Organ-specific biotransformation in salmonids: Insight into intrinsic enzyme activity and biotransformation of three micropollutants.

Aquatic ecosystems continue to be threatened by chemical pollution. To what extent organisms are able to cope with chemical exposure depends on their ability to display mechanisms of defense across different organs. Among these mechanisms, biotransformation processes represent key physiological responses that facilitate detoxification and reduce the bioaccumulation potential of chemicals. Biotransformation does not only depend on the ability of different organs to display biotransformation enzymes but also on the affinity of chemicals towards these enzymes. In the present study, we explored the ability of different organs and of two freshwater fish to support biotransformation processes through the determination of in vitro phase I and II biotransformation enzyme activity, and their role in supporting intrinsic clearance and the formation of biotransformation products. Three environmentally relevant pollutants were evaluated: the polycyclic aromatic hydrocarbon (PAH) pyrene (as recommended by the OECD 319b test guideline), the fungicide azoxystrobin, and the pharmaceutical propranolol. Comparative studies using S9 sub-cellular fractions derived from the liver, intestine, gills, and brain of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) revealed significant phase I and II enzyme activity in all organs. However, organ- and species-specific differences were found. In brown trout, significant extrahepatic biotransformation was observed for pyrene but not for azoxystrobin and propranolol. In rainbow trout, the brain appeared to biotransform azoxystrobin. In this same species, propranolol appeared to be biotransformed by the intestine and gills. Biotransformation products could be detected only from hepatic biotransformation, and their profiles and formation rates displayed species-specific patterns and occurred at different magnitudes. Altogether, our findings further contribute to the current understanding of organ-specific biotransformation capacity, beyond the expression and activity of enzymes, and its dependence on specific enzyme-chemical interactions to support mechanisms of defense against exposure.

Study of the influence of tributyrin-supplemented diets on the gut bacterial communities of rainbow trout (Oncorhynchus mykiss).

Dietary supplementation with triglyceride tributyrin (TBT), a butyrate precursor, has been associated with beneficial effects on fish health and improvements in the ability of carnivorous fish to tolerate higher levels of plant-based protein. In this study, we aimed to investigate the effects of a plant-based diet supplemented with TBT on the structural diversity and putative function of the digesta-associated bacterial communities of rainbow trout (Oncorhynchus mykiss). In addition to this, we also assessed the response of fish gut digestive enzyme activities and chyme metabolic profile in response to TBT supplementation. Our results indicated that TBT had no significant effects on the overall fish gut bacterial communities, digestive enzyme activities or metabolic profile when compared with non-supplemented controls. However, a more in-depth analysis into the most abundant taxa showed that diets at the highest TBT concentrations (0.2% and 0.4%) selectively inhibited members of the Enterobacterales order and reduced the relative abundance of a bacterial population related to Klebsiella pneumoniae, a potential fish pathogen. Furthermore, the predicted functional analysis of the bacterial communities indicated that increased levels of TBT were associated with depleted KEGG pathways related to pathogenesis. The specific effects of TBT on gut bacterial communities observed here are intriguing and encourage further studies to investigate the potential of this triglyceride to promote pathogen suppression in the fish gut environment, namely in the context of aquaculture.

Non-microcystin extracellular metabolites of Microcystis aeruginosa impair viability and reproductive gene expression in rainbow trout cell lines.

Microcystis aeruginosa is a ubiquitous freshwater cyanobacterium best known for producing hepatotoxic microcystins; however, this common bloom-forming species also produces myriad biologically active and potentially deleterious other metabolites. Our understanding of the effects of these non-microcystin metabolites on fish is limited. In this study, we evaluated cytotoxicity of extracellular metabolites harvested from both microcystin-producing (MC+) and non-producing (MC-) strains of M. aeruginosa on rainbow trout (Oncorhynchus mykiss) cell lines derived from tissues of the brain, pituitary, heart, gonads, gills, skin, liver, and milt. We also examined the influence of M. aeruginosa exudates (MaE) on the expression of critical reproduction-related genes using the same cell lines. We found that exudates of the MC- M. aeruginosa strain significantly reduced viability in RTBrain, RTgill-W1, and RT-milt5 cell lines and induced significant cellular stress and/or injury in six of the eight cell lines-highlighting potential target tissues of cyanobacterial cytotoxic effects. Observed sublethal consequences of Microcystis bloom exposure occurred with both MC+ and MC- strains' exudates and significantly altered expression of developmental and sex steroidogenic genes. Collectively, our results emphasize the contributions of non-MC metabolites to toxicity of Microcystis-dominated algal blooms and the need to integrate the full diversity of M. aeruginosa compounds-beyond microcystins-into ecotoxicological risk assessments.

