Interaction of Emerging Contaminants with the Cytochrome P450

Article pubs.acs.org/est

In Vitro Interaction of Emerging Contaminants with the Cytochrome P450 System of Mediterranean Deep-Sea Fish Carla Ribalta and Montserrat Solé* Institute of Marine Sciences (ICM-CSIC), Pg. Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain ABSTRACT: The interactions of emerging contaminants with the xenobiotic and endogenous metabolizing system of deep-sea fish were compared. The drugs diclofenac, fluoxetine, and gemfibrozil belong to different pharmaceutical classes with diverse mechanistic actions, and the personal care products triclosan, galaxolide, and nonylphenol are representative of antibacterial agents, nitro-musks, and surfactants, respectively. The fish compared are representative of the middle and lower slope of deep-sea habitats. The species were adults of Trachyrynchus scabrus, Mora moro, Cataetix laticeps, and Alepocehalus rostratus. The hepatic metabolic system studied were the activities associated with several cytochrome P450 isoforms (CYPs): 7-ethoxyresorufin-O-deethylase (EROD), benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD), and 7-ethoxycoumarin-O-deethylase (ECOD). Results showed differences in baseline activities and sensitivity to chemicals which were species, chemical, and pathway dependent. T. scabrous was the most sensitive species to chemical interactions with the xenobiotic and endogenous metabolizing (EROD and BFCOD) systems, especially in the case of diclofenac interference with BFCOD activity (IC50 = 15.7 ± 2.2 μM). Moreover, T. scabrous and A. rostratus possessed high basal ECOD activity, and this was greatly affected by in vitro exposure to diclofenac in T. scabrous also (IC50 = 6.86 ± 1.4 μM). These results highlight the sensitivity of marine fish to emerging contaminants and propose T. scabrous (middle slope) and A. rostratus (lower slope) as sentinels and the inclusion of ECOD activity as a sensitive biomarker to these exposures.

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INTRODUCTION Deep-sea habitats, especially those in close proximity to coastal areas and connected by submarine canyons, are likely to experience high inputs of organic matter and their associated contaminants during important hydrodynamic events. This phenomenon, known as dense shelf water cascading (DSWC), is triggered by very cold and windy winter conditions and periodically occurs every 6−10 years in the NW Mediterranean region,1 increasing the input of organic pollutants from the coastal margin to the deep-waters2 and affecting its fauna.3 Deep-sea environments and their inhabitants are under a considerable amount of threats,4 such as, the intention to exploit this fragile habitat for fish, mineral, and oil resources,5 the dumping of waste including pharmaceutical waste, the significant presence of litter on the sea bed6,7 and increasing amounts of microplastics in oceans worldwide.8 Legacy and emerging contaminants, such as polybrominated diphenyl ethers (PBDEs), have been detected in Mediterranean deep-sea species, and some of their associated biochemical responses have been measured.9−14 All these studies have indicated a high accumulation of organic and inorganic pollutants in their tissues at levels similar to or higher than those found in coastal species. Among the deep-sea fish we studied, the gadiforms Trachyrhynchus scabrous and Mora moro are species of great abundance in the middle slope with the highest percentages of biomass between 600 and 1100 m and 700−1100 m, respectively.15 Alepocephalus rostratus is more © XXXX American Chemical Society

abundant at greater depths (>1200m), and Cataetyx laticeps, although less represented, stands out for its high bioaccumulation of certain pollutants.13 In addition to the potential transfer of pollutants from the vicinal coastal regions, the biomagnification attained in these species in the NW Mediterranean can be due to their reported high trophic level values (δ15N = 9.0−11.8).16 Emerging contaminants, such as pharmaceuticals and personal care products (PPCPs), have raised growing environmental concern for their constant and increasing usage worldwide.17 The occurrence, fate, and ecotoxicological consequences of these exposures for nontarget species in the aquatic environment, where complex mixtures occur, have been comprehensively reviewed.18−21 Recent fish studies have also demonstrated the interaction of emerging contaminants with the endogenous and xenobiotic metabolism system in freshwater fish and selected species currently used as models in toxicology.22−25 The metabolism of drugs and other xenobiotics is carried out in part by several cytochrome P450 isoforms (mainly in mammals CYP1-4 families). Cytochrome P450 (CYP) enzymes catalyze the oxidative conversion of drugs and other lipophilic compounds to hydrophilic Received: June 18, 2014 Revised: September 9, 2014 Accepted: September 16, 2014

