The Interference of Nitro- and Polycyclic Musks with Endogenous and

Nov 20, 2009 - The Interference of Nitro- and Polycyclic Musks with Endogenous and Xenobiotic Metabolizing Enzymes in Carp: An In Vitro Study...
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Environ. Sci. Technol. 43, 9458–9464

The Interference of Nitro- and Polycyclic Musks with Endogenous and Xenobiotic Metabolizing Enzymes in Carp: An In Vitro Study SABINE SCHNELL, REBECA MARTIN-SKILTON, DENISE FERNANDES, AND CINTA PORTE* Environmental Chemistry Department, IDAEA-CSIC. C/ Jordi Girona, 18, 08034 Barcelona, Spain

Received July 16, 2009. Revised manuscript received November 5, 2009. Accepted November 6, 2009.

Synthetic musks are widely used as perfuming agents in products, such as cosmetics, detergents, and soaps. The increased detection of these substances in the aquatic environment and their high bioconcentration potential raises concerns about potential effects on aquatic species. This work aimed at assessing the interactions of the most widely used musks: nitromusks (musk xylene, musk ketone) and polycyclic musks (celestolide, galaxolide, and tonalide) with fish enzymatic systems involved in both xenobiotic and endogenous metabolism. Therefore, CYP catalyzed pathways were investigated in carp liver microsomes (CYP1A, CYP3A), ovarian microsomes (CYP19) and testicular mitochondria (CYP17 and CYP11β) using standard substrates. Phase II activities (UDPglucuronosyltransferases and sulfotransferases) were determined in carp liver microsomes and cytosol, respectively. Polycyclic musks (galaxolide and tonalide) were stronger inhibitors of CYP3A- (IC50: 68-74 µM), CYP17- (IC50: 213-225 µM), CYP11βand CYP19-catalyzed activities than nitromusks, while the latter showed higher ability to interfere with CYP1A (IC50: 35-37 µM). The sulfation of estradiol was also significantly inhibited by tonalide and galoxolide (IC50: 140-294 µM). Overall, polycyclic musks showed the highest potential to interfere with those activities involved in the synthesis and metabolism of steroids while nitromusks mainly interfered with xenobiotic metabolism (CYP1A-catalyzed reactions). The obtained data suggest that CYP isoforms are potentially sensitive targets of synthetic musk substances in fish.

Introduction Synthetic musks are man-made chemicals produced in large quantities and extensively used in the fragrance industry for the production of a vast array of scented consumer products, including detergents, cleaning agents, air fresheners, perfumes, aftershaves, and cosmetics (1). They were created as inexpensive substitutes of natural musks and comprise two major classes of compounds: nitromusks and polycyclic musks. Nitromusks, e.g., 1-tert-butyl-3,5-dimethyl-2,4,5trinitrobenzene (musk xylene, MX) and 1-tert-butyl-3,5dimethyl-2,6-dinitro-4-acetylbenzene (musk ketone, MK) are highly substituted benzenes with at least two of the substituents being nitro groups. In contrast, polycyclic musks, * Corresponding author phone: +34 934006175; fax: +34 932045904; e-mail: [email protected]. 9458

