The Interference of Pharmaceuticals with Endogenous and Xenobiotic

Jul 7, 2006 - strongly inhibited CYP2M-catalyzed activity (91% inhibition) and other CYP isoforms (CYP1A and CYP3A-like). Additionally, glucuronidatio...
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Environ. Sci. Technol. 2006, 40, 5154-5160

The Interference of Pharmaceuticals with Endogenous and Xenobiotic Metabolizing Enzymes in Carp Liver: An In-Vitro Study R EÄ M I T H I B A U T , * S A B I N E S C H N E L L , A N D CINTA PORTE* Environment Chemistry Department, IIQAB-CSIC. C/Jordi Girona, 18, 08034 Barcelona, Spain

The interactions of fibrate (clofibrate, fenofibrate, bezafibrate, gemfibrozil), antiinflammatory (ibuprofen, diclofenac, naproxen, ketoprofen), and anti-depressive (fluoxetine, fluvoxamine, paroxetine) drugs with CYP catalyzed pathways (CYP1A, CYP3A-, CYP2K-, and CYP2M-like) and Phase II activities (UDP-glucuronosyltransferases and sulfotransferases), involved in both xenobiotic and endogenous metabolism in fish, were investigated in-vitro by incubating carp liver subcellular fractions in the presence of the substrate and the selected drug. Antidepressive drugs were strong inhibitors of CYP1A (9294% inhibition), CYP3A-like (69-80% inhibition), and CYP2Klike (36-69% inhibition) catalyzed activities, while antiinflammatory drugs were potent CYP2M-like inhibitors (3274% inhibition). Among the lipid regulators, gemfibrozil strongly inhibited CYP2M-catalyzed activity (91% inhibition) and other CYP isoforms (CYP1A and CYP3A-like). Additionally, glucuronidation of naphthol and testosterone were targeted by antiinflammatory drugs, and to a lesser extent, by fibrate drugs (48-78% inhibition). No significant alteration on sulfotransferase activities was observed, apart from a minor inhibitory effect of clofibrate, gemfibrozil, and fluoxetine on the sulfation of estradiol. Overall, gemfibrozil, diclofenac, and the three anti-depressive drugs appear to be the pharmaceuticals with the highest potential to interfere with fish metabolic systems.

Introduction Pharmaceuticals are a class of emerging environmental contaminants that are extensively and increasingly used in human and veterinary medicine. Human pharmaceutical usage leads to the excretion of unchanged drugs and their metabolites into urban wastewaters, and this, together with the improper disposal of unused and expired drugs by users, leads to considerable concentrations of various pharmaceuticals in municipal sewages (1); the discharge from sewage treatment plants is one of the main sources of pharmaceuticals in the aquatic environment (2, 3). Pharmaceuticals generally occur at low concentrations in the aquatic environment (ng-µg/L), and as they are designed to exert a biological activity with the lowest toxicity for humans, they do not exert acute toxicity for the aquatic fauna (1-4). In fact, acute toxicity values are in the mg/L * Address correspondence to either author. Phone: +34 934006175 (R.T.). Fax +34 932045904 (R.T.). E-mail: [email protected] (R.T.); [email protected] (C.P.). 5154