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dx.doi.org/10.1021/es5029603 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

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Table 1. Phylogeny and Depth of Occurrence of the Mediterranean Fish Species Selected for the in Vitro Studya fish weight species (M:F)

order

depth of occurrence

Solea solea (4:4) Dicentrarchus labrax (2:2) Trachyrinchus scabrus (4:4) Mora moro (4:4) Cataetyx laticeps (3:3) Alepocephalus rostratus (4:4)

Pleuronectiform Perciform Gadiform Gadiform Ophidiiform Osmeriform

coastal coastal middle slope middle slope lower slope lower slope

EROD

g 360.8 225.0 171.0 530.4 430.0 332.4

± ± ± ± ± ±

BFCOD

ECOD

pmol/min/mg prot 76.3 19.2 20.6 59.2 47.8 37.9

23.4 83.1 17.5 22.9 53.8 4.2

± ± ± ± ± ±

6.1* 33.9 3.0 4.8* 16.1 0.9

30.7 87.2 24.0 6.7 4.4 22.1

± ± ± ± ± ±

5.6 20 3.3 1.7 0.9 3.4

4.5 18.4 19.2 18.6 8.8 87.0

± ± ± ± ± ±

0.3 2.9 1.9 1.5 0.9 10.3

Their total weight reported as mean ± SD. Activities of 7-ethoxyresorufin-O-deethylase (EROD), benzyloxy-4-[trifluoromethyl]-coumarin-Odebenzyloxylase (BFCOD), and 7-ethoxycoumarin-O-deethylase (ECOD) are presented as mean ± SEM. In brackets, number of male (M) and female (F) used. The asterisk (∗) (p < 0.05) denotes statistical difference between male and female basal activities in a given species. a

action on aquatic species in vitro23 and in vivo on mating, feeding, and predator avoidance behavior.39 It is often used as a model for assessing the impact of SSRI in the aquatic environment. Gemfibrozil belongs to the fibrate group of lipid regulators. In fish it has been reported to disrupt sex hormone balance33 and induce embryonic malabsorption syndrome in zebrafish.40 Among personal care products (PCPs), galaxolide is a polycyclic musk that interacts with the xenobiotic but mostly endogenous (steroid) metabolism of fish.25,41 Triclosan is an aromatic hydrocarbon used as an antibacterial agent whose metabolites possess toxicological properties and may act as an endocrine disruptor.42 Nonylphenol, included in the WFD priority list,38 is the final degradation product of alkylphenol surfactants. Although its use has been banned for years, it has been found to be present in the NW Mediterranean at depths well over 1000 m, affecting the xenobiotic and reproductive system of the deep-sea fish species A. rostratus.43 All these chemicals are classed as holding an environmental concern level of between 3 and 4 owing to their persistence and bioaccumulation potential, rating among the highest published scores.44 The aim of the study was to assess the potential interaction of six PPCPs of environmental concern on particular enzyme activities within the CYP family involved in the xenobiotic and endogenous metabolism in fish representatives of the middle and lower deep-sea Mediterranean waters. Coastal species, Solea solea and Dicentrarchus labrax, representative of the benthic and pelagic realm, respectively, and both of great commercial interest in fisheries and aquaculture were included for comparisons.

