Comparison of three fluorescent CYP3A substrates in two vertebrate models: pig and Atlantic salmon.

We investigated in vitro inhibitory effects of ketoconazole (KTZ) on cytochrome P450 activity in microsomes from pigs and Atlantic salmon. The following enzymatic reactions were studied: 7-benzyloxyresorufin and 7-ethoxyresorufin O-dealkylation (BROD and EROD, respectively), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD) and 7-benzyloxyquinoline O-debenzylation (BQOD). KTZ was a potent non-competitive inhibitor of BROD and BQOD in the microsomes from pigs, whereas in the microsomes from Atlantic salmon, these reactions were competitively inhibited by KTZ. BFCOD activity was inhibited by KTZ in a non-competitive manner in both species. KTZ non-competitively inhibited EROD in Atlantic salmon, but not in porcine microsomes. The activity of BROD and BQOD was higher in male than that in female pigs, but the activity of BFCOD showed no sex-related differences.

In vitro and in vivo association of porcine hepatic cytochrome P450 3A and 2C activities with testicular steroids.

The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17ß-estradiol, 17α-estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism-based inhibitions were examined. 7-benzyloxyresorufin (BR) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7-benzyloxyresorufin O-dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism-based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17ß-oestradiol. Mechanism-based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α-oestradiol through the mechanism-based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.

Verapamil does not modify catalytic activity of CYP450 in rainbow trout after long-term exposure.

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 µg l(-1)) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Influence of dietary sesamin, a bioactive compound on fatty acids and expression of some lipid regulating genes in Baltic Atlantic salmon (Salmo salar L.) juveniles.

The effects of inclusion of sesamin / episesamin in Baltic Atlantic salmon (Salmo salar L.) diets based on vegetable oils were studied. The study was designed as a dose response study with two control diets, one diet based on fish oil (FO) and one diet based on a mixture of linseed and sunflower oil (6:4 by vol.) (MO). As experimental diets three different levels of inclusion of sesamin / episesamin (hereafter named sesamin) to the MO based diet and one diet based on sesame oil and linseed oil (SesO) (1:1 by vol.) were used. The dietary oils were mirrored in the fatty acid profile of the white muscle. Sesamin significantly decreased the levels of 18:3n-3 in the white muscle phospholipid (PL) fraction of all groups fed sesamin, no significant differences were found in the triacylglycerol fraction (TAG). Slightly increased levels of docosahexaenoic acid (22:6n-3, DHA) in PL and TAG were found in some of the sesamin fed groups. Sesamin significantly affected the expression of peroxisome proliferator-activated receptor alpha, scavenger receptor type B and hormone sensitive lipase, in agreement with previous studies on rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar L.) hepatocytes published by our group. No significant effects on toxicological response measured as ethoxyresorufin O-deethylase activity was found. The total cytochrome P450 enzymes were significantly higher in MO 0.29 and SesO group. The amount of alpha- and gamma-tocopherols in liver and the amount of gamma-tocopherol in white muscle were significantly lower in fish fed the FO diet compared to the MO diet, but no difference after inclusion of sesamin was found in this study. Increased inclusion of sesamin increased the levels of sesamin and episesamin in the liver, but did not affect the amounts in white muscle.

Post-release growth and dispersal of pond and hatchery-reared European grayling Thymallus thymallus compared with their wild conspecifics in a small stream.

The growth, and dispersal of stocked European grayling Thymallus thymallus, reared in a hatchery (fed dry food pellets) or in a pond (fed natural food), compared with their wild conspecifics was assessed from the recapture of individually tagged fish 168 days after their release into the Blanice River, Czech Republic. Recapture rates and site fidelity were higher for wild T. thymallus than for artificially reared fish. Specific growth rate and upstream or downstream dispersal did not significantly differ between any of the groups of fish. An influence of rearing conditions (pond v. hatchery) on the overall performance of stocked fish was not demonstrated. Initially, lower condition factors of reared T. thymallus were equal to wild fish after recapture, suggesting adaptation of artificially reared fish that remained in the sections studied.

Hepatic ethoxy-, methoxy- and pentoxyresorufin O-dealkylase activities in Landrace and Duroc pigs stimulated with HCG.

