ii) The effect was differentially affected by the ionic composition of the medium. Implications and limitations of the observations towards practical Zn nutrition of salmonids are discussed.

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.

Investigations on the metabolism and potentially adverse effects of ethoxyquin dimer, a major metabolite of the synthetic antioxidant ethoxyquin in salmon muscle.

The feed additive ethoxyquin (EQ) is a commonly used synthetic antioxidant preservative in animal feeds. In farmed Atlantic salmon fillets, EQ residues are present, both as the parent compound and as EQ derivatives. One of the main EQ derivates in fish muscle is an ethoxyquin dimer (EQDM), and the potential toxicity of this metabolite is not known. The aim of this study was to evaluate the metabolism and potentially toxicological effects of EQDM. A 90-day subchronic exposure study with repeated dietary exposure to EQDM at 12.5 mg/kg of body weight per day was performed with male F344 rats. Hepatic Cyp1a1 mRNA was significantly reduced to <3% of the control in rats fed EQDM, and hepatic Cyp2b1 mRNA was increased to 192%. EQDM increased Gstpi1 mRNA expression to 144% that of the control, but the activity level of this phase II enzyme was reduced. Biomarkers of liver and kidney function did indicate adverse effects of EQDM when F344 rats were fed 12.5 mg/kg of body weight per day. The present study revealed that EQDM produces responses that are comparable to those produced by the parent compound (EQ) in terms of activating the same enzyme systems.

Dietary fatty acids and inflammation in the vertebral column of Atlantic salmon, Salmo salar L., smolts: a possible link to spinal deformities.

Vegetable oils (Vo) are an alternative to fish oil (Fo) in aquaculture feeds. This study aimed to evaluate the effect of dietary soybean oil (Vo diet), rich in linoleic acid, and of dietary fish oil (Fo diet) on the development of spinal deformities under bacterial lipopolysaccharide (LPS)-induced chronic inflammation conditions in Atlantic salmon, Salmo salar L. Fish [25 g body weight (BW)] were fed the experimental diets for 99 days. On day 47 of feeding (40 g BW), fish were subjected to four experimental regimes: (i) intramuscular injections with LPS, (ii) sham-injected phosphate-buffered saline (PBS), (iii) intraperitoneally injected commercial oil adjuvant vaccine, or (iv) no treatment. The fish continued under a common feeding regime in sea water for 165 more days. Body weight was temporarily higher in the Vo group than in the Fo group prior to immunization and was also affected by the type of immunization. At the end of the trial, no differences were seen between the dietary groups. The overall prevalence of spinal deformities was approximately 14% at the end of the experiment. The Vo diet affected vertebral shape but did not induce spinal deformities. In groups injected with LPS and PBS, spinal deformities ranged between 21% and 38%, diet independent. Deformed vertebrae were located at or in proximity to the injection point. Assessment of inflammatory markers revealed high levels of plasma prostaglandin E2 (PGE2) in the Vo-fed and LPS-injected groups, suggesting an inflammatory response to LPS. Cyclooxigenase 2 (COX-2) mRNA expression in bone was higher in fish fed Fo compared to Vo-fed fish. Gene expression of immunoglobulin M (IgM) was up-regulated in bone of all LPS-injected groups irrespective of dietary oil. In conclusion, the study suggests that Vo is not a risk factor for the development of inflammation-related spinal deformities. At the same time, we found evidence that localized injection-related processes could trigger the development of vertebral body malformations.

Retinoic acid cross-talk with calcitriol activity in Atlantic salmon (Salmo salar).

Vitamins A (VA) and D (VD) are metabolised by vertebrates to bioactive retinoic acid (RA) and calcitriol (CTR). RA and CTR involvement in bone metabolism requires fine-tuned regulation of their synthesis and breakdown. In mammals antagonism of VA and VD is observed, but the mechanism of interaction is unknown. We investigated VA-VD interactions in Atlantic salmon (Salmo salar L.) following i.p. injection of RA and/or CTR. VA metabolites, CTR, calcium (Ca), magnesium (Mg) and phosphorus (P) were determined in plasma. Expression of bone matrix Gla protein (mgp), collagen 1 alpha2 chain (col1a2) and alkaline phosphatase (alp) mRNA was quantified to reflect osteogenesis. Branchial epithelial Ca channel (ecac listed as trpv6 in ZFIN Database) mRNA levels and intestinal Ca and P influx were determined to study Ca/P handling targets of RA and CTR. RA-injection (with or without CTR) decreased plasma CTR-levels three- to sixfold. CTR injection did not affect RA metabolites, but lowered CTR in plasma 3 and 5 days after injection. Lowered plasma CTR correlated with decreased mgp and col1a2 expression in all groups and with decreased alp in CTR-injected fish. RA-treated salmon had enhanced alp expression, irrespective of reduced plasma CTR. Expression of ecac and unidirectional intestinal influx of Ca were stimulated following RA-CTR treatment. Plasma Ca, Mg and P were not affected by any treatment. The results suggest cross-talk of RA with the VD endocrine system in Atlantic salmon. Enhanced Ca flux and osteogenesis (alp transcription) in RA-treated fish and inhibition of mgp expression revealed unprecedented disturbance of Ca physiology in hypervitaminosis A.