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e.g., 7-acetyl-1,1,3,4,4,6-hexamethyl-tetrahydronaphtalene (Tonalide), 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran (Galaxolide), and 4-acetyl-1,1dimethyl-6-tert-butylindane (Celestolide) are indan and tetralin derivatives highly substituted mainly by methyl groups (see Supporting Information (SI) Figure S1). Nitromusks have been used for many decades, but due to concerns about their toxicological effects, their production and use is now greatly reduced in Europe (2). Musk xylene and musk ketone are the only two nitromusks of commercial importance today; the industrial usage in Europe in year 2000 was of 102 tons. In contrast, the polycyclic musks galaxolide and tonalide are widely used (1785 tons in year 2000) and have in recent years become the most important commercial synthetic musks. MK, MX, tonalide, and galaxolide account for 95% of the European market for synthetic musks (2). Since the first report of detection of musk xylene and musk ketone in water and biota from Japan (3), the concentration and distribution of these compounds has been investigated in various environmental matrices and locations (4-6). The polycyclic musks galaxolide and tonalide have been detected in sewage effluents (0.6-4.2 µg/L) and sludge (2.0-4.8 ng/g w · w.) and in freshwater fish sampled around the facilities (0.03-3.6 µg/g w · w.) (4, 7), demonstrating that major sources of synthetic musks to the environment are sewage treatment plants. Nevertheless, musk fragrances have been detected in many other areas and environmental matrices (air, freshwater, seawater, and sediments) suggesting that these compounds are widespread contaminants in the environment (8, 9). Because of their highly lipophilic nature, MX (Log Kow ) 4.90), MK (Log Kow ) 4.30) as well as the polycyclic musks, galaxolide (Log Kow ) 5.90), tonalide (Log Kow ) 6.35) and celestolide (Log Kow ) 6.60) have been detected in a number of aquatic organisms, such as mussels, crustaceans, and fish at concentrations from 0.1 up to 3600 ng/g w · w (7, 10). Hitherto toxicological studies on synthetic musks indicate no severe health risks for invertebrates and fish since acute and chronic toxicity thresholds are much higher than environmentally measured levels (11). Nonetheless, longterm carcinogenic effects for compounds such as MK cannot be ruled out (12). Furthermore, antagonizing effects toward the estrogen receptor in zebrafish (Danio rerio) have been reported for tonalide and galaxolide (13), and both, nitromusks and polycyclic musks, were potent inhibitors of the activity of multidrug efflux transporters in marine mussels (14). In earlier studies, MX and MK were found to be effective inducers of toxifying liver enzymes in mice and rats (15, 16) and as a consequence, they could amplify the genotoxicity of a number of well-known pregenotoxicants (17, 18). However, despite the wide occurrence of synthetic musks in the aquatic environment and their bioaccumulation by aquatic fauna, the interaction of these compounds with the enzymatic system of aquatic organisms has not been thoroughly investigated. Endogenous enzymatic systems are involved in the metabolism of both xenobiotics and endogenous compounds (i.e., fatty acids, steroids) and any interference of synthetic musks with those enzymatic systems will have consequences in terms of xenobiotic metabolism and toxicity and/or may impair steroid synthesis and metabolism and alter hormonal balance within the organism. Among those enzymatic systems, CYP1A is a cytochrome P450 isoform involved in the detoxification and often bioactivation of common environmental pollutants. Other CYP isoforms, such as CYP3A, CYP17 (C17,20-lyase), CYP11β (11β-hydroxylase) and CYP19 (P450 aromatase) are involved 10.1021/es902128x

 2009 American Chemical Society

Published on Web 11/20/2009

in endogenous metabolism. CYP17 catalyzes the conversion of 17R-hydroxyprogesterone (17P4) to androstenedione (AD), a precursor of testosterone (T) in male gonads; both T and AD can be transformed into their respective 11-hydroxylated metabolites by CYP11β, and further metabolized to 11ketotestosterone (11-KT) which is considered to be the main androgen in male teleosts, being more effective than T in stimulating secondary sexual characteristics, influencing spermatogenesis and stimulating reproductive behavior (19). CYP19 is a key enzymatic pathway involved in estrogen synthesis. Phase II enzymes (UDP-glucuronosyltransferases and sulfotransferases) play a key role catalyzing the conjugation and potential excretion of both xenobiotics and endogenous compounds (e.g., steroids). The present study was designed to further investigate the interaction of synthetic musks with different fish cytochrome P450 catalyzed reactions and phase II activities, in order to elucidate the modes of action of these compounds on nontarget organisms and tentatively predict potential in vivo effects. Several CYP isoenzymes (CYP1A, CYP3A-like, CYP19, CYP17, CYP11β) together with UDP-glucuronosyltransferases and sulfotransferases were selected for the study. Enzymatic activities were determined in liver and gonads of carp (Cyprinus carpio) following preincubation of the corresponding subcellular fraction in the presence and absence of the selected musk compound. Widely used synthetic musks, namely nitromusks (musk xylene and musk ketone) and polycyclic musks (galaxolide, celestolide and tonalide) were selected for the study.