The effect of human chorionic gonadotropin (hCG) stimulation on the activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD) and pentoxyresorufin O-depentylase (PROD) was studied in intact male pigs of purebred Landrace and Duroc breeds. Pigs were divided into four groups: two control groups of each breed, without hCG stimulation (n = 20 for each breed), and two experimental groups (n = 18 for each breed), with hCG stimulation (Pregnyl(®); N.V. Organon, Oss, The Netherlands, 30 IU/kg live weight). Pigs were slaughtered 3 days after hCG stimulation and enzyme activities were measured in hepatic microsomes using two approaches. First, only one substrate concentration was used for the analysis of each enzyme activity. We found that EROD activity was suppressed by hCG-stimulation in Landrace (p = 0.004), but not Duroc pigs (p > 0.05). Generally, EROD activity was higher in Duroc pigs compared with Landrace (p = 0.017). Methoxyresorufin O-demethylase and PROD activities did not differ between groups (p > 0.05). To further characterize EROD, MROD and PROD, enzyme kinetic studies were performed. V(max) values for EROD and MROD in both breeds were lower after hCG stimulation (p < 0.001 for Landrace and p < 0.05 for Duroc). Additionally, V(max) values for EROD significantly differed between Landrace and Duroc pigs being higher in Duroc pigs (p < 0.05). We concluded that both hCG stimulation and breed differences may be important in the regulation of EROD and MROD activities. This study provides the first data on the effect of hCG stimulation and thus high testicular steroids, on EROD, MROD and PROD activities. Further studies are needed to investigate individual CYP450 enzymes and their regulation in porcine tissues.

Effects of pollution on chub in the River Elbe, Czech Republic.

The Elbe River is one of the most polluted aquatic ecosystems in the Czech Republic. The effect of three major chemical plants located on the Elbe River (at Pardubice, Neratovice, and Usti nad Labem) on fish was studied in 2004. Health status, chemical concentrations (Hg, PCB, DDT, HCH, HCB, OCS, 4-tert-nonylphenols, 4-tert-octylphenol) in muscle, and biomarkers (hepatic ethoxyresorufin-O-deethylase (EROD), plasma vitellogenin, and plasma 11-ketotestosterone) were assessed in male chub (Leuciscus cephalus L.). Differences between localities upstream (US) and downstream (DS) from the monitored source of pollution were identified. Fish from DS sampling sites showed significantly higher levels of contaminants than fish from US sampling sites. Generally, the concentrations of pollutants in fish from the Elbe sites were significantly higher compared to the reference site. Reduced gonad size, decreased plasma levels of 11-ketotestosterone, EROD and vitellogenin induction, and histopathologies of male gonads indicated harmful effects of aquatic pollution in fish from the Czech portion of the Elbe River.

Modulation of porcine cytochrome P450 enzyme activities by surgical castration and immunocastration.

The present study aimed to evaluate some cytochrome P450 metabolic enzyme activities in hepatic microsomes prepared from entire male pigs (uncastrated pigs), surgically castrated pigs and pigs immunized against gonadotropin-releasing hormone (immunocastrated pigs). The activities of the following enzymes were measured: ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2), methoxyresorufin O-deethylase (MROD, CYP1A2), pentoxyresorufin O-depentylase (PROD, CYP2B), coumarin hydroxylase (COH, CYP2A) and p-nitrophenol hydroxylase (PNPH, CYP2A/2E1). The total cytochrome P450 contents were not affected by either surgical or immunocastration. Hepatic microsomal activities for EROD, PROD, COH and PNPH were lower in entire male pigs compared with surgically castrated and immunocastrated pigs (P < 0.05). Surgically and immunocastrated male pigs were similar with respect to EROD, MROD, PROD and COH activities (P > 0.05), whereas surgically castrated pigs exhibited lower PNPH activity compared with immunocastrated pigs (P = 0.029). The effect of different concentrations of testicular steroids - testosterone, 17ß-estradiol, free estrone and androstenone - on enzyme activities was evaluated by in vitro microsomal study. Testosterone at the concentration of 8 pmol/ml inhibited EROD activities and estradiol-17ß at the concentration of 1.8 pmol/ml inhibited PROD activities in hepatic microsomes from surgically castrated pigs. The highest concentration of androstenone (7520 pmol/ml) inhibited COH activities, whereas a 42-fold lower concentration of androstenone (180 pmol/ml) stimulated COH activities in surgically castrated pigs. Both free estrone (3.5 pmol/ml) and androstenone (55 pmol/ml) inhibited EROD activities in microsomes from entire male pigs. Stimulation of COH activities by the highest dose of free estrone (18 pmol/ml) was recorded in microsomes from entire male pigs. However, these effects of steroids were not concentration-dependent and the maximum extent did not exceed ±15% variation compared with the controls. There was no inhibition of PNPH activities in the hepatic microsomes from either entire or castrated pigs. In conclusion, we showed that EROD, PROD, COH and PNPH activities were lower in entire male pigs compared with those in surgically and immunocastrated pigs. Direct inhibition by the testicular steroids - testosterone, 17ß-estradiol, free estrone and androstenone - was not the primary cause of the reduced enzyme activities.