metabolites. Thus, CYPs play a dominant role in the elimination of drugs from the body. Nonetheless, inhibitory interactions occur when drugs compete for oxidation by specific CYPs. The correspondence between mammalian and fish CYPs is a subject under study and some important differences in both vertebrate groups are evident.26,27 In fish, the families of CYP1A (mostly involved in xenobiotic metabolism) and CYP3A (involved in the metabolism of drugs but also of endogenous compounds, such as steroids) are among the most widely studied and are measured as 7-ethoxyresorufin-Odeethylase (EROD) and 7-benzyloxy-4-trifluoromethyl-coumarin-O-dibenzyloxylase (BFCOD) activities, respectively. Less attention has been paid to ethoxycoumarin-O-deethylase (ECOD) activity in groups other than mammals, where it is mostly associated with CYP2A6 activity. To date, it is not known which CYP isoforms are involved in 7-ethoxycoumarin (EC) deethylation in fish. Few studies are available on the action of emerging contaminants on the xenobiotic and endogenous metabolism of marine fish,25 and none has been carried out on deep-sea fish. The most likely reason for this lack of information is the fact that PPCPs are not reported or under-represented in the marine environment. Nonetheless, coastal and estuarine areas situated near anthropogenic inputs of human discharges are already exposed to trace amounts of emerging contaminants, including those derived from fish farming usage.28,29 Moreover, an increasing worldwide anthropogenic impact is forecast on marine and deep waters, including emerging contaminants, such as the already reported illegal dumping of pharmaceuticals.30 Bearing in mind that deep habitats (>200 m) represent the largest ecosystems on earth and in many countries coastal sites and deep-waters are tightly connected, species inhabiting this realm might be vulnerable to increasing inputs of emerging contaminants. Standing out among the PPCPs of environmental concern are those with higher bioaccumulation potential (log Kow > 4− 5), in widespread and continuous use, and with potential toxic chronic effects on aquatic fauna. Some have already been reported to accumulate in fish: diclofenac,31 fluoxetine,32 gemfibrozil,33 galaxolide,34 nonylphenol,35 and triclosan.36 To a certain extent, they have already been detected in the marine environment in the vicinity of outfalls.37 Among pharmaceuticals, diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) of ample use worldwide that affects fish at environmentally realistic doses31 and has recently been included in the Water Framework Directive (WFD) watch list.38 Fluoxetine is the active ingredient in Prozac, an antidepressant drug that acts as a selective serotonin reuptake inhibitor (SSRI). It has been reported to exhort a noxious

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MATERIALS AND METHODS Sampling and Tissue Preparation. Fish from the middle and lower deep waters were obtained by trawling the Blanes canyon region (NW Mediterranean) within the PROMETREO project framework (sampling coordinates 41°15′N; 2°50′E). The coastal species S. Solea was obtained in the frame of the DEPURAMAR project (sampling coordinates 41°10′N; 1°53′E) and D. labrax was from the aquaculture facilities of our Institute (41°23′N; 2°11′E). The biological traits of the fish selected for the study are presented in Table 1. Microsomes for CYPs activity determinations were prepared from the individual livers of adult males and females at balanced sex ratios of the fish species detailed in Table 1. Livers (≈1.5 g) were homogenized in ice-cold 100 mM phosphate buffer (pH 7.4) containing 150 mM KCl, 1 mM dithiothreitol (DTT), 0.1 mM phenatroline, 0.1 mg/mL trypsin inhibitor, and 1 mM ethylenediaminetetraacetic acid (EDTA) in a 1:4 (w:v) ratio using a polytron blender. The homogenate was centrifuged at B



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Table 2. Cytochrome P450 (CYP) Related Activities Using Different Fluorometric Substrates, Assay Conditions, and Metabolite Formation Measured at Specific Excitation/Emission (Ex/Em). The Correspondence between Activity and CYP Isoform Is Derived from Mammalian Studies activity

mammalian CYPs [ref]

EROD; 7-ethoxyresorufin-O-deethylase BFCOD; 7-benzyloxy-4trifluoromethylcoumarin-Odebenzyloxylase ECOD; 7-ethoxycoumarin-O-deethylase

substrate (μM)

substrate

1A1 > 1A2, 1B1 [46] 3A4, 2C19 [46]

