Corrigendum: Oleaginous yeast Rhodotorula toruloides biomass effect on the metabolism of Arctic char (Salvelinus alpinus).

[This corrects the article DOI: 10.3389/fmolb.2022.931946.].

Oleaginous yeast Rhodotorula toruloides biomass effect on the metabolism of Arctic char (Salvelinus alpinus).

Sustainability issues arise when using fish oil and vegetable oils in fish feed production for aquaculture purposes. Microbial production of single cell oil is a potential alternative as a lipid ingredient in the production of fish feed. In this study, we replaced the vegetable oils with the oleaginous yeast R. toruloides biomass in the diet of Arctic char (S. alpinus) and investigated the effects on health and composition. Measurement of fish growth parameters showed a higher liver weight and hepatosomatic index in the experimental group of fish fed partly with yeast biomass compared to a control group fed a diet with vegetable oils. No significant differences in the lipid content of muscle and liver tissues were found. The fatty acid profiles in the muscle of both fish groups were similar while the experimental fish group had a higher amount of monounsaturated fatty acids in the liver. Histology of livers showed no significant difference in the number of lipid droplets. The size of hepatic lipid droplets seemed to be related to liver fat content. Quantification of metabolites in the liver revealed no differences between the fish groups while plasma metabolites involved in energy pathways such as alanine, 3-hydroxybutyrate, creatinine, serine, betaine, and choline were significantly higher in the experimental fish group.

Curcumin and quercetin modify warfarin-induced regulation of porcine CYP1A2 and CYP3A expression and activity in vitro.

The anticoagulant drug warfarin is used treat atrial fibrillation. Several cases of drug-drug and drug-food interactions have been reported for warfarin.The aim of this study was to investigate the interaction between simultaneous administration of warfarin with the two ubiquitous flavonoids quercetin and curcumin.Using porcine primary hepatocytes we demonstrated that warfarin treatment increased the mRNA and protein expression of CYP3A(29), while no changes in CYP1A2 were observed. Co-treatment with quercetin and/or curcumin decreased the warfarin-induced CYP3A protein expression. Moreover, when quercetin and curcumin were co-administrated to warfarin-exposed hepatocytes the protein expression of CYP1A2 was decreased. In hepatic microsomes, curcumin inhibited the activity of both CYP1A2 and CYP3A, while warfarin had no effect. Both quercetin and curcumin decreased the CYP1A2 and CYP3A activity when co-administrated with warfarin.The results clearly demonstrated that quercetin and curcumin can cause food-drug interactions with warfarin, and that the cocktail effect of exposure to more compounds than one can further enhance these interactions.

Metabolome adaptation and oxidative stress response of common carp (Cyprinus carpio) to altered water pollution levels.

Treated wastewater ponds (TWPs) serve as recipients and passive tertiary treatment mediators for recycled water. These nutrient-rich habitats are increasingly utilised in aquaculture, nevertheless multiple loads of various contaminants with adverse effects on aquatic fauna, including fish, have been recorded. In the present study, we investigated the effects of fish transfer in response to altered levels of pollution on liver metabolic profiles and tissue-specific oxidative stress biomarkers during short- and long-term exposure. In a field experiment, common carp (Cyprinus carpio) originating in severely polluted TWP were restocked after one year to a reference pond with a background pollutant concentration typical of the regional river. In contrast, fish that originated in the reference pond were restocked to TWP. Fish were sampled 0, 7, 14, 60, and 180 days after restocking and fish liver, kidney, intestine, and gill tissues were subjected to biomarker analysis. Pharmaceutically active compounds (PhACs) and metabolic profiles were determined in fish liver using liquid chromatography high-resolution mass spectrometry (LC-HRMS). Fish transferred from reference to polluted pond increased the antioxidant response and absorbed PhACs into metabolism within seven days. Fish liver metabolic profiles were shifted rapidly, but after 180 days to a lesser extent than profiles in fish already adapted in polluted water. Restocked fish from polluted to reference pond eliminated PhACs during the short phase within 14 days, and the highest antioxidant response accompanied the depuration process. Numerous elevated metabolic compounds persisted in such exposed fish for at least 60 days. The period of two weeks was suggested as sufficient for PhACs depuration, but more than two months after restocking is needed for fish to stabilise their metabolism. This study contributed to determining the safe handling with marketed fish commonly restocked to wastewaters and clarified that water pollution irreversibly altered fish metabolic profile.