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range for most of the pharmaceuticals detected in the environment (5). Nevertheless, aquatic organisms might be chronically exposed to these bioactive substances, and because of their continuous introduction in surface waters, exposure even to the environmental nonpersistent compounds may be significant. Pharmaceuticals are designed to target specific metabolic and molecular pathways in humans and animals, but could also have numerous effects on nontarget systems, and possibly cause side effects in the target organism. Basic mechanisms such as signal transduction, cell division, and key metabolizing enzymes such as different cytochrome P450 isoforms (CYP) are conserved in a large variety of organisms (6), and as a consequence, when introduced in the environment, pharmaceuticals might have identical or similar targets in humans, mammals, and aquatic vertebrates, and they may have similar adverse side effects, but certainly, interspecies differences in their mechanisms of action might be expected. In humans, approximately 20 CYP isoforms among the CYP1, CYP2, CYP3, and CYP4 subfamilies are involved in the metabolism of drugs. CYP1A2 and CYP3A4 are among the most important CYP isoforms in human drug metabolism (7), and CYP1A and CYP3A orthologs exist in fish with similar substrate specificity than in humans (8). CYP2 isoforms (2B6, 2C9, 2C19, 2D6, and 2E1) are induced by barbiturate-like drugs and they play a significant role in human drug metabolism (7). In fish, enzymes belonging to the CYP2 family (CYP2K- and CYP2M-like) are not orthologs of human CYP2 isoforms, and they do not respond to barbiturates (8). Nevertheless, polyclonal antibodies raised against purified rainbow trout CYP2M1 and CYP2K1 cross-reacted with rat CYP2B1 protein, suggesting the similarity of the trout CYP isoforms with mammalian members of the CYP2 family (8). However, the substrate specificity for the CYP2M- and CYP2Klike fish isoenzymes is close to that of human CYP4A11 and CYP2E1, since they are involved in ω- and (ω-1)-hydroxylation of fatty acids (8-10). Oxidations and conjugations catalyzed by cytochrome P450 monooxygenases and glyco- and sulfotransferases respectively, are among the most important metabolic processes involved in human drug metabolism. Similarly, those reactions play a key role in the metabolic fate of both endogenous and xenobiotic compounds in fish (11, 12). Thus, the present study was designed to investigate the potential interactions of selected pharmaceuticals with those fish enzymatic systems in order to elucidate the modes of action of those compounds on nontarget organisms and to tentatively predict possible in-vivo effects. The enzymatic systems selected for the study were several CYP catalyzed pathways (CYP1A, CYP3A-, CYP2K-, and CYP2M-like), together with Phase II activities (UDP-glucuronosyltransferases and sulfotransferases) involved in both xenobiotic and endogenous (steroid and fatty acid) metabolism in fish. Three classes of widely used human pharmaceuticals were selected for the study, namely lipid regulators from the fibrate group (clofibrate, fenofibrate, bezafibrate, gemfibrozil), analgesics/ antiinflammatory drugs (ibuprofen, diclofenac, naproxen, ketoprofen), and anti-depressives (fluoxetine, fluvoxamine, paroxetine).

Materials and Methods Chemicals. Testosterone (T), 17β-estradiol (E2), lauric acid (LA), R-naphthol, 7-ethoxyresorufin, 7-hydroxyresorufin, 7-hydroxy-4-(trifluoromethyl)-coumarin, UDPGA, and NADPH were obtained from Sigma (Steinheim, Germany). PAPS (adenosine 3′-phosphate 5′-phosphosulfate, tetralithium salt) 10.1021/es0607483 CCC: $33.50