Materials and Methods Chemicals. Testosterone (T), 17R-hydroxyprogesterone (17P4), androst-4-ene-3,17-dione (AD), 17β-estradiol (E2), R-naphthol, 7-ethoxyresorufin, 7-hydroxyresorufin, 7-hydroxy-4-(trifluoromethyl)-coumarin, UDP-glucuronic acid, and NADPH were obtained from Sigma (Steinheim, Germany). PAPS (adenosine 3′-phosphate 5′-phosphosulphate, tetralithium salt) was from Calbiochem (Darmstadt, Germany). We purchased 7-benzyloxy-4-trifluoromethyl-coumarin from Cypex (Dundee, Scotland, UK). Musk ketone (purity >98%), musk xylene, and galaxolide were from SigmaAldrich (Steinheim, Germany); celestolide and tonalide (purity >98%) were from LGC Promochem Gmbh (Wesel, Germany), and Promochem Iberia (Barcelona, Spain), respectively. [6,7-3H]-Estradiol, [4-14C]-testosterone were purchased from Amersham Biosciences (Buckinghamshire, England), [1-14C]-R-naphthol was from American Radiolabeled Chemical Inc. (St. Louis, MO), and [3H]17R-hydroxyprogesterone and [1β-3H]-androst-4-ene-3,17-dione from Perkin-Elmer Life Sciences (Boston, MA). The radio-chemical purity of labeled compounds was analyzed by Radio-HPLC and found to be >97%. All solvents and reagents were of analytical grade from Merck (Darmstadt, Germany). Organisms. Carp (Cyprinus carpio) were collected by direct current electrofishing from the Segre River, a tributary of the Ebro River (Spain) in April 2003. Fish were collected from a relative unpolluted site, 20 km downstream the city of Le´rida (20). Immediately after collection (n ) 15; nine males and six females), animals were killed, total length (40.2 ( 6.5 cm) and weight recorded (1252 ( 526 g), and the liver and gonads immediately dissected, frozen in liquid nitrogen, and stored at -80 °C until preparation of subcellular fractions. A subsample of the central part of the gonad was fixed in 10% buffered formalin for histological examination (20). Females had ovaries with vitellogenic oocytes of various sizes and development. Only testes classified as early spermatogenic were used for the determination of CYP17 and CYP11β activities. Preparation of Liver and Gonad Subcellular Fractions. Gonads and livers were flushed with ice cold 1.15% KCl to wash out the blood and homogenized in 1:5 w/v of cold 100