A model naphthenic acid decouples oxidative phosphorylation through selective inhibition of mitochondrial complex activity.

The naphthenic acid fraction compound (NAFC), 3,5-dimethyladamantane-1-acetic acid, was tested for its ability to uncouple mitochondrial oxidative phosphorylation. Mitochondria isolated from rainbow trout (Oncorhynchus mykiss) liver were exposed to 3,5-dimethyladamantane-1-acetic acid in state 3 and 4 respiration, and mitochondrial membrane potential were quantified. Electron transport chain (ETC) protein complexes were isolated using pharmacological agents and inhibitors, and their activities measured. The NAFC compound completely inhibited states 3 and 4 respiration with an IC50 of 0.77 and 1.01 mM, respectively. The NAFC compound partially uncoupled mitochondrial membrane potential in state 3 and 4 respiration with an IC50 of 2.19 and 1.73 mM, respectively. The NAFC impaired the activities of ETC protein complexes with a 9.5-fold range in sensitivity. The relative inhibitory effect of the ETC protein complexes to NAFC was CIV≥CI>CIII>CII. The impairment of mitochondrial oxidative phosphorylation by adamantane 3,5-dimethyladamantane-1-acetic acid is mediated via its inhibition of ETC protein complexes.

A cell line derived from heart of rainbow trout is refractory to Tilapia lake virus.

Cell lines are important in vitro models to answer biological mechanisms with less genetic variations. The present study was attempted to develop a cell line from rainbow trout, where we obtained a cell line from the heart, named "RBT-H." The cell line was authenticated using karyotyping and cytochrome c oxidase subunit I (COI) gene sequencing. The karyotype demonstrated diploid chromosome number (2n) as 62 and the sequence of partial COI gene was 99.84% similar to rainbow trout COI data set, both suggesting the origin of RBT-H from the rainbow trout. The heart cell line was mycoplasma-free and found to be refractory to infection with the Tilapia lake virus. The RBT-H cell line is deposited in the National Repository of Fish Cell Line (NRFC) at ICAR-NBFGR, Lucknow, India, with Accession no. NRFC0075 for maintenance and distribution to researchers on request for R&D.

Interspecific competitive interactions affect body size and oxidative status of two nonnative salmonid species.

In fish, interspecific interactions between nonnative and other sympatric species are considered determinants in shaping species assemblages. Such interactions can also arise between nonnative fish species only, including salmonids such as the brown trout (Salmo trutta, Linnaeus, 1758) and the rainbow trout (Oncorhynchus mykiss, Walbaum, 1792), returning contrasting outcomes. The present manipulative experiment was aimed at exploring the effect of interspecific competition on the body growth and the oxidative status of parr (2 + -year-old individuals) of the brown trout and the rainbow trout. Allopatric (intraspecific competition) and sympatric (interspecific competition) populations of these species were experimentally recreated in two wild streams. At the end of a 2-month-long experiment, changes in specific growth rate (SGR), oxidative status (i.e., levels of reactive oxygen species and activity of antioxidant enzymes such as superoxide dismutase - SOD, catalase - CAT and glutathione peroxidase - GPx) and oxidative damage (i.e., lipid peroxidation) were investigated in brown and rainbow trout individuals maintained in allopatric or sympatric populations. Sympatric interactions between rainbow and brown trout parr resulted in a significant decrease in SGR of brown trout individuals only. Moreover, an overall modulation of the oxidative status, in terms of an increase in ROS levels coupled with the activation of SOD and CAT activity, occurred in brown trout individuals under sympatric conditions. These findings might suggest that, under sympatric conditions, parr of the rainbow trout are more competitive than brown trout for food acquisition. However, this competition affected the antioxidant defenses of the brown trout only, probably because of reduced ingestion of dietary antioxidants or increased physical activity and aggressive behavior. Thus, interspecific interactions can induce physiological and phenotypic effects on parr of nonnative salmonids, with potential consequences on the establishment of populations of these species in freshwater ecosystems.