 2006 American Chemical Society Published on Web 07/07/2006

was from Calbiochem (Darmstadt, Germany). 7-Benzyloxy4-trifluoromethyl-coumarin was purchased from Cypex (Dundee, Scotland, UK). Pharmaceuticals and positive control inhibitors were purchased from Sigma (Steinheim, Germany) except paroxetine and ketoconazole obtained from Tocris (Bristol, UK). [6, 7-3H]-Estradiol, [4-14C]-testosterone and [1-14C]-lauric acid were purchased from Amersham Biosciences (Buckinghamshire, England) and [1-14C]-R-naphthol was from American Radiolabeled Chemical Inc. (St. Louis, MO). The radio-chemical purity of labeled compounds was analyzed by Radio-HPLC and found to be >97%. Preparation of Liver Subcellular Fractions. Carp (Cyprinus carpio) were collected by direct current electrofishing from the Ebro River (Spain). Fish were sacrificed and the liver immediately dissected, frozen in liquid nitrogen, and stored at -80°C until preparation of subcellular fractions. Livers from two females and one male (length 48 ( 6 cm, weight 2008 ( 465 g) were flushed with ice cold 1.15% KCl, and homogenized in 1:5 w/v of cold 100 mM potassiumphosphate buffer pH 7.4, containing 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenantroline, and 0.1 mg/mL trypsine inhibitor. Microsomal and cytosolic fractions were prepared by differential ultracentrifugation as described in Thibaut and Porte (13). Proteins were measured according to Lowry et al. (14), using bovine serum albumin as standard. In-Vitro Incubations. The interaction of pharmaceuticals with different fish enzymatic systems was investigated invitro by incubating the corresponding subcellular fraction (microsomes or cytosol) in the presence of the substrate and the selected drug. Enzymatic analyses were carried out in duplicate on individual livers. Pharmaceuticals were tested at a concentration of 1 mM except for estradiol sulfotransferase, where a concentration of 0.1 mM was assayed, because of the very low concentration of substrate in the assay (50 nM). When a significant effect (>50% inhibition) was detected, lower concentrations of pharmaceuticals were assayed in order to calculate the corresponding IC50. Pharmaceuticals solutions were prepared in ethanol, and 10 µL of solution was added per assay tube. The solvent was removed by evaporation under a nitrogen stream before starting the incubation. The reactions were initiated by addition of the substrate in a small volume of solvent (final concentration in the assay was below 2%). 7-Ethoxyresorufin O-Deethylase (EROD) Activity. EROD activity was assayed by incubating 100 µg of liver microsomal proteins with 3.7 µM of 7-ethoxyresorufin and 225 µM of NADPH in 100 mM potassium phosphate buffer pH 7.4 (final volume 250 µL) at 30°C for 10 min. Reaction was stopped by the addition of 400 µL of acetonitrile and after centrifugation (2000-g/10 min) an aliquot of supernatant (200 µL) was transferred to a 96-multiwell plate. Fluorescence was read at the excitation/emission wavelengths pars of 537/583 nm using a Gemini XPS SpectraMax Plus microplate reader (Molecular Devices Corporation). Quantification was made by calibration with 7-hydroxyresorufin. A blank containing the tested pharmaceutical was performed in every assay to remove the possible fluorescence emitted by the drug. BFC-O-Debenzyloxylase (BFCOD) Activity. BFCOD activity was analyzed according to the procedure described by BD Gentest and optimized for carp liver microsomes. The assay consisted in incubating 25 µg of liver microsomal protein with 200 µM of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) and 22.5 µM of NADPH in 100 mM potassium phosphate buffer pH 7.4 (final volume 250 µL), at 30°C for 10 min. The reaction was stopped by addition of 75 µL of 0.5 M Tris-base/acetonitrile (20:80, v/v), and the fluorescence was directly read in a 200 µL aliquot transferred to a 96multiwell plate at the excitation/emission wavelength pairs of 409 and 530 nm, using a Gemini XPS SpectraMax Plus microplate reader (Molecular Devices Corporation). Quan-

tification was made using the calibration curve of the 7-hydroxy-4-(trifluoromethyl)-coumarin authentic standard and several blanks containing the tested substances were done to remove possible fluorescence emitted by the drug. Laurate Hydroxylase Activities. Laurate hydroxylase activities were analyzed according to a procedure adapted from Thibaut et al. (15). Liver microsomal proteins (100 µg) were incubated with 50 µM [1-14C]-lauric acid (LA) and 1 mM NADPH in 50 mM Tris-HCl buffer pH 7.4 (final volume 250 µL), at 30°C for 30 min. The reaction was stopped by adding 250 µL of methanol and after centrifugation (2000g/10 min) 250 µL of the supernatant was analyzed by reversephase-HPLC as described by Thibaut et al. (15). Chromatographic peaks were monitored by on-line radioactivity detection with a Radioflow detector LB 509 (Berthold Technologies, Bad Wildbad, Germany) using Flo-Scint 3 (Packard BioScience, Groningen, The Netherlands) as scintillation cocktail. Metabolites were identified on the basis of retention time and quantified by integrating the area under the radioactive peaks. UDP-Glucuronosyltransferase (UGT) Activities. UGT activities were measured using testosterone and R-naphthol as model substrates for endogenous and xenobiotic metabolism, respectively. Liver microsomal proteins (0.25 mg) were incubated with 100 µM [4-14C]testosterone or 300 µM [1-14C]R-naphthol, and 3 mM UDPGA in 50 mM Tris-HCl buffer pH 7.4, 10 mM MgCl2 (final volume 250 µL), at 30°C for 30 min. The reaction was stopped by adding 2 mL of ethyl acetate, and the extraction of nonmetabolized substrate was further completed by 2 × 2 mL of ethyl acetate. An aliquot (50 µL) of the remaining aqueous phase containing glucuronides was quantified by liquid scintillation counting. Sulfotransferase (SULT) Activities. SULT activities were measured using estradiol and R-naphthol as endogenous and xenobiotic model substrates. The sulfation of estradiol (E2-SULT activity) was assayed as previously reported by Thibaut and Porte (13) with some modifications in the procedure. Liver cytosolic proteins (25 µg) were incubated with 50 nM [6,7-3H]-E2 and 10 µM PAPS in 100 mM potassium phosphate buffer pH 7.4 (final volume 170 µL), at 30°C for 30 min. This substrate concentration was used in order to work specifically with the sulfotransferase isoform showing the greatest affinity for E2 (13) and leading to the formation of E2 3-sulfate (unpublished results). The reaction was stopped with 3 mL of dichloromethane after the addition of 200 µL of ice cold water. The extraction of nonmetabolized E2 was completed by 3 mL of dichloromethane, and an aliquot (200 µL) of the remaining aqueous phase containing E2 3-sulfate was quantified by liquid scintillation counting. Naphthol sulfotransferase activity (naphthol-SULT) was assayed by incubating liver cytosolic protein (100 µg) with 2.18 µM [1-14C]R-naphthol and 10 µM PAPS in 100 mM potassium phosphate buffer pH 7.4 (final volume 160 µL), at 30 °C for 20 min. The reaction was stopped with 320 µL of ethyl acetate. The extraction of nonmetabolized naphthol was completed by 2 × 320 µL of ethyl acetate and an aliquot (50 µL) of the remaining aqueous phase containing naphtholsulfate was quantified by liquid scintillation counting. Statistical Analyses. The results are reported as mean ( standard deviation. Statistical significance was assessed by using one-way analysis of variance (Dunnett’s test for differences from control). Level of significance was set at p e 0.05.