Tissue-specific expression and activity of cytochrome P450 1A and 3A in rainbow trout (Oncorhynchus mykiss).

Piscine cytochrome P450 (CYP) enzymes play an important role in the metabolism of xenobiotics. Xenobiotics often act as inducers of CYP1A1 and CYP3A expression and activity in fish. We compared constitutive mRNA expression of CYP1A1, CYP3A27, and CYP3A45 and catalytic activity of CYP1A (7-ethoxyresorufin-O-deethylation, EROD) and CYP3A-like (benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation, BFCOD) enzymes in the following six rainbow trout tissues: liver, gill, heart, brain, intestine, and gonad. mRNA expression and activity were present in all investigated tissues. The CYP1A1 mRNA expression was higher in the liver, gill, heart, and brain compared to gonad and intestine. The intestine was the main site of CYP3A27 and CYP3A45 expression. The highest EROD and BFCOD activity was observed in liver tissue followed in descending order by heart, brain, gill, intestine, and gonad. Such differences might be related to the role of CYP physiological functions in the specific tissue. Rainbow trout exposure to 50 mg/kg of ß-naphthoflavone for 48 h resulted in a 7.5- and 5.9-fold increase in liver EROD and BFCOD activity, respectively. In vitro EROD activity inhibition with ellipticine showed tissue-specific inhibition, while ketoconazole decreased BFCOD activity by 50-98 % in all tissues. Further studies are needed to identify all CYP isoforms that are responsible for these activities and modes of regulation.

In Vitro Metabolic Transformation of Pharmaceuticals by Hepatic S9 Fractions from Common Carp (Cyprinus carpio).

Water from wastewater treatment plants contains concentrations of pharmaceutically active compounds as high as micrograms per liter, which can adversely affect fish health and behavior, and contaminate the food chain. Here, we tested the ability of the common carp hepatic S9 fraction to produce the main metabolites from citalopram, metoprolol, sertraline, and venlafaxine. Metabolism in fish S9 fractions was compared to that in sheep. The metabolism of citalopram was further studied in fish. Our results suggest a large difference in the rate of metabolites formation between fish and sheep. Fish hepatic S9 fractions do not show an ability to form metabolites from venlafaxine, which was also the case for sheep. Citalopram, metoprolol, and sertraline were metabolized by both fish and sheep S9. Citalopram showed concentration-dependent N-desmethylcitalopram formation with Vmax = 1781 pmol/min/mg and Km = 29.7 µM. The presence of ellipticine, a specific CYP1A inhibitor, in the incubations reduced the formation of N-desmethylcitalopram by 30-100% depending on the applied concentration. These findings suggest that CYP1A is the major enzyme contributing to the formation of N-desmethylcitalopram. In summary, the results from the present in vitro study suggest that common carp can form the major metabolites of citalopram, metoprolol, and sertraline.

In vitro investigations of the metabolism of Victoria pure blue BO dye to identify main metabolites for food control in fish.

Although banned, dyes, such as Victoria pure blue BO (VPBO), are illicitly used in aquaculture to treat or prevent infections due to their therapeutic activities. The present study examined the formation of phase I and phase II metabolites derived from VPBO using trout liver microsomes and S9 proteins. The well-known malachite green (MG) dye was also studied as a positive control and to compare its metabolism with that of VPBO. First, we optimised the incubation conditions for the detection of VPBO and MG metabolites by studying the formation of cytochrome P450 (CYP) substrates. Using the determined conditions (2 h at 20 °C), we incubated VPBO with trout microsomal and S9 fractions induced with ß-naphtoflavone, and analysed the supernatant in a LC-LTQ-Orbitrap-HRMS system. The in vitro assays led to the detection of 16 VPBO metabolites from Phase I reactions, arising in particular from reactions with CYP1A. No metabolites were detected from Phase II reactions. The main metabolite detected, deethyl-VPBO, was CID-fragmented to determine its chemical structure, and thus recommend a potential biomarker for the control of VPBO in farmed fish foodstuffs.