Inhibition effect of epicatechin gallate on acid phosphatases from rainbow trout (Oncorhynchus mykiss) liver by multispectral and molecular docking.

Inhibition of acid phosphatase, which significantly contributes to inosine 5'-monophosphate (IMP) degradation, is crucial for preventing flavor deterioration of aquatic products during storage. In this study, the inhibitory effect of epicatechin gallate (ECG) on the activity of acid phosphatase isozymes (ACPI and ACPII) was analyzed using inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry, and molecular simulation. ACPI and ACPII with molecular weights of 59.5 and 37.3 kDa, respectively, were purified from rainbow trout liver. ECG reversibly inhibited ACPI and ACPII activities via mixed-type inhibition, with half maximal inhibitory concentration (IC50) of 0.24 ± 0.01 mmol/L and 0.27 ± 0.03 mmol/L, respectively. Fluorescence spectra indicated that ECG statically quenched the intrinsic fluorescence of ACPI and ACPII. ECG could spontaneously bind to ACPI and ACPII through hydrogen bonding and van der Waals forces and exhibited a higher affinity for ACPI than for ACPII. In addition, molecular dynamic simulation revealed that ECG-ACPI and ECG-ACPII complexes were relatively stable during the entire simulation process. Our findings provide a theoretical basis for the use of ECG as an inhibitor of ACP to improve the flavor of aquatic products.

Bioconcentration Assessment of Three Cationic Surfactants in Permanent Fish Cell Lines.

Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid-water partitioning coefficient, DMLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Dietary sulfated polysaccharides extracted from Caulerpa sp. and Padina sp. modulated physiological performance, antibacterial activity and ammonia challenge test in juvenile rainbow trout (Oncorhynchus mykiss).

Nowadays, the use of seaweed derivatives in aquaculture has drawn attention for their potential as an immunostimulant and growth promotor. The sulfated polysaccharide extracted (SPE ) from green (Caulerpa sp.; SPC) and brown (Padina sp.; SPP) seaweeds with two concentrations (0.05% and 0.1%); nominated in four groups: SPC0.05 , SPC0.1 , SPP0.05 , SPP0.1 and control group (free of SPE ) were used for juvenile rainbow trout (Oncorhynchus mykiss) diet. Fish (N: 150; 8.5 ± 0.2 g) were selected aleatory distributed in 15 circular tanks (triplicate for the group) and fed test diets for 56 days. The outcomes revealed that the supplementation of SPE up to 1 g kg-1 failed to show significant differences in the organosomatic indices as compared to the control group. The most inferior protein value of dress-out fish composition was observed in the fish fed the control diet, which was statistically lower than the SCP0.1 group (p < 0.05), while no significant difference was observed in other macronutrient composition among the treatments. Total monounsaturated fatty acid (MUFA) had lower trend in the carcass of fish fed SPE supplemented diets, so that lowest MUFA were observed in SPC0.05 group (p < 0.05; 25.22 ± 4.29%). The lowest value of docosahexaenoic acid was observed in the control diet compared to the SPE -supplemented diets (p < 0.05). The serum alternative complement pathway levels in all treatments tend to promote compared to the control treatment. A similar trend was observed for lysozyme activity. According to the results, the superoxide dismutase (SOD) value were highest in SPC0.05 and SPC0.1 compared to the other treatments (p < 0.05), while a further elevation of the SPE Padina sp. extracted level (SPP0.1 ) leads to a decrease in SOD value. Thiobarbituric acid reactive substances of plasma was indicated not to influence by sulfated polysaccharide extracts in the refrigerated storage. The lowest serum stress indicators were observed in fish fed SPP0.05 group postchallenge test. Taken together, our outcomes revealed that SPE of two species of seaweeds bestows benefits in some of the immunity and antioxidant system. Also, notable elevations in HUFA were observed in juvenile rainbow trout fed supplemented with SPE .