ER; 7-ethoxyresorufin BFC; 7-benzyloxy-4trifluoromethylcoumarin

2 200

1A, 2A, 2B [59]

EC; 7-ethoxycoumarin

100

metabolite

Ex/Em

NADPH (μM)

Resorufin sodium salt 7-OH-4trifluoromethyl coumarin 7-OHcoumarin

537/583 410/538

200 20

370/450

200

Table 3. Properties of the Pharmaceuticals (Diclofenac, Fluoxetine, And Gemfibrozil) and Personal Care Products (Galaxolide, Nonylphenol and Triclosan) Used for in Vitro Testing. The Bioconcentration Factor (BCF) Was Reported from Published Fish Studies compound

use

family

log Kow

BCF

matrix

species

ref

diclofenac fluoxetine gemfibrozil galaxolide nonylphenol triclosan

analgesic antidepressant lipid regulator fragance detergent antibacterial agent

nonsteroidal antiinflamatory drug (NSAID) serotonine reuptake inhibitors (SSRI) fibrates polycyclic musk alkyphenol ethoxylates chlorinated aromatic hydrocarbon

4.5 4.1 4.8 5.9 4.5 4.7

1372 580 113 862 605 3407

liver liver blood plasma muscle liver muscle

O. mykiss Oryzias latipes Carassius auratus Anguilla anguilla Rutilus rutilus Danio rerio

31 32 33 60 61 36

consecutive steps of 10 000g × 30′ and the supernatant obtained was further centrifuged at 100 000g × 60′ at 4 °C. The final microsomal pellet obtained was dissolved in homogenization buffer containing 20% glycerol in a 2:1 (w:v) ratio.45 CYP Determinations. Catalytic activities of hepatic CYPs were determined in the microsomal fraction using three fluorometric substrates at assay conditions as listed in Table 2, based on the method described elsewhere.46 A 10 μL sample of microsomes (100 μg of protein) was incubated with their respective substrates and NADPH (Table 2) for 10 min at 30 °C, and the metabolite formation rate was recorded at their specific wavelengths. Quantification was contrasted with a calibration curve for each specific metabolite (ranging from 0 to 160 nM). CYP assays were carried out in 100 mM phosphate buffer (pH 7.4), except ECOD determination, which was in 100 mM Tris buffer (pH 7.4). All reactions were run in 96-well format microplates and were linear for the recorded period using the kinetic assay mode (Magellan v6.0) in a TECAN Infinite 200 microplate reader. CYP-related activities were expressed as pmol/min/mg prot. Total protein content was determined according to the method by Bradford47 adapted to the microplate, using bovine serum albumin (BSA) as standard (0.1−1 mg/mL). Inhibition Determinations. For the inhibition studies, a stock solution of the chemical was dissolved in either dimethyl sulfoxide (DMSO) or ethanol and further diluted to the concentration desired, for each particular case, in assay buffer. The maximal solvent concentration in the incubation mixture was 0.025% and it was seen not to affect these enzymatic activities. Incubations were made at room temperature (23 °C) for 30 min and the enzymatic measurements lasted 10 min and were conducted at 30 °C as previously described.45 All reagents ́ and PPCPs were from Sigma-Aldrich Quimica, SA, Madrid, Spain. Only the substrate BFC was purchased at CYPEX Ltd., Dundee, Scotland. Model CYP Inhibitors. The CAS number of the selected inhibitors was α-naphthoflavone (αNF; 604-59-1) and ketoconazole (KETO; 65277-42-1). Three concentrations of each inhibitor were assayed 1, 5, and 10 μM.