mM potassium-phosphate buffer pH 7.4, containing 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenantroline, and 0.1 mg/mL of trypsine inhibitor. Ovarian microsomal fractions and hepatic microsomal and cytosolic fractions were prepared by differential ultracentrifugation as described in Lavado et al. (20). Mitochondrial fractions were isolated from testes as indicated in Fernandes et al. (21). Proteins were measured according to Bradford (22), using bovine serum albumin as standard. In Vitro Incubations. The interaction of synthetic musks with different fish enzymatic systems was investigated in vitro by preincubating the corresponding subcellular fraction (microsomes, cytosol, or mitochondria) in the presence of the selected musk for 10 min prior to the addition of the reaction substrate as indicated below. For assays with a short incubation time (EROD and BFCOD), preincubation time was of 20 min instead of 10 min (23). Synthetic musk solutions were prepared in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the assay was always below 1%. Control reactions consisted of incubation of the corresponding subcellular fraction with the carrier alone; the final concentrations of DMSO used were shown not to affect enzyme activities. Enzymatic analyses were carried out in duplicate on individual gonads and individual livers of three to five different fish. Synthetic musks were tested at a concentration of 0.1 and 1 mM. When a significant effect (>50% inhibition) was detected, lower concentrations (0.01, 0.1, 1, 10, and 50 µM) were assayed in order to calculate the concentrations resulting in 50% inhibition (IC50). 7-Ethoxyresorufin O-deethylase (EROD) Activity (CYP1A). EROD activity was assayed by incubating 100 µg of liver microsomal proteins with 3.7 µM 7-ethoxyresorufin and 225 µM NADPH in 100 mM potassium phosphate buffer pH 7.4 (final volume 250 µL) at 25 °C for 10 min. Reaction was stopped by addition of 400 µL of acetonitrile and after centrifugation (2000g/10 min), an aliquot of supernatant (200 µL) was transferred to a 96-multiwell plate. Fluorescence was read at the excitation/emission wavelengths pairs of 537/ 583 nm using a Varioskan plate reader (Thermo Electron Corporation). Quantification was made by calibration with 7-hydroxyresorufin. BFC-O-debenzyloxylase (BFCOD) Activity (CYP3A). BFCOD activity was analyzed according to the procedure described by BD Gentest and optimized for carp liver microsomes (23). The assay consisted of 25 µg of liver microsomal protein, 200 µM 7-benzyloxy-4-trifluoromethylcoumarin, and 22.5 µM NADPH in 100 mM potassium phosphate buffer pH 7.4. The mixture was incubated at 30 °C for 10 min, and the reaction was stopped by addition of 75 µL of 0.5 M tris-base/acetonitrile (20:80, v/v). The fluorescence was directly read in a 200 µL aliquot that was transferred to a 96-multiwell plate at the excitation/emission wavelength pairs of 409 and 530 nm using a Varioskan plate reader (Thermo Electron Corporation). Quantification was made using the calibration curve of the 7-hydroxy-4(trifluoromethyl)-coumarin. Ovarian Aromatase Activity (CYP19). Aromatase activity was determined by the tritiated-water release method as described in Lavado et al. (20). Ovarian microsomal proteins (0.4 mg) were incubated at 25 °C for 1 h in the presence of 100 mM tris-HCl, pH 7.6, [1β-3H]-androstenedione (42.5 pmol, 1 µCi) and 0.2 mM NADPH. The reaction was stopped by placing the tubes on ice and organic metabolites and the excess of substrate were immediately eliminated from the aqueous phase by extracting with dichloromethane. The remaining tritiated steroids were further eliminated by the addition of a suspension of 2.5% (w/v) activated charcoal and 0.25% dextran in milli-Q water. The solution was centrifuged for 1 h at 3600 rpm, and two aliquots of the supernatant (1 mL) were counted for 3H radioactivity. VOL. 43, NO. 24, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Testicular Metabolism of 17r-Hydroxyprogesterone (CYP17) and Androstenedione (CYP11β). The metabolism of 17R-hydroxyprogesterone (17P4) was assessed by incubating mitochondrial fractions (0.25 mg protein) in 50 mM trisHCl buffer pH 7.4 with 0.2 µM [3H]17R-hydroxyprogesterone (166,500 dpm), 10 mM MgCl2, and 1 mM NADPH in a total volume of 250 µL. The reaction was initiated by the addition of NADPH and incubated in constant shaking for 30 min at 30 °C. Similarly, the metabolism of androstenedione (AD) was assessed by incubating mitochondrial proteins (0.5 mg) in 50 mM tris-HCl buffer pH 7.4 with 0.2 µM [3H]androstenedione (166,500 dpm), 10 mM MgCl2, and 1 mM NADPH in a total volume of 250 µL. The reaction was initiated by the addition of NADPH and incubated in constant shaking for 30 min at 30 °C. Both incubations were stopped by adding 250 µL of acetonitrile and after centrifugation (1500g, 10 min), 200 µL of supernatant was injected into a reversed-phase HPLC system coupled with a radiometric detector (21). UDP-Glucuronosyltransferase (UGT) Activity. UGT activity was measured by using testosterone and R-naphthol as model substrates. 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 (20). Sulfotransferase (SULT) Activity. SULT activity was assessed by using estradiol and R-naphthol as model substrates (24). 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 (24). The reaction was stopped with 3 mL of dichloromethane after 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 aqueous phase was quantified by liquid scintillation counting. Naphthol sulfotransferase activity (naphthol-SULT) was assayed by incubating liver cytosolic protein (100 µg) with 1.9 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 300 µL of ethyl acetate. The extraction of nonmetabolized naphthol was completed by 2 × 300 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 error (SEM). Statistical significance was assessed by using one-way ANOVA analysis of variance (Dunnett’s test for differences from control). Data was tested for normality and homogeneity of variance. Level of significance was set at p e 0.05. The concentrations of musk compounds resulting in 50% inhibition (IC50) were calculated using Prism 4 (GraphPad Sofware, San Diego, CA).