Results EROD Activity. The interaction of pharmaceuticals with CYP1A was assessed by measuring EROD activity. R-Naphthoflavone (ANF) was used as a positive control, and at a concentration of 10 µM, a 76% significant inhibition of EROD activity was observed. Among the tested pharmaceuticals VOL. 40, NO. 16, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Percentage of inhibition of different cytochrome P450 related activities after incubation of hepatic microsomal fractions of Cyprinus carpio with different pharmaceuticals at a concentration of 1 mM. Values expressed as percentage of control activity are means ( SD (n ) 3). * signifies a value significantly different from the control (p < 0.05). EROD specific activity: 27.8 ( 22.0 pmol/min/mg; BFCOD: 247.1 ( 64.0 pmol/min/mg; (ω-1)- and (ω-2)-laurate hydroxylase: 408.9 ( 67.8 pmol/min/mg; ω-laurate hydroxylase: 118.5 ( 47.0 pmol/ min/mg. White bars correspond to positive controls. ANF ) r-naphthoflavone. (1 mM), six of them were found to significantly inhibit EROD activity (Figure 1). The anti-depressive drugs (fluoxetine, paroxetine, and fluvoxamine) were the most potent inhibitors with a mean of 93% inhibition, followed by clofibrate (73% inhibition). Gemfibrozil and fenofibrate were less potent inhibitors than clofibrate (the percentage of inhibition was below 50%). For anti-depressive drugs, assays were repeated at lower concentrations (10, 50, and 100 µM) to determine the concentrations resulting in 50% inhibition (IC50). IC50 values of 11.4 ( 1.2, 22.5 ( 1.0, and 46.5 ( 1.3 µM were found for paroxetine, fluvoxamine, and fluoxetine, respectively. BFCOD Activity. The effects of pharmaceuticals on CYP3A-like catalytic activity were measured using BFCOD activity. Ethynyl-estradiol (EE2) was used as positive control, and at a concentration of 200 µM EE2, an inhibition of 93% of BFCOD activity was observed. Significant inhibitors of BFCOD activity were found in each pharmaceutical class (Figure 1). Again, the most potent inhibitors were the antidepressive drugs paroxetine, fluvoxamine and fluoxetine leading to 80, 78, and 69% inhibition, respectively. The antiinflammatory drug diclofenac, and the lipid regulators -gemfibrozil and clofibrate- reduced BFCOD activity by 67, 55, and 46%, respectively. IC50 values were calculated at concentrations of 2, 1, 0.5, and 0.1 mM of the pharmaceutical tested. Paroxetine and fluvoxamine had IC50 values at 262.5 ( 1.2 and 274.0 ( 1.3 µM, respectively. IC50 for gemfibrozil and fluoxetine were 616.3 ( 1.1 and 643.0 ( 1.0 µM. Diclofenac and clofibrate had IC50 values of 805.3 ( 1.5 and 1047.0 ( 1.0 µM. Laurate Hydroxylase Activities. The HPLC metabolic profile obtained for LA in control carp liver microsomes 5156

