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.

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.

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.

Sub-lethal effects and bioconcentration of the human pharmaceutical clotrimazole in rainbow trout (Oncorhynchus mykiss).

The aim of this study was to characterize biomarker responses, haematological profiles, structural changes and uptake in juvenile rainbow trout exposed to clotrimazole (CLO) at three concentrations (0.01 - [lowest environmentally relevant concentration], 1.0 [highest environmentally relevant concentration] and 10 µg L(-1)) in a semi-static system over a period of 42 days. Antioxidant defence enzymes, which responded to CLO exposure, changed the oxidative stress status of cells, but no differences were observed in lipid peroxidation. Clotrimazole triggered a biphasic response of CYP3A-like activity in liver microsomes, which may indicate a detoxification process in the liver. Histopathological alterations were most pronounced in kidneys and testes in the group exposed to 10 µg L(-1). Structural changes in the kidney included tubulonephrosis and hyaline droplet degeneration in the tubular epithelial cells. The relative proportions of germ cells in testes were changed: The number of spermatozoa was reduced, and the spermatogonia and spermatocytes were increased. The highest CLO concentration was detected in fish liver (3710 ng per gram wet tissue) and kidney (4280 ng per gram wet tissue). Depuration half-life was estimated to be 72, 159, and 682 h in liver, muscle, and kidney, respectively. Taken together, these results provide valuable toxicological data on the effects of CLO on aquatic non-target organisms, which could be useful for further understanding of the potential risks in the real aquatic environment.

Phase I metabolism of 3-methylindole, an environmental pollutant, by hepatic microsomes from carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

We studied the in vitro metabolism of 3-methylindole (3MI) in hepatic microsomes from fish. Hepatic microsomes from juvenile and adult carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were included in the study. Incubation of 3MI with hepatic microsomes revealed the time-dependent formation of two major metabolites, 3-methyloxindole (3MOI) and indole-3-carbinol (I3C). The rate of 3MOI production was similar in both species at both ages. No differences in kinetic parameters were observed (p = 0.799 for Vmax, and p = 0.809 for Km). Production of I3C was detected only in the microsomes from rainbow trout. Km values were similar in juvenile and adult fish (p = 0.957); Vmax was higher in juvenile rainbow trout compared with adults (p = 0.044). In rainbow trout and carp, ellipticine reduced formation of 3MOI up to 53.2% and 81.9% and ketoconazole up to 65.8% and 91.3%, respectively. The formation of I3C was reduced by 53.7% and 51.5% in the presence of the inhibitors ellipticine and ketoconazole, respectively. These findings suggest that the CYP450 isoforms CYP1A and CYP3A are at least partly responsible for 3MI metabolism. In summary, 3MI is metabolised in fish liver to 3MOI and I3C by CYP450, and formation of these metabolites might be species-dependent.

Effects of acetone, acetonitrile, ethanol, methanol and DMSO on cytochrome P450 in rainbow trout (Oncorhynchus mykiss) hepatic microsomes.

In vitro impacts of five organic solvents on cytochrome P450 (CYP450) enzyme activity were investigated using hepatic microsomes of rainbow trout. The rates of several CYP450-mediated reactions were investigated at solvent concentrations ranging from 0.01% to 3%. The solvents greatly affected all tested reactions. In at least 0.8% ethanol, 2% methanol or acetone, 1% acetonitrile or 3% dimethyl sulfoxide (DMSO), 7-ethoxyresorufin-O-deethylase (EROD) activity decreased and at 3% acetonitrile or ethanol, it was undetected. At 3%, all tested solvents except methanol reduced 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylase (BFCOD) activity, but at low concentrations of ethanol (2% and lower) or DMSO (1% and lower), it was induced. This was not seen with the inclusion of a pre-incubation step. p-Nitrophenolhydroxylase (PNPH) activity was not affected at concentrations below 1% DMSO, and at 2% acetonitrile it was reduced, as it was above 1% methanol or 0.5% ethanol. Acetone did not affect PNPH activity with or without a pre-incubation step. In general, the degree of inhibition was similar with and without the pre-incubation step. We conclude that the concentration of organic solvent for solubilizing the substrate and inhibitor in in vitro microsomal studies should be minimized.

Does dexamethasone affect hepatic CYP450 system of fish? Semi-static in-vivo experiment on juvenile rainbow trout.

Effects of aquatic pollutants on fish are of increasing concern. Pharmaceutical-based contaminants are prioritized for further study in environmental risk assessment using several approaches. Dexamethasone (DEX) was one such contaminant recognised for its effect on fish health status. Thus, we carried out an in vivo experiment to identify potential effects of DEX on rainbow trout. Fish were exposed to 3, 30, 300 and 3000ngL(-1) DEX in a semi-static system over a period of 42d. The concentrations of DEX that fish were exposed to was confirmed by LC-LC-MS/MS. Using hepatic microsomes, we determined cytochrome P450 content, activities of ethoxyresorufin O-deethylase (EROD), p-nitrophenol hydroxylase (PNPH), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) and benzyloxyquinoline O-debenzylase (BQOD), as well as protein expression. Our results showed that fish do not change the catalytic activity of CYP450-mediated reactions after high DEX concentration exposure. These results disagree with mammalian studies, where DEX is a well-known inducer of CYP450. We showed a significant effect of DEX exposure on CYP450-mediated reactions (EROD, BCFOD, BQOD and PNPH) when expressed as amount of product formed per min per nmol total CYP450 at 3, 30 and 300ngL(-1) after 21d exposure. Moreover, BFCOD and BQ activities showed matching trends in all groups. Western blot analysis showed induction of CYP3A-like protein in the presence of the lowest environmentally relevant concentration of DEX. Based on these findings, continued investigation of the effect of DEX on fish using a battery of complementary biomarkers of exposure and effect is highly relevant.

Histological damage and inflammatory response elicited by Monobothrium wageneri (Cestoda) in the intestine of Tinca tinca (Cyprinidae).

BACKGROUND: Among the European cyprinids, tench, Tinca tinca (L.), and the pathological effects their cestodes may effect, have received very little or no attention. Most literature relating to Monobothrium wageneri Nybelin, 1922, a common intestinal cestode of tench, for example, has focused on aspects of its morphology rather than on aspects of the host-parasite interaction. RESULTS: Immunopathological and ultrastructural studies were conducted on the intestines of 28 tench, collected from Lake Piediluco, of which 16 specimens harboured tight clusters of numerous M. wageneri attached to the intestinal wall. The infection was associated with the degeneration of the mucosal layer and the formation of raised inflammatory swelling surrounding the worms. At the site of infection, the number of granulocytes in the intestine of T. tinca was significantly higher than the number determined 1 cm away from the site of infection or the number found in uninfected fish. Using transmission electron microscopy, mast cells and neutrophils were frequently observed in close proximity to, and inside, the intestinal capillaries; often these cells were in contact with the cestode tegument. At the host-parasite interface, no secretion from the parasite's tegument was observed. Intense degranulation of the mast cells was seen within the submucosa and lamina muscularis, most noticeably at sites close to the tegument of the scolex. In some instances, rodlet cells were encountered in the submucosa. In histological sections, hyperplasia of the mucous cells, notably those giving an alcian blue positive reaction, were evident in the intestinal tissues close to the swelling surrounding the worms. Enhanced mucus secretion was recorded in the intestines of infected tench. CONCLUSIONS: The pathological changes and the inflammatory cellular response induced by the caryophyllidean monozoic tapeworm M. wageneri within the intestinal tract of an Italian population of wild tench is reported for the first time.