Inhibitory effects of Oncorhynchus mykiss lipids in LPS-induced RAW264.7 cells via suppression of NF-κB and MAPK pathways.

Oncorhynchus mykiss, a significant aquaculture species, possesses compounds with numerous biological and pharmacological functions, including antioxidant, anticancer, anti-microbial, and anti-obesity effects. However, possible anti-inflammatory effects of lipids extracted from O. mykiss eggs on RAW264.7 cells induced by LPS have not been elucidated yet. The current study identified 13 fatty acids in lipids extracted from O. mykiss eggs that contained high amounts (51.92% of total fatty acids) of polyunsaturated fatty acids (PUFAs), especially DHA (33.66%) and EPA (7.77%). These O. mykiss lipids (100-400 µg/mL) showed significant anti-inflammatory effects by inhibiting NO and iNOS expression in LPS-stimulated RAW264.7 cells. They also inhibited expression of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α, while upregulating anti-inflammatory cytokines IL-10, IL-11, and TGF-ß. These lipids from O. mykiss effectively inhibited LPS-induced expression CD86 as a surface biomarker on RAW264.7 cells. Additionally, O. mykiss lipids suppressed phosphorylation of p38, JNK, and ERK1/2 and the expression of phosphorylated NF-κB subunit p65. These findings indicate that O. mykiss lipids possess anti-inflammatory properties by inhibiting NF-κB and MAPK signaling pathways.

Dual effect of polyaromatic hydrocarbons on sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity of a teleost fish (Oncorhynchus mykiss).

Polycyclic aromatic hydrocarbons (PAHs) are embryo- and cardiotoxic to fish that might be associated with improper intracellular Ca2+ management. Since sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of intracellular Ca2+, the SERCA activity and the contractile properties of rainbow trout (Oncorhynchus mykiss) ventricle were measured in the presence of 3- and 4-cyclic PAHs. In unfractionated ventricular homogenates, acute exposure of SERCA to 0.1-1.0 µM phenanthrene (Phe), retene (Ret), fluoranthene (Flu), or pyrene (Pyr) resulted in concentration-dependent increase in SERCA activity, except for the Flu exposure, with maximal effects of 49.7-83 % at 1 µM. However, PAH mixture did not affect the contractile parameters of trout ventricular strips. Similarly, all PAHs, except Ret, increased the myotomal SERCA activity, but with lower effect (27.8-40.8 % at 1 µM). To investigate the putative chronic effects of PAHs on SERCA, the atp2a2a gene encoding trout cardiac SERCA was expressed in human embryonic kidney (HEK) cells. Culture of HEK cells in the presence of 0.3-1.0 µM Phe, Ret, Flu, and Pyr for 4 days suppressed SERCA expression in a concentration-dependent manner, with maximal inhibition of 49 %, 65 %, 39 % (P < 0.05), and 18 % (P > 0.05), respectively at 1 µM. Current findings indicate divergent effects of submicromolar PAH concentrations on SERCA: stimulation of SERCA activity in acute exposure and inhibition of SERCA expression in chronic exposure. The depressed expression of SERCA is likely to contribute to the embryo- and cardiotoxicity of PAHs by depressing muscle function and altering gene expression.

Involvement of miR-495 in the skin pigmentation of rainbow trout (Oncorhynchus mykiss) through the regulation of mc1r.

MicroRNAs (miRNAs) play crucial roles in skin pigmentation in animals. Rainbow trout (Oncorhynchus mykiss) is a key economic fish species worldwide, and skin color directly affects its economic value. However, the functions of miRNAs in rainbow trout skin pigmentation remain largely unknown. Herein, we overexpressed and silenced miR-495 in vitro and in vivo to investigate its functions. The analysis of spatial and temporal expression patterns suggested that miR-495 is a potential regulator during the process of skin pigmentation. In vitro, mc1r was validated as a direct target for miR-495 by dual-luciferase reporter assay, and overexpression of miR-495 significantly inhibited mc1r expression; in contrast, mc1r and its downstream gene mitf levels were markedly upregulated by decreased miR-495. In vivo, overexpressed miR-495 by injecting agomiR-495 led to a substantial decrease in the expression of mc1r and mitf in dorsal skin and liver, while the opposite results were obtained after miR-495 silencing by antagomiR-495. These findings suggested that miR-495 can target mc1r to regulate rainbow trout skin pigmentation, which provide a potential basis for using miRNAs as target drugs to treat pigmentation disorders and melanoma.