Results EROD Activity. The interaction of synthetic musks with CYP1A was assessed by measuring EROD activity. All tested synthetic musks were found to significantly inhibit EROD activity when tested at a concentration of 100 µM (Figure 1A). The nitromusks, MK and MX, were the most potent inhibitors (69 ( 5 and 71 ( 5% inhibition, respectively), followed by tonalide (39 ( 1% inhibition), galaxolide (28 ( 4%), and celestolide (27 ( 4%). Nitromusks were further assayed at lower concentrations (0.01, 0.1, 1, 10, and 50 µM) to determine the concentrations resulting in 50% inhibition 9460

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FIGURE 1. Inhibitory effect on (A) 7-ethoxyresorufin O-deethylase (EROD) and (B) BFC-O-debenzyloxylase (BFCOD) after incubation of hepatic microsomal fractions of Cyprinus carpio with different synthetic musks at a concentration of 100 µM. Values expressed as percentage of control activity are presented as mean ( SEM (n ) 3). * indicates values significantly different from control activities (p < 0.05). EROD specific activity: 20.1 ( 8.6 pmol/min/mg protein; BFCOD: 17.4 ( 2.6 pmol/min/mg protein. (IC50) of the EROD assay. IC50 values of 35 ( 10 and 37 ( 7 µM were found for MK and MX, respectively. BFCOD Activity. The effects of synthetic musks on CYP3Alike catalytic activity were measured using 7-benzyloxy-4trifluoromethyl-coumarin as a substrate. All the compounds, except MX, acted as inhibitors of BFCOD activity (Figure 1B). The most potent inhibitors were galaxolide (57 ( 7% inhibition when tested at a concentration of 100 µM) and tonalide (48 ( 15% inhibition), followed by celestolide (32 ( 7% inhibition) and the nitromusk, MK (31 ( 1%). Galaxolide, tonalide and celestolide were further assayed at lower concentrations (0.1, 1, 10, and 50 µM) to obtain the IC50 values that were of 68 ( 12, 74 ( 21 and 279 ( 79 µM, respectively. Ovarian Aromatase Activity. The aromatization of androstenedione to estrone catalyzed by P450 aromatase (CYP19) was significantly inhibited by four out of the five musks when tested at a concentration of 1 mM (Figure 2A). The most potent inhibitor was galaxolide (86 ( 3% inhibition), followed by tonalide (59 ( 9% inhibition). Celestolide and MK yielded to 33 ( 12 and 21 ( 8% inhibition of aromatase activity, respectively (Figure 2A). When tested at a concentration of 100 µM, only galaxolide acted as a significant inhibitor (48 ( 8% inhibition) of ovarian aromatase activity (data not shown). Testicular CYP17 and CYP11β. The in vitro effect of synthetic musks on 17P4 metabolism is shown in Figure 2B. All five musks tested at a concentration of 1 mM did significantly alter the mitochondrial metabolism of 17P4 in male gonads. Galaxolide and tonalide revealed the strongest inhibitory effect upon CYP17 (C17,20-lyase): the metabolism

FIGURE 3. In vitro interference of synthetic musks with liver microsomal UDP-glucuronosyltransferase activity toward (A) testosterone and (B) r-naphthol. Values expressed as percentage of control activity are means ( SEM (n ) 3). * indicates values significantly different from control activities (p < 0.05). Testosterone-UGT specific activity: 135.47 ( 34.35 pmol/ min/mg protein; naphthol-UGT: 711.84 ( 90.11 pmol/min/mg protein.