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showed the presence of three metabolites that could be identified as ω-, (ω-1)-, and (ω-2)-hydroxy-LA by comparison of their retention times with authentic standards. Microsomes formed 364.9 ( 59.5 pmol/mg/min of (ω-1)-hydroxy-LA as the major metabolite, and 118.5 ( 47.0 and 44.0 ( 15.0 pmol/ mg/min of ω- and (ω-2)-hydroxy-LA, respectively. In trout, the CYP2M1 form (LMC1) catalyzes the ω-hydroxylation of LA, while the CYP2K1 form (LMC2) catalyzes the formation of (ω-1)- and (ω-2)-hydroxylates (8). CYP2M- and CYP2Klike enzymes with these same region-specific hydroxylation properties exist in other fish species, (15); and ketoconazole -an inhibitor of both isoformss and EE2 san inhibitor of CYP2K-like activity- have been used as demonstrative tools. In the present study, we observed that CYP2M- and CYP2Klike enzymes also exist in carp liver and are responsible of ω- and (ω-1)- and (ω-2)-hydroxylations of LA, respectively. The effects of pharmaceuticals were tested on CYP2K- (sum of (ω-1)- and (ω-2)-hydroxy-LA) and CYP2M-like (ω-hydroxyLA) related activities (Figure 1). All the tested substances except clofibrate and naproxen significantly reduced CYP2Klike related activity, but only four compounds yielded to more than 50% inhibition, namely paroxetine (69%), fluoxetine (57%), diclofenac (52%), and gemfibrozil (50%). For CYP2Mlike related activity, fluoxetine and paroxetine were the only two substances that had no effect. The most efficient inhibitors were gemfibrozil (91% inhibition), diclofenac (74%), ibuprofen (64%), and fluvoxamine (59%). UGT Activities. Only 2 out of the 11 drugs tested, namely gemfibrozil and diclofenac, significantly inhibited testosterone-UGT activity (Figure 2). These two compounds yielded to 51 and 45% inhibition, respectively.

FIGURE 2. Percentage of inhibition of UDP-glucuronyltransferases (UGT) and sulfotransferases (SULT) after incubation of hepatic microsomal or cytosolic fractions of Cyprinus carpio with different pharmaceuticals at a concentration of 0.1 mM (estradiol-SULT) and 1.0 mM (UGT activities and naphthol-SULT). Values expressed as percentage of control activity are means ( SD (n ) 3). * signifies a value significantly different from the control (p < 0.05). Testosterone-UGT specific activity: 200.3 ( 75.2 pmol/min/mg; naphthol-UGT: 91.7 ( 61.6 pmol/min/mg; estradiol-SULT: 1.2 ( 0.1 pmol/min/mg; naphthol-SULT: 94.7 ( 41.9 pmol/min/mg. In contrast, the four antiinflammatory drugs tested and two lipid regulators significantly inhibited the glucuronidation of naphthol (Figure 2). Among the lipid regulators, gemfibrozil was the most potent inhibitor (78% inhibition), followed by bezafibrate (64.2%). Regarding the antiinflammatory drugs, ketoprofen was the most active (69% inhibition) followed by diclofenac, ibuprofen, and naproxen (54, 52, and 46% inhibition, respectively). IC50 values were calculated at concentrations of 2, 1, 0.5, 0.1, 0.05, and 0.01mM of pharmaceutical compound. The IC50 value obtained for gemfibrozil was of 62.7 ( 1.1 µM. Intermediate IC50 were obtained for ibuprofen, diclofenac, ketoprofen, and bezafibrate (408.7 ( 1.0, 531.5 ( 1.3, 645.3 ( 1.0, 807.0 ( 1.0 µM respectively). The IC50 obtained for naproxen was of 1628.0 ( 1.1 µM. SULT Activities. Pharmaceuticals were shown to be weak inhibitors of E2 sulfonation (Figure 2), even more, if we consider that they were tested at a concentration 2000-fold higher than the substrate. Three substances, fluoxetine, clofibrate, and gemfibrozil acted as cytosolic E2-SULT inhibitors, percentages of inhibition being 28, 23, and 21%, respectively. None of the tested pharmaceuticals had a significant inhibitory effect on SULT activity when naphthol was used as a substrate (Figure 2).