Sesamin increases alpha-linolenic acid conversion to docosahexaenoic acid in atlantic salmon (Salmo salar L.) hepatocytes: role of altered gene expression.

In vitro cultivated Atlantic salmon (Salmo salar L.), hepatocytes were incubated without or with a mixture of sesamin and episesamin in order to test for possible effects on lipid metabolism. Sesamin/episesamin exposure (0.05 mM, final concentration) led to increased elongation and desaturation of (14)C 18:3n-3 to docosahexaenoic acid ((14)C 22:6n-3, DHA, P < 0.01) and down regulated gene expression of Delta6 and Delta5 desaturases compared to control treatment. Sesamin/episesamin further increased the hepatocytes capacity for fatty acid beta-oxidation of (14)C 18:3n-3 (P < 0.01) to the (14)C acid soluble products, acetate, malate and oxaloacetate, in agreement with an increased gene expression of carnitine palmitoyltransferase I. Also the gene expression of cluster of differentiation 36 was upregulated and the expression of scavenger receptor type B, peroxisome proliferator-activated receptors alpha and gamma were downregulated. The amount of triacylglycerols secreted by the cells tended to be lower in the sesamin/episesamin incubated hepatocytes than the control cells. This study shows that sesamin has favourable effects on lipid metabolism leading to increased level of DHA, which may be of interest for aquaculture use.

Sesamin supplementation increases white muscle docosahexaenoic acid (DHA) levels in rainbow trout (Oncorhynchus mykiss) fed high alpha-linolenic acid (ALA) containing vegetable oil: metabolic actions.

The effects of including an equi-mixture of sesamin and episesamin in fish diets based on vegetable oils of different fatty acid composition were examined. Sesamin/episesamin (hereafter named sesamin) was included at 0.58 g/100 g diet. The oil used in the feed was either a mixture of linseed and sunflower oils (6:4, by vol) or 100% linseed oil. Addition of sesamin increased the percentages of docosahexaenoic acid (DHA) in white muscle phospholipid and triacylglycerol fraction by up to 37% but the fatty acids in red muscle and liver were not affected. The expression of the peroxisome proliferator-activated receptor PPARalpha was significantly down regulated in the liver of the fish fed sesamin and mixed oil diet (P < 0.05). Sesamin and episesamin were detected in liver and muscle tissues of the fish that had been fed sesamin. Fish fed sesamin had elevated levels of total cytochrome P450 (CYP) enzymes and EROD activity in the liver, indicating an induction of CYP1A in this tissue. Our conclusion was that supplementation of fish feed with sesamin increased the proportions of DHA in the white muscle.

Chub (Leuciscus cephalus) as a bioindicator of contamination of the Vltava River by synthetic musk fragrances.

Synthetic musk fragrances, which are contained in almost all scented consumer products, enter aquatic environment mainly by way of wastewater paths. To monitor contamination of the Vltava River by these relatively persistent chemicals in the surroundings of Prague industrialized agglomeration, chub (Leuciscus cephalus) was employed as a bioindicator. Validated gas chromatography-mass spectrometry method was used for fish sample examination. Polycyclic musks, represented by 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-(gamma)-2-benzopyran (galaxolide) and 1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthalenyl)-ethanone (tonalide) were the most abundant representatives of this group; their levels in fillets were in the range of 1.7 to 105.9 microg/kg and 0.9 to 19.3 microg/kg wet weight, respectively. Nitro-musks, musk ketone, and musk xylene were also detected in most samples; nevertheless, their levels were lower,