Emerging Contaminants. The selected PPCPs CAS numbers were diclofenac (15307-79-6), fluoxetine (56296-787), gemfibrozil (25812-30-0), galaxolide (1222-05-5), nonylphenol (84852-15-3) and triclosan (3380-34-5), and some of their properties are detailed in Table 3. All these chemicals were tested at 100 μM, and when the inhibition attained at this dose was well over 50%, another three lower concentrations were also considered (0.1, 1, and 10 μM) and their IC50 values were calculated. Inhibition incubations and further CYP determinations were as described above. Statistics. Data were tested for normality (Shapiro-Wilk’s test) and homogeneous variance (Levene's test) and were log10 (X)-transformed, when necessary, to comply with normality and homoscedasticity assumptions. The Student's t test was used to compare differences in enzyme activities per sex and the inhibitory effect of a particular PPCP in relation to its control. One-way ANOVA followed by post hoc T3 Dunnet's was applied to test the effect of a chemical assayed at several concentrations in relation to its control. Activities are expressed as mean ± SEM and the number of individuals contrasted is indicated in each case. IC50 calculations were made using the PROBIT regression test from IBM SPSS Statistics 19 program with the log transformed data. The significance level was set at α = 0.05.

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RESULTS Basal EROD, BFCOD, and ECOD Activities and the Action of Model Inhibitors. Basal activities calculated for all the species are listed in Table 1. Sex differences in EROD activity were seen for S. solea and M. moro with males displaying higher activities than females (p < 0.05), whereas for BFCOD and ECOD no sex-related trends were evidenced (p > 0.05). For species characterization of CYPs, the model CYP1A inhibitor αNF was used in EROD determination. The fungicide KETO is considered to be a specific CYP3A4 inhibitor and was used in BFCOD measures (Figure 1). The inhibition on EROD activity was dose-dependent and reached 95% in S. solea, whereas in T. scabrous and A. rostratus it reached only 50% of the controls at the highest dose assayed. As for BFCOD (Figure 1), the inhibition at the highest dose of KETO was similar in all C



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achieved after incubations with triclosan: 20.9 ± 2.3% (M. moro), 36.3 ± 2.7% (D. labrax), and up to 49.4 ± 13.0% (T. scabrous). Nonylphenol incubations inhibited this same activity in D. labrax (22.7 ± 2.2%) and more significantly in T. scabrous (38.8 ± 7.9%). In vitro microsomal incubations affected EROD activity in T. Scabrous after diclofenac (48.7 ± 8.7%) and galaxolide (35.6 ± 8.7%) exposures. Gemfibrozil mean EROD inhibition, although significant in some cases, was under 10% in all the species tested. Out of all species, T. scabrous was the most affected by chemical interactions, whereas M. moro was somewhat irresponsive. PPCPs Interaction on BFCOD Activity. As with EROD inhibition contrasts, only females were chosen, except in the case of C. laticeps and D. labrax for which both sexes were considered (Figure 2). No inhibition was tested further in C. laticeps because of very low basal BFCOD activity. In M. moro, the metabolism of BFC was affected by incubation with all the drugs (20−45% inhibition) except gemfibrozil. D. labrax similarly responded to incubations with all the chemicals (20−39%) and T. scabrous inhibitions of BFCOD activity reached up to 75.6 ± 5.9% with diclofenac and about 20% for galaxolide, nonylphenol, and triclosan. Owing to the high inhibition of this activity with diclofenac in T. scabrous, lower concentrations were tested, and the IC50 found was 15.7 ± 2.2 μM. The deep-sea fish A. rostratus responded with an inhibition of this activity to diclofenac (11%) but more significantly to fluoxetine incubations (37.4 ± 5.0%). For the benthic coastal fish S. solea, BFCOD activity was somewhat insensitive to the chemicals tested in vitro although a maximal 17% significant inhibition was attained after galaxolide incubation. PPCPs Interaction on ECOD Activity. The inhibition on the degree of deethylation of the substrate EC was measured as ECOD activity. Only the two species with significant basal activities and consistent inhibition values were considered. In A. rostratus ECOD was more strongly inhibited 48.6 ± 7.7% by fluoxetine incubation (Figure 3), whereas T. scabrous responded to a similar extent to nonylphenol (44.8 ± 2.2%) and triclosan (47.7 ± 1.4%) but more significantly to diclofenac (91.2 ± 3.3%) with an IC50 value as low as 6.86 ± 1.4 μM.