The possible involvement of oxidative stress in the oocyte ageing process in goldfish Carassius auratus (Linnaeus, 1758).

Decreasing egg quality following oocyte ageing is a major restricting factor for the breeding programs. The mechanisms behind this process has not yet been clarified. To examine the possible involvement of oxidative stress in the oocyte ageing process, the relative mRNA abundance of specific transcripts were determined in oocytes collected from 6 females and incubated in vitro for 18 hours post stripping at 20 °C in goldfish Carassius auratus. During the 18 hour-post-stripping ageing of the oocytes, relative mRNA levels of candidate transcripts involved in oxidative injury, mitochondrial function and stress response, cell cycles, apoptosis, reproduction and germ line speciation and developmental competence were measured by real-time PCR. None of the relative mRNA abundance of the examined genes were significantly altered through oocyte ageing. In addition, the amount of thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, did not change over time following stripping. The activity of the antioxidant enzymes also remained constant during oocyte ageing. The results of the current study indicated that oxidative stress unlikely plays a role as an initiator or promotor in the progress of oocyte ageing in goldfish.

Alteration of mRNA abundance, oxidation products and antioxidant enzyme activities during oocyte ageing in common carp Cyprinus carpio.

Oocyte ageing is the most important factor affecting egg quality of several fish species after ovulation. Oxidative stress has been proposed as the initiator of the oocyte ageing process in other vertebrates. To identify the role of oxidative stress and apoptosis on the progress of oocyte ageing in the common carp Cyprinus carpio, changes in the relative mRNA abundance of selected transcripts were examined. The possible alteration in the oxidation status of the oocytes during ageing was also studied. In addition, the activity of antioxidant enzymes during oocyte ageing was evaluated. Oocytes from 6 females were incubated in vivo for 14 hours post-ovulation (HPO) and in vitro for 10 hours post-stripping (HPS) at 20°C before fertilization. Hatching rates were over 65% up to 4-6 HPO, finally dropping to 1.3% at 12-14 HPO.Hatching rates were over 65% up to 4-6 HPO, finally dropping to 1.3% at 12-14 HPO. Hatching rates were more than 70% for the eggs stored in vitro up to 6 HPS and then decreased to 21.3% at 10 HPS. The results demonstrated no significant changes in the relative mRNA levels of oxidative stress-related genes or genes involved in the cell cycle during the progress of oocyte ageing in common carp. Additionally, the amount of TBARS and carbonyls did not change as time elapsed following ovulation. The apoptosis-related genes however, were significantly altered following the prolonged time interval between ovulation and fertilization. The lack of response of both activities of antioxidant enzymes and oxidation products during oocyte ageing strengthens the conclusion that oxidative stress is unlikely to be a main factor determining the progress of oocyte ageing in common carp. However, an increase in the mRNA abundance of apoptosis-related genes demonstrates that apoptotic pathway might be involved in the progress of oocyte ageing.

End-product inhibition of skatole-metabolising enzymes CYP1A, CYP2A19 and CYP2E1 in porcine and piscine hepatic microsomes.

The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.

Effects of Multi-Component Mixtures from Sewage Treatment Plant Effluent on Common Carp (Cyprinus carpio) under Fully Realistic Condition.