Effects of Acute and Subchronic Waterborne Thallium Exposure on Ionoregulatory Enzyme Activity and Oxidative Stress in Rainbow Trout (Oncorhynchus mykiss).

The mechanisms of acute (96-hour) and subchronic (28-day) toxicity of the waterborne trace metal thallium (Tl) to rainbow trout (Oncorhynchus mykiss) were investigated. Specifically, effects on branchial and renal ionoregulatory enzymes (sodium/potassium adenosine triphosphatase [ATPase; NKA] and proton ATPase) and hepatic oxidative stress endpoints (protein carbonylation, glutathione content, and activities of catalase and glutathione peroxidase) were examined. Fish (19-55 g) were acutely exposed to 0 (control), 0.9 (regulatory limit), 2004 (half the acute median lethal concentration), or 4200 (acute median lethal concentration) µg Tl L-1 or subchronically exposed to 0, 0.9, or 141 (an elevated environmental concentration) µg Tl L-1 . The only effect following acute exposure was a stimulation of renal H+ -ATPase activity at the highest Tl exposure concentration. Similarly, the only significant effect of subchronic Tl exposure was an inhibition of branchial NKA activity at 141 µg Tl L-1 , an effect that may reflect the interaction of Tl with potassium ion handling. Despite significant literature evidence for effects of Tl on oxidative stress, there were no effects of Tl on any such endpoint in rainbow trout, regardless of exposure duration or exposure concentration. Elevated basal levels of antioxidant defenses may explain this finding. These data suggest that ionoregulatory perturbance is a more likely mechanism of Tl toxicity than oxidative stress in rainbow trout but is an endpoint of relevance only at elevated environmental Tl concentrations. Environ Toxicol Chem 2024;43:87-96. © 2023 SETAC.

Long-term regulation of fat sensing in rainbow trout (Oncorhynchus mykiss) fed a vegetable diet from the first feeding: focus on free fatty acid receptors and their signalling.

Taste plays a fundamental role in an animal's ability to detect nutrients and transmits key dietary information to the brain, which is crucial for its growth and survival. Providing alternative terrestrial ingredients early in feeding influences the growth of rainbow trout (RT, Oncorhynchus mykiss). Thus, the present study aimed to assess the influence, via long-term feeding (from the first feeding to 8 months), of alternative plant ingredients (V diet for vegetable diet v. C diet for a control diet) in RT on the mechanism of fat sensing at the gustatory level. After the feeding trial, we studied the pathways of the fat-sensing mechanism in tongue tissue and the integrated response in the brain. To this end, we analysed the expression pattern of free fatty acid receptors (ffar) 1 and 2, markers of calcium-signalling pathways (phospholipase Cß, Orai, Stim or Serca), the serotonin level (a key neurotransmitter in taste buds) and the expression pattern of appetite-regulating neuropeptides in the hypothalamus (central area of appetite regulation). The results revealed that the V diet modified the expression pattern of ffar1 and paralogs of ffar2 genes in tongue tissue, along with differential regulation of calcium-signalling pathways and a defect in serotonin level and brain turnover, without influencing neuropeptide expression. This study is the first to support that changes in feeding behaviour of RT fed a V diet could be due to the difference in nutrient sensing and a decrease in hedonic sensation. We revealed that RT have similar fat-detection mechanisms as mammals.

Bioaccumulation of Linear Siloxanes in Fish.