Discussion CYP-Catalyzed Activities. CYP1A is uniformly distributed in fish liver and is also expressed in other tissues such as kidney, olfactory organ, brain, pituitary, and endothelial cells, where it is believed to contribute to the biotransformation of

chemicals in the blood (8). CYP1A is involved in both, metabolic detoxification of toxins such as aflatoxin B1 and bioactivation of environmental procarcinogens, like PAHs and PCBs. Thus, any interaction of pharmaceuticals with CYP1A-catalyzed activities is likely to disrupt hepatic and extra-hepatic xenobiotic metabolism in fish (16). In this study, we have observed that fluoxetine, fluvoxamine, and paroxetine were the most potent inhibitors of EROD activity in carp liver microsomes. The efficiency of paroxetine as a CYP1A inhibitor (50% inhibition at 11.4 µM) was comparable to that of R-naphthoflavone (76% inhibition at 10 µM), which was used as a positive control. In fact, the three anti-depressive drugs are known inhibitors of the human CYP1A2 isoform (17,18), and fluoxetine was previously shown to act as an inhibitor of EROD activity in primary rainbow trout hepatocytes (19). Apart from anti-depressive drugs, EROD activity was significantly inhibited by clofibrate, the most potent among the tested fibrates, followed by gemfibrozil and fenofibrate, whereas bezafibrate did not show any effect. These in-vitro observations are in agreement with data on primary rainbow trout hepatocytes, where clofibrate and fenofibrate acted as inhibitors of EROD activity (19). The inhibitory potential of fibrates on EROD activity was previously reported in rat liver S9 fractions, where fenofibrate (10 µM), gemfibrozil (100 µM), and bezafibrate (1 mM) were potent in-vitro inhibitors of EROD activity, but no effect was observed for clofibrate (20). Although no significant effect of nonsteroidal antiinflammatory drugs on EROD activity was observed in the present study, ibuprofen and ketoprofen were reported to decrease VOL. 40, NO. 16, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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EROD activity in rat liver when co-administered with the inducer 3-methylcholanthrene, but the mechanism remained unknown (21). Also, diclofenac acted as an inhibitor of EROD activity in primary rainbow trout hepatocytes (19). In fact, it might be possible that a diclofenac metabolite produced in the cells, rather than diclofenac itself, is acting as an inhibitor (19). Regarding CYP3A catalyzed activities (BFCOD); the antidepressive drugs (paroxetine, fluvoxamine, and fluoxetine) were the most potent inhibitors. These results were expected since these compounds are well-known substrates and inhibitors of human CYP3A4 (7, 17, 18). Among the fibrates, gemfibrozil, and clofibrate were inhibitors of BFCOD activity, but not fenofibrate and bezafibrate. The phase I biotransformation of fibrates (clofibrate, bezafibrate, fenofibrate, gemfibrozil) occurs predominantly via a CYP3A4 isoenzyme in humans (22), and drug interactions have been described with other compounds that are also metabolized by CYP3A4 (23). In rats, fenofibrate, bezafibrate, gemfibrozil, and to a much lesser extent, clofibrate, inhibit the in-vitro N-demethylation of ethylmorphine, a CYP3A-mediated activity (20). Nevertheless, gemfibrozil is not an inhibitor of the 1′hydroxylation of midazolam, another CYP3A4-mediated activity in human liver microsomes (24, 25), suggesting that the inhibitory effects of pharmaceuticals often depends on the substrate used as a catalytic probe, and that significant inter-species differences in sensitivity are likely to exist. Human CYP3A4 is known to hydroxylate the antiinflammatory drug diclofenac (26), and diclofenac reduces significantly BFCOD activity in carp liver microsomal fractions, an indication that diclofenac is also a CYP3A substrate in fish. To our knowledge, this is one of the first studies reporting the effect of pharmaceuticals on CYP3A-like activity in fish. The antifungal agents ketoconazole, miconazole, and clotrimazole, and EE2 were shown to inhibit CYP3A catalyzed activities (progesterone 6β-hydroxylase and BFCOD activities) in different fish species (27-29). Ketoconazole acted as a noncompetitive inhibitor while EE2 acted as an uncompetitive inhibitor of BFCOD activity (27, 28). CYP3A-like plays an important role in the metabolism of both steroid hormones (progesterone and testosterone) and xenobiotic substances (N-demethylation of benzphetamine) in fish (8). Consequently, those pharmaceuticals interfering with CYP3A-like activities can potentially disrupt both steroid hormone and xenobiotic metabolism in fish. Regarding CYP2K-like, most of the studied pharmaceuticals (9 out of 11) had an inhibitory effect on the catalytic activity. Paroxetine, fluoxetine, gemfibrozil, and diclofenac were among the most potent inhibitors of CYP2K-like catalyzed activity, and gemfibrozil, ibuprofen, diclofenac, and fluvoxamine had a strong inhibitory effect on CYP2Mlike activity. Interestingly, clofibrate and naproxen, substances without effect on CYP2K-like, were inhibitors of CYP2M-like related activity, whereas fluoxetine and paroxetine that were the most potent inhibitors of CYP2K-like activity had no significant effect on CYP2M-like activity. The three anti-depressive drugs tested, gemfibrozil, and fenofibrate are well-known inhibitors of human CYP2 isoforms (2C9, 2C8, 2C19, and 2D6); diclofenac, ibuprofen, naproxen, and the anti-depressive drugs are also substrates of these CYP2 forms (7, 17, 18, 25). Since it has been suggested that trout CYP2M1 and CYP2K1 present similarities with mammalian members of the CYP2 family (8), our results are consistent with data on human isoforms. Surprisingly, lipid regulators, being peroxisome proliferating agents and, therefore, capable of interfering with fatty acid metabolism (30, 31), were not the most potent inhibitors of lauric acid hydroxylation. Thus, gemfibrozil, fenofibrate, and bezafibrate were able to inhibit both terminal and sub-terminal LA 5158