This study characterized changes in biomarker responses in common carp (Cyprinus carpio) upon exposure to effluent water discharged from a sewage treatment plant (STP) under real conditions. Fish were exposed to contamination in Cezarka pond, which receives all of its water input from the STP in the town of Vodnany, Czech Republic. Five sampling events were performed at day 0, 30, 90, 180, and 360 starting in April 2015. In total, 62 pharmaceutical and personal care products (PPCPs) were detected in the polar organic chemical integrative sampler. Compared to a control pond, the total concentration of PPCPs was 45, 16, 7, and 7 times higher in Cezarka pond at day 30, 90, 180, and 360, respectively. The result of oxidative stress and antioxidant enzyme biomarkers indicated alterations in the liver and intestine tissues of fish from Cezarka pond at day 30 and 360, respectively. High plasma vitellogenin levels were observed in both exposed females (180 and 360 days) and males (360 days) compared with their respective controls. However, only exposed female fish had higher vitellogenin mRNA expression than the control fish in these periods. Exposed female fish showed irregular structure of the ovary with scattered oocytes, which further developed to a vitellogenic stage at day 360. Low white blood cell levels were indicated in all exposed fish. Despite numerous alterations in exposed fish, favorable ecological conditions including high availability of food resulted in a better overall condition of the exposed fish after 1 year of exposure compared to the controls.

Correction to: Effects of multi-component mixtures from sewage treatment plant effluent on common carp (Cyprinus carpio) under fully realistic condition.

The original version of this Article unfortunately contained an error. The authors' given and family names were transposed erroneously. It has been corrected now in this Erratum.

Porcine cytochrome 2A19 and 2E1.

Cytochrome P450 (CYP) is a major group of enzymes, which conduct Phase I metabolism. Among commonly used animal models, the pig has been suggested as the most suitable model for investigating drug metabolism in human beings. Moreover, porcine CYP2A19 and CYP2E1 are responsible for the biotransformation of both endogenous and exogenous compounds such as 3-methylindole (skatole), sex hormones and food compounds. However, little is known about the regulation of porcine CYP2A19 and CYP2E1. In this MiniReview, we summarise the current knowledge about the regulation of porcine CYP2A19 and CYP2E1 by environmental, biological and dietary factors. Finally, we reflect on the need for further research, to clarify the interaction between active feed components and the porcine CYP system.

Complex effects of pollution on fish in major rivers in the Czech Republic.

Monitoring the contamination level in aquatic environments and assessing the impact on aquatic life occurs throughout the world. In the present study, an approach based on a combination of biomarkers and the distribution of various industrial and municipal pollutants was used to investigate the effect of aquatic environmental contamination on fish. Monitoring was performed in ten rivers in the Czech Republic (Berounka, Dyje, Elbe, Luznice, Odra, Ohre, Otava, Sázava, Svratka, and Vltava rivers, with one or two locations in each river) at the same sites that were regularly monitored within the Czech National Monitoring Program in 2007-2011. Health status, hepatic ethoxyresorufin-O-deethylase (EROD) activity, total cytochrome P450 content, and the plasma vitellogenin concentration were assessed in wild chub (Squalius cephalus) males caught at the monitored sites. The contamination level was the highest in the Svratka River downstream of Brno. Among all measured persistent organic pollutants (POPs), polychlorinated biphenyls and dichlorodiphenyltrichloroethane and its metabolites were the major contributors of POPs in fish muscle. Elbe, Odra, and Svratka rivers were identified as the most polluted. Fish from these locations showed reduced gonad size, increased vitellogenin concentration in male plasma, EROD, and total cytochrome P450 content. These biomarkers can be used for future environmental monitoring assessments. Overall, this study improves our understanding of the relationship between human activities and pollutant loads and further contributes to the decision to support local watershed managers to protect water quality in this region.

CYP1A1 activity in rainbow trout is inhibited by the environmental pollutant p-cresol.

Naturally- and anthropogenically-produced cresols could pose serious risks to fish health. In this study, three piscine CYP isoforms were investigated for their abilities to interact with p-cresol. Therefore, the activity of 7-ethoxyresorufin-O-deethylase (EROD), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD), and p-nitrophenol hydroxylase (PNPH) were evaluated in the hepatic microsomes of juvenile rainbow trout. Results showed that EROD activity was inhibited in a competitive manner, BFCOD activity was inhibited in presence the highest tested p-cresol concentration and PNPH activity was not affected. These results indicate that p-cresol might affect the ability of fish to metabolize numerous aromatic hydrocarbons and dioxin compounds, which are present in the aquatic environment.