The bioaccumulation behavior, including the uptake, internal distribution, depuration, and biotransformation rates, of three widely used linear methyl-siloxanes was investigated in rainbow trout. Dietary uptake efficiencies of octamethyltrisiloxane (L3), decamethyltetrasiloxane (L4), and dodecamethylpentasiloxane (L5) were 15% (3.3% standard error [SE]), 8.6% (1.4% SE), and 15% (1.8% SE), respectively, and for L3 and L4 were well below those of nonmetabolizable reference chemicals with similar octanol-water partition coefficients, suggesting significant intestinal biotransformation of L3 and L4. Somatic biotransformation rate constants were 0.024 (0.003 SE) day-1 for L3 and 0.0045 (0.0053 SE) day-1 for L4 and could not be determined for L5. Lipid-normalized biomagnification factors for L3, L4, and L5 were 0.24 (0.02 SE), 0.24 (0.01 SE), and 0.62 (0.05 SE) kg-lipid kg-lipid-1 , respectively. Bioconcentration factors standardized to a 5% lipid content fish for water in Canadian oligotrophic lakes with a dissolved organic carbon content of 7.1 mg L-1 were 2787 (354 SE) for L3, 2689 (312 SE) for L4, and 1705 (418 SE) L kg-wet weight-1 , respectively, and 3085 (392 SE) for L3, 4227 (490 SE) for L4, and 3831 (938 SE) L kg-wet weight-1 in water with a dissolved organic carbon content of 2.0 mg L-1 . A comparison of 238 bioaccumulation profiles for 166 different chemicals shows that the bioaccumulation profiles for L3, L4, and L5 are vastly different from those of other very hydrophobic compounds found in the environment. Environ Toxicol Chem 2024;43:42-51. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Interactive Effects of Copper-Silver Mixtures at the Intestinal Epithelium of Rainbow Trout: An In Vitro Approach.

While metals are present in mixture in the environment, metal toxicity studies are usually conducted on an individual metal basis. There is a paucity of data in the existing literature regarding specific metal-metal interactions and their effect on metal toxicity and bioavailability. We studied interactions of a silver (Ag)-copper (Cu) mixture at the intestinal epithelium using an intestinal cell line derived from rainbow trout (Oncorhynchus mykiss), the RTgutGC. Exposures were conducted in media containing different chloride concentrations (low chloride, 1 mM; high chloride, 146 mM), thus resulting in different metal speciation. Cytotoxicity was evaluated based on two endpoints, cell metabolic activity and cell membrane integrity. The Ag-Cu mixture toxicity was assessed using two designs: independent action and concentration addition. Metal mixture bioavailability was studied by exposing cells to 500 nM of Ag or Cu as a single metal or a mixture (i.e., 500 nM of Cu plus 500 nM of Ag). We found an antagonistic effect in the low-chloride medium and an additive/synergistic effect in the high-chloride medium. We found that Cu dominates over Ag toxicity and bioavailability, indicating a competitive inhibition when both metals are present as free metal ions in the exposure media, which supports our hypothesis. Our study also suggests different mechanisms of uptake of free metal ions and metal complexes. The study adds valuable information to our understanding of the role of metal speciation on metal mixture toxicity and bioavailability. Environ Toxicol Chem 2024;43:105-114. © 2023 SETAC.

Environmental enrichment improves growth and fillet quality in rainbow trout.

BACKGROUND: Some environmental enrichment methods, such as occupational enrichment (OE), can improve fish growth, but little is known about its effects on fillet quality. In this study, we evaluated the effects of OE using underwater currents on different aspects of fillet quality and muscle metabolism in rainbow trout (Oncorhynchus mykiss), before and after a handling procedure (fasting). The trout were placed in groups of 30 in separate tanks in three treatments for 30 days: no artificial currents (CON), randomly fired underwater currents (RFC), and continuous underwater currents (CUC). Additionally, half of the individuals in each treatment were fasted (5 days, 45.2 °C days). RESULTS: Slaughter weight, condition factor, and relative growth were lower in CON fish, indicating a positive effect of OE on growth. Rigor mortis, muscle pH, and muscle glycogen levels were similar among treatments, indicating no effect of OE on classical measures of fillet quality. However, significant differences were found regarding fillet colour and muscle enzymes. The fillets of RFC fish were more salmon-pink in colour, which is favoured by consumers. Also, activity levels of pyruvate kinase and glycogen phosphorylase in muscle were significantly higher in CUC fish, probably due to increased energy demands, as pumps were on continually in that treatment. CONCLUSION: Overall, RFC fish seemed to have received enough stimulation to improve growth while not being excessive in terms of exhausting the animals (avoiding negative effects on muscle metabolism), whereas OE may have provided a hormetic effect, allowing fish to better adjust to fasting. © 2023 Society of Chemical Industry.