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hydroxylations while clofibrate only inhibited the terminal hydroxylation. Gemfibrozil was a strong inhibitor of LA ω-hydroxylation. Comparable results have been reported in rat, where gemfibrozil, fenofibrate, and bezafibrate were invitro inhibitors of CYP2E-mediated LA (ω-1)-hydroxylation, while only gemfibrozil was an inhibitor of CYP4A-mediated LA ω-hydroxylation, and clofibrate had no significant effect (20). In fact, in mammals, fibrates act as ligands of the peroxisome proliferator activated receptor R (PPARR), and they modulate the expression and activity of several CYP, including CYP2E and CYP4A isoforms involved in fatty acid hydroxylation (32). Moreover, some positively regulated PPARR target genes encode enzymes, among them CYP2C8, that are inhibited by PPARR activators (32). CYP2K- and CYP2M-like are involved in the metabolism of fatty acids, steroid hormones and xenobiotics such as aflatoxin B1 and nonylphenol (8, 15), and CYP2K1 is the major rainbow trout hepatic P450 isoform (8). As for CYP3A, the consequences of interactions of pharmaceuticals with CYP2Kand CYP2M-like related activities might be multiple, e.g., disruptions of the steroid hormone, fatty acid, and xenobiotic metabolism. Phase II Activities. Clark et al. (33) proposed the existence of a polymorphism for UGT in fish and it has been suggested that distinct isoforms are involved in the glucuronidation of testosterone and phenolic substrates (34). Hence, we investigated the effect of the pharmaceuticals on the glucuronidation of both, testosterone and naphthol, as model substrates. Diclofenac and gemfibrozil were the only compounds that had an inhibitory effect on the glucuronidation of testosterone, while the four antiinflammatory drugs (diclofenac, ketoprofen, ibuprofen, and naproxen) and two lipid regulators (gemfibrozil and bezafibrate) were inhibitors of naphthol glucuronidation. The anti-depressive drugs did not have any effect on UGT activity. To date, only few examples of drug-drug interactions at the level of glucuronidation have been reported. Diclofenac is a substrate of human UGT 1A9 and 2B7, and therefore, an inhibitor of the glucuronidation of other substrates catalyzed by these isoforms (35-37). Recently, diclofenac was shown to be an inhibitor of UGT 1A1-, 1A3, 1A6-, 1A7-, 1A8-, 1A10-, 2B15-, and 2B17, which catalyzed the glucuronidation of 4-methylumbelliferone and 1-naphthol (38). Accordingly, our results also suggest that diclofenac is a nonselective UGT inhibitor that might interfere with several fish UGT isoforms. Additionally, human UGTs 1A3, 1A9, 2B7 catalyze the glucuronidation of many nonsteroidal antiinflammatory drugs, including ketoprofen, ibuprofen, and naproxen(3942). However, only naproxen (500 µM) is shown to be a weak inhibitor of a UGT2B7 related activity (43). In the present study, naproxen, but also ibuprofen and ketoprofen, were potent inhibitors of naphthol glucuronidation suggesting a higher sensitivity of fish phenol-UGT than human’s isoforms to antiinflammatory drugs. Regarding gemfibrozil, the compound showing the strongest inhibitory effect on the glucuronidation of both testosterone and naphthol, it has been reported that glucuronidation is a major pathway in the metabolism of gemfibrozil in humans (25). Besides, in-vitro studies have indicated that gemfibrozil, but not fenofibrate, significantly interferes with the glucuronidation of cerivastatin and simvastatin by UGT1A1 and UGT1A3 (44, 45). Our data suggest that fish UGT enzymes are even more sensitive to interactions with antiinflammatory and lipid regulators than human UGTs. This finding is of great relevance, since glucuronidation is a key pathway in the metabolism and homeostasis of endogenous molecules in fish and play a key role in the detoxification and excretion of contaminants. Thus, antiinflammatory and lipid regulators drugs are likely to interfere with the glucuronidation of both endogenous and