Biomarker response, health indicators, and intestinal microbiome composition in wild brown trout (Salmo trutta m. fario L.) exposed to a sewage treatment plant effluent-dominated stream.

Concerns about the effect of sewage treatment plant (STP) effluent on the health of freshwater ecosystems have increased. In this study, a unique approach was designed to show the effect of an STP effluent-dominated stream on native wild brown trout (Salmo trutta L.) exposed under fully natural conditions. Zivny stream is located in South Bohemia, Czech Republic. The downstream site of Zivny stream is an STP-affected site, which receives 25% of its water from Prachatice STP effluent. Upstream, however, is a minimally polluted water site and it is considered to be the control site. Native fish were collected from the upstream site, tagged, and distributed to both upstream and downstream sites. After 30, 90, and 180days, fish were recaptured from both sites to determine whether the downstream site of the Zivny stream is associated with the effects of environmental pollution. Several biomarkers indicating the oxidative stress and antioxidant enzyme activities, cytochrome P450 activity, xenoestrogenic effects, bacterial composition, and lipid composition were investigated. Additionally, polar chemical contaminants (pharmaceuticals and personal care products (PPCPs)) were quantified using polar organic chemical integrative samplers (POCIS). Fifty-three PPCPs were detected in the downstream site; 36 of those were constantly present during the 180-day investigation period. Elevated hepatic 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylase (BFCOD) (after 90days) and blood plasma vitellogenin concentrations in males were detected in fish downstream of the STP effluent during all sampling events. An increase in the fishes' total fat content was also observed, but with low levels of ω-3 fatty acid in muscle tissue. Two bacterial taxa related to activated sludge were found in the intestines of fish from downstream. Our results show that Prachatice STP is a major source of PPCPs in the Zivny stream, which has biological consequences on fish physiology.

The effects of sewage treatment plant effluents on hepatic and intestinal biomarkers in common carp (Cyprinus carpio).

Sewage treatment plants (STPs) are one of the major source of pharmaceuticals and personal care products in the aquatic environment. Generally, the effects of individual chemicals on fish are studied under laboratory conditions, which leads to results that are potentially not realistic regarding the effects of these chemicals under environmental conditions. Therefore, in this study, common carps were held in exposed pond that receive water from STP effluents for 360 days under natural conditions. Elimination of xenobiotics starts in the fish intestine, in which the microbial community strongly influences its function. Moreover, the fish intestine functions as crucial organ for absorbing lipids and fatty acids (FA), with consequent transport to the liver where their metabolism occurs. The liver is the primary organ performing xenobiotic metabolism in fish, and therefore, the presence of pollutants may interact with the metabolism of FA. The catalytic activity of CYP1A and CYP3A-like enzymes, their gene expression, FA composition and intestinal microbiome consortia were measured. The catalytic activity of enzymes and their gene and protein expression, were induced in hepatic and intestinal tissues of fish from the exposed pond. Also, fish from the exposed pond had different compositions of FA than those from the control pond: concentration of 18:1 n-9 and 18:2 n-6 were significantly elevated and the longer chain n-3 FA 20:5 n-3, 22:5 n-3 and 22:6 n-3 were significantly lowered. There were clear differences among microbiome consortia in fish intestines across control and exposed groups. Microbiome taxa measured in exposed fish were also associated with those found in STP activated sludge. This study reveals that treated STP water, which is assumed to be clean, affected measured biomarkers in common carp.

Effect of human pharmaceuticals common to aquatic environments on hepatic CYP1A and CYP3A-like activities in rainbow trout (Oncorhynchus mykiss): An in vitro study.