FIGURE 2. Inhibitory effect on (A) ovarian P450 aromatase activity, (B) metabolism of 17r-hydroxyprogesteronesCYP17-, and (C) metabolism of androstenedionesCYP11β- after incubation of ovarian microsomal fractions (A) and testicular mithocondrial fractions (B,C) of Cyprinus carpio with different synthetic musks at a concentration of 1 mM. Values expressed as percentage of control activity are presented as mean ( SEM (n ) 3-5). * indicates values significantly different from control activities (p < 0.05). Specific activity P450 aromatase activity: 1.81 ( 0.33 pmol/h/mg protein; CYP17: 10.6 ( 0.50 pmol/h/mg protein; CYP11β: 9.84 ( 2.87 pmol/h/mg protein. of 17P4 to form AD decreased by 76 ( 11 and 66 ( 9%, respectively. Celestolide, musk xylene, and musk ketone yielded to a 43 ( 1, 21 ( 7, and 20 ( 6% decrease in the synthesis of AD. IC50 values for galaxolide, tonalide, and celestolide were of 225 ( 20, 213 ( 70, and 1757 ( 282 µM, respectively. The hydroxylation of AD to form 11β-androstenedione (βAD), a CYP11β catalyzed reaction, was significantly inhibited by all tested musks at a concentration of 1 mM (Figure 2C). The polycyclic musk galaxolide showed the strongest potential to inhibit the CYP11β enzyme (48 ( 7%), followed by celestolide with 37 ( 6%. MX, MK, and tonalide showed an inhibition of 32 ( 5, 28 ( 1, and 22 ( 3%, respectively.

UGT Activity. Only one out of the five musks tested interfered with the glucuronidation of testosterone: galaxolide yielded to a 12 ( 3% inhibition of UGT-T activity when tested at a concentration of 100 µM and a 33 ( 3% inhibition when tested at 1 mM (Figure 3A). In contrast, when using naphthol as a substrate, a stimulatory effect (68-82%) on UGT activity was observed for 100 µM MX and MK, whereas no significant effect was observed for celestolide, galoxolide, and tonalide (Figure 3). The stimulatory effect was observed for all five compounds when tested at a higher concentration (1 mM); this was particularly evident for MX (3.6-fold increase over control) and MK (2.6-fold) (Figure 3B). SULT Activity. The polycyclic musks galaxolide, tonalide, and celestolide acted as inhibitors of E2-sulfonation: percentages of inhibition of 72, 78, and 23% were observed when tested at a concentration of 1 mM (Figure 4A). IC50s of 141 ( 33 µM and 294 ( 69 µM were recorded for tonalide and galaxolide, respectively. In contrast, no significant effect was detected for the nitromusks (Figure 4A). Conversely, none of the tested synthetic musks had a significant effect on SULT activity when naphthol was used as a substrate, with the exception of 1 mM galaxolide (Figure 4B).