The vitamin D receptor and its ligand 1alpha,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar).

Seaward migration of Salmo salar is preceded by preparatory physiological adaptations (parr-smolt transformation) to allow for a switch from freshwater (FW) to seawater (SW), which also means a switch in ambient calcium from hypocalcic (<1 mM Ca(2+)) to the plasma (~1.25 mM Ca(2+)) and to strongly hypercalcic (8-12 mM Ca(2+)). Uptake, storage (skeleton, scales) and excretion of calcium need careful regulation. In fish, the vitamin D endocrine system plays a rather enigmatic role in calcium physiology. Here, we give direct evidence for calcitriol involvement in SW migration. We report the full sequence of the nuclear vitamin D receptor (sVDR0) and two alternatively spliced variants resulting from intron retention (sVDR1 and sVDR2). In FW parr, SW adapting smolts, and in SW adults, plasma concentrations of 25(OH)D(3) and 24,25(OH)(2)D(3) did not change significantly. Plasma calcitriol concentrations were lowest in FW parr, doubled during smoltification and remained elevated in SW adults. Increased calcitriol coincided with a twofold decrease in sVDR mRNA levels in gill, intestine, and kidney of FW smolts and SW adults, when compared with parr. Clearly, there was a negative feedback and dynamic response of the vitamin D endocrine system during parr-smolt transformation. The onset of these dynamic changes in FW parr warrants a further search for the endocrines that initiate these changes. We speculate that the vitamin D system plays a crucial role in calcium and phosphorus handling in Atlantic salmon.

Teratogenicity of elevated egg incubation temperature and egg vitamin A status in Atlantic salmon, Salmo salar L.

The present study was undertaken to investigate the possibility that high egg vitamin A (VA) status in combination with elevated egg incubation temperatures may cause deformities in Atlantic salmon, Salmo salar L. Egg batches selected for their total VA concentration were exposed to low (normal, 8 degrees C) or elevated (14 degrees C) egg incubation temperatures. Temperature was the main factor causing bone deformities such as warped gill opercula, fin and jaw deformities, but not for the development of spinal deformities where all groups displayed a 'baseline' occurrence of mild deformity (decreased vertebral size in the cephalic region) and no systematic variation in the occurrence of serious spinal deformities (fused vertebrae). A possible effect of egg incubation temperature fluctuation was found for the groups reared at low temperatures. An indication of a negative effect of elevated egg VA status for the development of organ deformities such as missing septum transversum and situs inversus was found in addition to temperature effects, however, no firm conclusions could be drawn from the present data. The phenotypes for temperature-induced deformities resembled the phenotype of VA-induced deformities, but no clear conclusions on the causality of the deformities found in the present study could be drawn. Egg incubation temperatures, both absolute temperature and temperature variations, should therefore be strictly controlled.

Determination of carotenoid and vitamin A concentrations in everted salmonid intestine following exposure to solutions of carotenoid in vitro.

Carotenoid (astaxanthin and canthaxanthin) concentrations in everted intestine from rainbow trout (Oncorhynchus mykiss, Walbaum) and Atlantic salmon (Salmo salar, L.) exposed to micelle solubilised carotenoid, have been determined. Following exposure (1 h) to astaxanthin solution (5 mg l(-1)), trout pyloric caeca and mid intestine had higher (P<0.05) mean tissue astaxanthin concentrations (0.50+/-0.08 microg g(-1) and 0.54+/-0.09 microg g(-1), respectively) compared to hind intestine (0.04+/-0.01 microg g(-1); n=11+/-S.E.). Furthermore, the astaxanthin concentration in pyloric caeca (0.50+/-0.08 microg g(-1)) was greater (P<0.05) than that of canthaxanthin (0.11+/-0.01 microg g(-1); n=11, +/-S.E.) when exposed to solutions of similar carotenoid concentration (5.11+/-0.16 mg l(-1) and 5.35+/-0.16 mg l(-1), respectively; n=3+/-S.E.). However, no differences (P>0.05) were recorded between trout and salmon intestinal tissue in terms of astaxanthin concentration following exposure. Trout caeca exposed to astaxanthin solution had significantly (P<0.05) more vitamin A (514.1+/-36.4 microg g(-1)) compared to control tissues (316.5+/-61.7 microg g(-1); n=8+/-S.E.). Vitamin A(1) concentrations in caeca (287.7+/-11.0 microg g(-1)) exposed to astaxanthin solution were significantly higher (P<0.05) compared to controls (174.9+/-26.9 microg g(-1)). However, vitamin A(2) concentrations were not significantly (P>0.05) different (226.3+/-28.2 microg g(-1) and 141.6+/-35.2 microg g(-1), respectively).