This study examined the ability of several human pharmaceuticals to modulate hepatic piscine CYP-mediated monooxygenase activities. Effects of six pharmaceuticals: diclofenac, sulfamethoxazole, tramadol, carbamazepine, venlafaxine and nefazodone, were investigated in vitro in rainbow trout hepatic microsomes. The reactions of 7-ethoxyresorufin-O-deethylase (EROD) and benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylase (BFCOD), were used as markers for hepatic CYP1A and CYP3A-like activities, respectively. Our results showed that EROD and BFCOD activities were both affected by nefazodone. Nefazodone inhibited EROD in a dose dependent manner and was found to be a potent non-competitive inhibitor of EROD with a Ki value of 6.6 µM. BFCOD activity was inhibited non-competitively in the presence of nefazadone with Ki value of 30.7 µM. BFCOD activity was slightly reduced only by the highest concentration of carbamazepine. Diclofenac, sulfamethoxazole, tramadol, and venlafaxine did not affect the activity of either EROD or BFCOD. We further exposed microsomal fraction to mixtures of six pharmaceuticals to investigate potential inhibition. The results showed that EROD and BFCOD activity was inhibited on 94% and 80%, respectively at higher tested concentration. To our knowledge, this is the first report to demonstrate an inhibitory effect of nefazodone on hepatic CYP1A and CYP3A-like proteins in rainbow trout.

In vitro effects of diosmin, naringenin, quercetin and indole-3-carbinol on fish hepatic CYP1A1 in the presence of clotrimazole and dexamethasone.

Phytochemicals are widely present in fruits, vegetables and other plants and have great health benefits owing to their antioxidant properties. They are naturally found in the aquatic environment as well as discharged from sewage treatment plants after their large consumption. Little is known about their impact on fish; particularly in light of their interactions with pharmaceuticals. Therefore, this study was designed to determine the effects of diosmin, naringenin, quercetin and idole-3-carbinol on CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity on rainbow trout hepatic microsomes in the presence of two pharmaceuticals: clotrimazole and dexamethasone. The interactions between the phytochemicals and pharmaceuticals used in this study were determined using a combination index. Hepatic microsomes were exposed to two concentrations (1-or 50 µM) of phytochemicals and pharmaceuticals separately and in combinations. Singly, clotrimazole inhibited EROD activity 40% and 90% of control, while dexamethasone did not. Naringenin and diosmin inhibited EROD activity alone up to 90% and 55% respectively, but activities were further inhibited in the presence of either pharmaceutical. The preliminary study of combinations of clotrimazole with phytochemicals primarily showed synergistic effects. While EROD activity was not inhibited in the presence of quercetin or indole-3-carbinol, significant and synergistic inhibition was detected when either of these was combined with clotrimazole or dexamethasone.

Ecotoxicity of two organic UV-filters to the freshwater caddisfly Sericostoma vittatum.

Organic ultraviolet filters (UV-filters) used for protection against radiation in personal care products and other materials (e.g. textiles, plastic products) are considered emerging contaminants of aquatic ecosystem. Benzophenone-3 (BP3) and 3-(4-methylbenzylidene)camphor (4-MBC) are the most commonly used organic UV-filters and have been reported in freshwater environments due to contamination through discharges from wastewater treatment plants and swimming pools or by direct contamination from recreational activities. Our aim was to evaluate the ecotoxicological effects of these UV-filters using the freshwater caddisfly Sericostoma vittatum' biochemical biomarkers and energy processing related endpoints (feeding behaviour, energy reserves and cellular metabolism). In laboratory trials, both compounds induced feeding inhibition of S. vittatum at 3.55 mg/kg of BP3 and at concentrations ≥2.57 mg/kg of 4-MBC, decreased carbohydrates content at 3.55 and 6.95 mg/kg of BP3 and 4-MBC respectively, and increased total glutathione levels at concentrations ≥1.45 and 1.35 mg/kg of BP3 and 4-MBC respectively. No significant effects were observed on endpoints associated with oxidative stress, antioxidant defences, phase II biotransformation or neurotoxicity after exposure to the two UV-filters. Our results show that environmental relevant concentrations of BP3 and 4-MBC, can negatively impact freshwater insects and demonstrate the importance of monitoring the ecological effects of organic UV-filters using non-model invertebrate species.