xenobiotic compounds in carp, while anti-depressive drugs have no significant effect. Regarding sulfotransferase activities, apart from a minor inhibitory effect of clofibrate, gemfibrozil, and fluoxetine on E2-SULT, no other significant effects were observed. These findings are in agreement with a previous work where we observed a lower sensitivity of E2-SULT than E2-UGT or T-UGT when tested in-vitro in the presence of several environmental contaminants (13), and only 3 out of the 12 tested xenobiotics were potent inhibitors of E2-SULT, while seven compounds acted as UGT inhibitors. Moreover, sulfonation of endogenous and exogenous substances is not a dominant phase II metabolism pathway in fish, and sulfotransferase enzymes are probably less abundant and have less polymorphisms and substrate diversity in fish than in humans, which makes this pathway not so susceptible to xenobiotic interferences. In summary, the present study reveals that pharmaceuticals are able to inhibit the catalytic activity of different CYP and UGT isoforms in fish. Anti-depressive drugs were strong inhibitors of CYP1A, CYP3A-like, and CYP2K-like catalyzed activities, while antiinflammatory drugs were potent CYP2Mlike inhibitors. Among the lipid regulators, it is worth mentioning the strong inhibitory effect of gemfibrozil on CYP2M-catalyzed activity, and on the other CYP isoforms. Additionally, glucuronidation was targeted by antiinflammatory and to a lesser extend by fibrate drugs. Among the tested compounds, gemfibrozil, diclofenac, and the three anti-depressive drugs appear to be the pharmaceuticals with the highest potential to interfere with fish enzymatic systems. The observed interactions occurred at concentrations 2-16fold lower than the enzymatic substrate (see IC50 for BFCOD and naphthol-UGT), suggesting that significant alterations of those key enzymatic systems are likely to occur in-vivo. Overall, the obtained data suggest that CYP isoforms are potential and sensitive targets of pharmaceutical substances in fish, and useful endpoints when designing specific in-vivo studies.

Acknowledgments This study was supported by the Spanish Ministry of Science and Education under Project ref. CGL2005-02846. S.S. acknowledges a predoctoral fellowship from the Ministerio de Educacio´n y Ciencia. R.T. acknowledges an I3P contract from the Spanish Government.

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Received for review March 29, 2006. Revised manuscript received June 2, 2006. Accepted June 7, 2006. ES0607483