Figure 1. Percentage of activity in relation to controls (100%) after in vitro incubation with three concentrations (1, 5, and 10 μM) of the CYP1A inhibitor α-naphtoflavone in EROD activity and the CYP3A4 inhibitor ketoconazole for BFCOD activity measures. Species codes: SS = S. solea, DL = D. labrax, TS = T. scabrus, MM = M. moro, CL = C. laticeps and AR = A. rostratus. Complete names are listed in Table 1. Number of individuals used are in brackets. The asterisk (∗) denotes p < 0.05 with respect to the control.

species (about 90%), with the exception of T. scabrous (78%) and M. moro (44%). Basal ECOD activity was generally low or not consistent after inhibition exposures, except for A. rostratus and T. scabrous fish species (Table 1). Therefore, further inhibitory studies were carried out in these two species only. CYP1A and CYP3A involvement in the oxidation of the substrate EC was tested by the incubation of the microsomes with αNF and KETO. ECOD activity was not significantly suppressed after αNF (1− 10 μM) incubations in A. rostratus or T. scabrus with maximal inhibition < 7% at the highest dose. Nonetheless, it was significantly inhibited after KETO incubations (1−10 μM) at the highest dose only, and this was species-dependent (23.3% for A. rostratus and 36.8% for T. scabrus). PPCPs Interaction on EROD Activity. Only females were chosen for in vitro interactions of the emerging contaminants in EROD activity as in most cases they were revealed to be more sensitive to αNF incubations. In the case of C. laticeps and D. labrax no sex differences in sensitivity were seen and, as sample sizes were smaller, individuals of both sexes were considered. This CYP1A-associated activity was affected by in vitro incubation of the six chemicals tested at the same 100 μM concentration in a species-dependent manner (Figure 2). The metabolism of the substrate ER was inhibited with 100 μM fluoxetine incubations in A. rostratus (30.7 ± 3.4%) and S. solea (23 ± 1.3%). Significant inhibitions of this activity were also

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DISCUSSION To date, there is no evidence that the currently reported concentrations of emerging contaminants in water bodies could pose a threat to marine species. Nonetheless, predictions of increasing production and usage of these chemicals, low removal rates for some of them in STWs, direct disposal from aquaculture activities, illegal dumping in some developing countries, and the high bioaccumulation properties of some should be an alert against the consequences of these exposures in marine species. Moreover, with diet being a likely route of exposure in fish, an additional factor of concern is the high trophic level of some deep-sea species. In fact, the middle- and lower-slope fish selected in this study express a high trophic level that makes them more vulnerable to the toxic consequences of the exposures. In vitro interference of PPCPs on the xenobiotic and steroid metabolism of fish has already been pointed out for some of the compounds selected in the present study. Nonetheless, to the best of our knowledge, this is the first report on interference of these chemicals in these same metabolic pathways in marine species currently living in a wide depth range (from 900 to 2000 m) in the Mediterranean. For all the fish species considered, emerging contaminants interfered with their D



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Figure 2. Percentage of 7-ethoxyresorufin-O-deethylase (EROD) and benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) activity in relation to controls (100%) after in vitro incubation with a 100 μM concentration of the pharmaceuticals (diclofenac, fluoxetine, and gemfibrozil) and the personal care products (galaxolide, nonylphenol, and triclosan). Species codes: SS = S. solea, DL = D. labrax, TS = T. scabrus, MM = M. moro, CL = C. laticeps, and AR = A. rostratus. Complete names are listed in Table 1. Number of individuals used are in brackets. The asterisk (∗) denotes p < 0.05 with respect to the control.