Discussion CYP-Catalyzed Activities. This study shows the higher ability of nitromusks (MK, MX) in comparison to polycyclic musks to interact with carp CYP1A. Both, MK and MX acted as in vitro inhibitors of EROD activity in carp liver microsomal fractions (IC50 values of 35-37 µM). The efficiency of both compounds as CYP1A inhibitors was lower than that of R-naphthoflavone (76% inhibition at 10 µM), often employed as a selective inhibitor of CYP1A (23), but to some extent comparable to that of TBT that acted as a CYP1A inhibitor on hepatic microsomes of Mullus barbatus at a concentration VOL. 43, NO. 24, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Inhibitory effect of synthetic musks on sulfotransferase activity (SULT) toward (A) estradiol and (B) r-naphthol after incubation of hepatic cytosolic fractions of Cyprinus carpio with different synthetic musks. Values expressed as percentage of control activity are means ( SEM (n ) 3). * indicates values significantly different from control activities (p < 0.05). Estradiol-SULT specific activity: 0.69 ( 0.12 pmol/min/mg protein; naphthol-SULT: 57.78 ( 8.75 pmol/min/mg protein. as low as 10 µM (25). CYP1A is involved in both, metabolic detoxification of toxins such as aflatoxin B1 and bioactivation of environmental procarcinogens, like polycyclic aromatic hydrocarbons, polychlorinated biphenyls, etc. Thus, any interaction of nitromusks with CYP1A-catalyzed activities is likely to alter hepatic and extra-hepatic xenobiotic metabolism in fish (26). This statement is supported by previous in vivo studies which identified MX as a CYP1A1 inducer, and particularly CYP1A2 inducer, in rat liver (15). Additionally, both MK and MX, were recognized as strong inducers of toxifying liver enzymes in rat and acted as cogenotoxicants with a number of well-known environmental chemicals, such as benzo(a)pyrene, 2-aminoanthracene, and aflatoxin B1 that are mutagenic in their metabolized form (18, 27). Regarding CYP3A catalyzed activities (BFCOD), the polycyclic musks (tonalide, galaxolide, and celestolide) were the most potent inhibitors (Figure 1). To our knowledge, this is the first study showing the inhibitory effect of galaxolide, celestolide, and tonalide on CYP3A-like activity in fish; the observed IC50 (68-279 µM) were in the lower range of those described for the antidepressive drugs paroxetine, fluvoxamine, and fluoxetine (262-643 µM) (23); all three antidepressive drugs are well-known substrates and inhibitors of human CYP3A4. So far, almost no data is available regarding in vivo interaction of polycyclic musks with either human, rats or fish CYP3A enzymes. Among the nitromusks, only MK had a minor inhibitory effect (31 ( 1%) when tested at a concentration of 100 µM, whereas MX was less effective (23 ( 10% inhibition). Interestingly, MX was reported to act as a CYP3A inducer in developing rats at postnatal day 14, but not in adult specimens, indicating differential sensitivity to MX in development (28). Other compounds have been shown to inhibit CYP3A catalyzed activities (progesterone 6Rhydroxylase and BFCOD activities) in carp and other fish 9462

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species (29). Thus, treatment with ketoconazole (100 mg/kg) decreased CYP3A-mediated BFCOD activities by 80% in rainbow trout 3 and 6 days post injection (30). Atlantic cod injected with ethynylestradiol (5 mg/kg) showed significantly reduced BFCOD activities as well as CYP3A protein levels (31). Furthermore, 200 µM ethynylestradiol was shown to act as an in vitro inhibitor of hepatic BFCOD in carp (93% inhibition at EE2) and atlantic cod (23, 31). CYP3A plays an important role in the metabolism of both steroid hormones (progesterone and testosterone) and xenobiotic substances (N-demethylation of benzphetamine) in fish (32); consequently, xenobiotics interfering with these activities can potentially disrupt steroid hormone and xenobiotic metabolism in fish. Additionally, galaxolide, the most active CYP3A inhibitor, shows three-dimensional structure similarities with the steroid hormone androstenone (5R-androst16-en-3-one), a mammalian steroid pheromone (33); this raises the question on whether galaxolide and other synthetic musks might have an effect on chemical communication and/or the hormone system of aquatic species (34). Interestingly, galoxolide was the most potent in vitro inhibitor of ovarian P450 aromatase (86 ( 3% inhibition at a concentration of 1 mM) followed by tonalide (59 ( 9% inhibition). Since P450 aromatase (CYP19) catalyzes the terminal step in the biosynthesis of estrogens, any alteration of CYP19 can disturb the local and systemic balance between estrogens and androgens and have consequences in terms of sexual differentiation, the regulation of female reproductive cycle, and male reproductive behavior (35). However, it is worth mentioning that the potency of galaxolide and tonalide as P450 aromatase inhibitiors is rather low if compared with that reported for several imidazole and triazole fungicides (clotrimazole, propiconazole, fenbuconazole, imazalil and prochloraz) with IC50 in the range of 0.016-1.0 µM on rainbow trout ovarian P450 aromatase activity (36). Additionally, CYP17 and CYP11β enzymes involved in the synthesis of active androgens in male fish, were investigated as potential targets for synthetic musks as they represent key ecotoxicological targets for environmental pollutants (21). In this study, the inhibitory effect of synthetic musks on CYP17 (C17,20-lyase) was more pronounced than that on CYP11β. Among the tested compounds, galaxolide and tonalide were the most potent inhibitors (IC50: 213-225 µM for CYP17). To our knowledge this is the first report on the effect of synthetic musks on CYP17 (C17,20-lyase) and CYP11β activity in fish gonads. Although the observed inhibitory effect is lower than reported for other environmental pollutants, e.g., nonylphenol and ketoconazole strongly inhibited CYP17 in sea bass mitochondrial fractions (21), it would be advisible to determine to which extent the inhibitory potency of synthetic musks, and particularly galaxolide and tonalide, on fish steroidogenic enzymes can be extrapolated to the in vivo situation. Phase II Activities. The ability of synthetic musks of altering metabolic clearance of hormones and xenobiotics was investigated by measuring the interference with phase II enzymes (UGTs and SULTs) using testosterone and estradiol as model endogenous substrates and naphthol as a model xenobiotic. Our in vitro studies demonstrated that the glucuronidation of testosterone was not a primary target for synthetic musks, as only a minor inhibitory effect of galaxolide was observed (34% inhibition at the highest dose tested, 1 mM) and no interaction was detected for the other musk compounds. Interestingly, this contrasts with a significant inhibitory effect of polycyclic musks on the sulfation of estradiol, with IC50s in the range of 140-294 µM for tonalide and galaxolide, respectively. 4-Nonylphenol and triphenyltin inhibited the sulfation of estradiol at micromolar concentrations (30-70 µM) in two marine fish species (24) and in carp (17-41 µM) (37). Comparatively, the efficiency