Temperature acclimation improves high temperature tolerance of rainbow trout (Oncorhynchus mykiss) by improving mitochondrial quality and inhibiting apoptosis in liver.

Global warming is threatening the survival and growth of cold-water fish, and the methods to improve the high-temperature adaptability of cold-water fish need to be explored urgently. This study aims to explore the mechanism of improving high temperature tolerance of rainbow trout by temperature acclimation (TA). Rainbow trout were acclimated by two modes at 22 °C, including fluctuating TA (FA) and constant TA (CA), and thereafter subjected to heat stress (HS) at 25 °C. Results showed that TA markedly increased the critical temperature maximum (CTmax) of rainbow trout. Secondly, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and malondialdehyde (MDA) in liver of CA + HS group significantly decreased compared to those in HS group without TA, indicating the reduction of liver injury by CA. Moreover, HS significantly induced ROS production and reduced mitochondrial membrane potential (MMP) in rainbow trout liver, but TA reduced the levels of ROS and increased the MMP in liver of rainbow trout after HS, indicating the reduced oxidative stress and mitochondrial damage. Furthermore, TA up-regulated the expression of genes related to mitochondrial autophagy, fusion, fission and biogenesis, as well as the expression of marker proteins of autophagy (LC3II) and mitophagy (Parkin) in the liver, so as to maintain mitochondrial homeostasis. Moreover, TA also inhibited the occurrence of apoptosis (decrease in bax/bcl-2), which may be owing to the reduced ROS and mitochondrial damage by TA. Interestingly, CA significantly up-regulated the genes expression of methyltransferase in the liver, which may inhibit the genes or transcription factors related to oxidative stress and apoptosis by DNA methylation. In conclusion, TA increased the upper limit of heat tolerance of rainbow trout by improving mitochondrial quality and inhibiting apoptosis in liver. This study will provide an effective solution to the risk of high temperature in cold-water fish culture.

Comparative subchronic toxicity of copper and a tertiary copper mixture to early life stage rainbow trout (Oncorhynchus mykiss): impacts on growth, development, and histopathology.

This research aimed to characterize and compare the subchronic impacts of Cu to a Cu, Cd, and Zn mixture in early life stages of rainbow trout (Oncorhynchus mykiss) by examining uptake, survival, growth, development, and histopathology parameters. To accomplish this, rainbow trout were exposed for 31 days from eyed embryos to the swim-up fry life stage to waterborne Cu (31, 47, 70, and 104 µg/L) individually or as mixture containing Cd (4.1, 6.2, 9.3, and 14 µg/L) and Zn (385, 578, 867, and 1300 µg/L). Exposures elicited pronounced effects on survival when Cu was administered as a mixture (LC25 = 32.9 µg/L Cu) versus individually (LC25 = 46.3 µg/L Cu). Mixtures of Cu, Cd, and Zn also elicited more pronounced sublethal toxicity relative to equivalent Cu treatments with respect to reduced yolk sac resorption and increased incidence and/or severity of gill, liver, and kidney lesions. Our findings of reduced body weight (EC10, Cu = 55.0 µg/L Cu; EC10, Cu+Cd+Zn = 58.9 µg/L Cu), yolk sac resorption (LOECCu = 70 µg/L Cu; LOECCu+Cd+Zn = 70 µg/L Cu), coelomic fat (LOECCu = 47 µg/L Cu; LOECCu+Cd+Zn = 70 µg/L Cu), and increased hepatocellular cytoplasmic vacuolation (LOECCu = 70 µg/L Cu; LOECCu+Cd+Zn = 47 µg/L Cu) collectively indicate a complicated metabolic interference by metals in exposed fish. These lethal and sublethal effects observed in the laboratory could translate to reduced survival and fitness of wild salmonid populations inhabiting waterbodies receiving wastewater or runoff containing multiple metals at elevated concentrations.