responsible for EC deethylation still remain unknown.48 In humans, diclofenac is mostly metabolized by CYP2C9 and by other isoforms from the same CYP2 family. Nonetheless, the existence of the homologous CYP2 family in fish is controversial and CYP2B, 2C and 2D are reported absent.49 In addition, αNF (CYP1A inhibitor) did not significantly inhibit ECOD activity in fish, as also found by Parente et al.,48 and KETO had only a moderate inhibitory effect on this activity. Thus, it seems plausible that other unknown CYPs from a related CYP2 family might be involved in the metabolism of both EC and diclofenac. We are not aware of any other studies addressing drug in vitro interactions due to diclofenac exposure using the hepatic microsomal system of fish. The toxicity and high consumption of this analgesic has raised enough environmental concern to warrant its recent inclusion, together with another two pharmaceuticals, in the European Directive of priority substances in surface water bodies.38 This fact together with its high bioconcentration factor in the liver of some fish species31,50 and the high drug interference observed in the two marine species with greater

xenobiotic and endogenous system (measured as EROD and BFCOD inhibitions) in a species- and chemical-dependent manner. The inclusion of ECOD activity in the two species that expressed higher basal and consistent enzyme activities, gave grounds to some interesting results which will be further discussed. Diclofenac is a NSAID that clearly interfered in the CYP1A and CYP3A system of the most sensitive middle slope species, T. scabrous, but also with BFCOD activity in the perciform D. labrax. To date, as far as we are aware, the IC50 of 15.7 ± 2.2 μM attained in T. scabrus for BFCOD activity after in vitro incubation with diclofenac is the lowest reported in fish. This drug also interfered in BFCOD activity of Cyprinus carpio hepatic microsomes, although with a much higher dose (IC50 = 805.3 μM) but not with the CYP1A related EROD activity.22 The interference of this drug on ECOD activity was even more significant as the IC50 value was as low as 6.86 ± 1.4 μM. No other studies have reported interaction of PPCPs on ECOD activity in fish. In humans, the substrate EC is mostly metabolized by CYP2A6 and, in rats, by several isoforms belonging to several subfamilies; in fish the CYP isoforms E



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Figure 3. Percentage of 7-ethoxycoumarin-O-deethylase (ECOD) activity in relation to controls (100%) after in vitro incubation with a 100 μM concentration of the pharmaceuticals (diclofenac, fluoxetine, and gemfibrozil) and the personal care products (galaxolide, nonylphenol, and triclosan). Complete names are listed in Table 1. Number of individuals used are in brackets. The asterisk (∗) denotes p < 0.05 with respect to the control.

pharmaceutical (1 mM) inhibited in vitro microsomal EROD and BFCOD activities by about 50% and at 55%, respectively, with an IC50 for the latter activity of 616.3 μM.22 In light of the present results and other reported studies, the toxicological consequences of drug interactions in the two tested pathways seem highly variable and species dependent. The polycyclic musk galaxolide was a poor CYP1A inhibitor in all species except T. scabrus (40% at 100 μM) and a moderate BFCOD activity inhibitor in D. labrax, T. scabrus, and M. moro (23−33%). This observation partly agrees with results obtained in a fresh water fish species, C. carpio, in which this musk was a poor CYP1A inhibitor (IC50 > 100 μM) but differed because of its strong inhibition in the CYP3A4 pathway (IC50 = 68 ± 12 μM).41 In an injection experiment with the marine fish D. labrax, it was revealed that this fragrance was metabolized, and mostly affected the mitochondrial CYPs (CYP17 and CYP11β) of male gonads, although to a lesser extent than the fungicide KETO.25 To the best of our knowledge, no other in vitro fish studies are available for contrast. Nonetheless, the high worldwide consumption of this fragrance, a reported high BCF in fish, its log Kow of 5.9 together with the interference observed with the xenobiotic and endogenous metabolism of the sensitive fish T. scabrus, warrant further ecotoxicological consideration. The surfactant derivate nonylphenol was responsible for a significant inhibition of EROD activity in T. scabrous (about 40%) but also interfered with BFCOD activity in D. labrax, T. scabrous, and M. moro (24−45% inhibition). Previous studies with fish have coincided with an inhibitory action on CYP1A and more significantly on CYP3A4- related activities.53 The results of this study partly support these observations and also highlight the fact that the inhibitory action of nonylphenol does not seem universal in fish. Despite being banned by the EU since 2005, because of its endocrine disruption properties, it has been found recently in the bile of the deep-sea fish A. rostratus in the same NW Mediterranean region and made responsible for VTG gene induction in males.43 Despite the regulations, it is still present in the marine sediment and biota and can also be