of tonalide and galaxolide as SULT-E2 inhibitors is 4-17fold lower than the one reported for classical endocrine disrupters, but even though, giving the high volume usage of these compounds and their relatively high persistence in the environment, the observed interference with hormone sulfation is a matter of concern. Surprisingly, when naphthol was used as a UGT substrate, the glucuronidation reaction was stimulated by the presence of nitro- and polycyclic musks in the incubation media. The mechanisms behind this stimulation are unknown; changes in UGT conformation caused by binding of the musk compounds to the enzyme may have enhanced the glucuronidation of naphthol (38). However, no such in vitro stimulation of piscine UGTs has been reported so far. In contrast, a strong inhibitory effect on the glucuronidation of naphthol was reported for the lipid regulator gemfibrozil (IC50 ) 63 µM) and the anti-inflammatory drugs ibuprofen, diclofenac, ketoprofen (IC50 ) 409-807 µM) (23). Overall, the obtained data provides further support to the hypothesis that different UGT isoforms are involved in the glucuronidation of testosterone and phenolic compounds (39). However, whether the stimulation of UGT-naphthol is just an in vitro artifact or on the contrary will also occur in vivo should be further investigated. Interestingly, the sulfonation of naphthol was not altered by any of the musk compounds tested, with the possible exception of galaxolide. In summary, the present study reveals that synthetic musks are able to inhibit the catalytic activity of different CYP, UGT and SULT isoforms in fish. Polycyclic musks acted as inhibitors of CYP3A, CYP19, CYP17, and SULT-E2 catalyzed activities, while nitromusks were CYP1A inhibitors (see SI Table S1). The obtained data suggest that CYP isoforms together with SULT-E2 could be potential sensitive targets for synthetic musk substances in fish, and useful end points to investigate when designing specific in vivo studies. Among the tested compounds, galaxolide had the highest potential to interfere with fish enzymatic systems. Overall, this work contributes to the better understanding of the impact of synthetic musks on fish species.

Acknowledgments This study was supported by the Spanish Ministry of Science and Education under Project ref. CGL2005-02846. Sabine Schnell and Rebeca Martin-Skilton acknowledge a predoctoral fellowship from the Ministerio de Educacio´n y Ciencia. Denise Fernandes acknowledges a postdoc fellowship (SFRH/ BPD/34289/2006) from the Portuguese Fundac¸a˜o para a Cieˆncia e Tecnologia.

Supporting Information Available Chemical structures of the selected musk compounds and percentage of inhibition and IC50 for the different musk compounds and enzymatic systems investigated. This material is available free of charge via the Internet at http:// pubs.acs.org.

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