ECOD activity in this study, further alerts against the long-term consequences of diclofenac exposure in marine waters. Fluoxetine belongs to the SSRIs drug group. In mammals it has been reported to inhibit the CYP1A2, CYP2C19, and CYP2D6 forms51 but it also acts as a substrate and inhibitor of human CYP3A4. In fish, fluoxetine maximal EROD inhibition was about 20% and it acted more selectively on BFCOD activity in A. rostratus and M. moro (33−37% inhibition). In fact, even if these two species differed in basal EROD and BFCOD activities and in their response to the inhibitors αNF and KETO, they shared a similar interaction on BFCOD activity, such as that described in humans. CYP1A2 activity, measured as the oxidation of the substrate 7-methoxyresorufin was very low (data not shown), which suggests the lack of this isoform in these fish species. In addition, the presence of orthologous to mammalian CYP2C19 and CYP2D6 enzymes has not yet been reported in fish.49 Furthermore, in marine and freshwater fish models, metabolism of fluoxetine has been described to occur, being more significant in CYP induced specimens and producing a range of metabolites other than norfluoxetine, the main one reported in mammals.52 Microsomal EROD activity in freshwater carp, C. carpio, was highly responsive to fluoxetine inhibition with a reported IC50 of 46.5 μM while this value for BFCOD activity was higher (643 μM).22 In another study, the same 100 μM dose of fluoxetine caused a 84% reduction in BFCOD activity in liver microsomes of trout, Oncorhynchus mykiss, whereas the inhibition of EROD activity, although significant, was more modest.24 As revealed by the in vitro studies available so far in fish, the action of fluoxetine on EROD and BFCOD activities appears to be somewhat species dependent. Gemfibrozil is a lipid regulator that did not greatly interfere with ER metabolism in any of the fish species studied (max inhibition 10%) and the interference with the CYP3A pathway was highly species dependent, at its highest in the perciform D. labrax (26%). By contrast, the same concentration of gemfibrozil caused a 45% reduction in EROD activity in microsomes of the trout O. mykiss with even greater significance in BFCOD activity.24 A 10-fold higher concentration of this F



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observations, we propose D. labrax (coastal), T. scabrous (middle slope), and A. rostratus (lower slope) as potential sentinels to test vulnerability to emerging contaminants in the marine environment. D. labrax is a valuable commercial species in fisheries and aquaculture and likely to be subjected to exposure to pharmaceutical drugs. T. scabrous and A. rostratus are of great ecological value as they represent a high biomass percentage of their respective habitats.

transferred through the trophic chain and interfere with the endocrine system of sensitive fish species. Triclosan is an antibacterial agent that was shown to interfere with the CYP1A mediated EROD activity in D. labrax (36%) and T. scabrous (50%) and on CYP3A related BFCOD measures in the former two species and M. moro (22−39%). As far as we know, there are no studies on the interaction of this PCP with the microsomal CYP system of fish. This drug has a high BCF and a log Kow of 4.7. Water-borne exposure to this PCP impaired swimming behavior in fish with significant ecological consequences.54 In fish, the interference of triclosan with CYPs is controversial, and the induction of EROD and BFCOD activities and associated genes in a freshwater fish study could not be clearly attributed to triclosan (and galaxolide) exposures.55 As indicated for the other emerging contaminants, more ecotoxicological research on marine species is needed, in light of the moderate interactions of triclosan with ER- and BFC-associated activities in fish. A noteworthy result of the present study was the interaction revealed for PPCPs in ECOD activity of the middle- (T. scabrous) and lower- (A. rostratus) slope species. Basal ECOD activities in these two fish were high and the IC50 of 6.9 ± 1.4 μM for diclofenac on this activity is among the lowest recorded in fish. Few fish studies have reported on the interactions with ECOD activity as EC is recognized as a broad CYP substrate with much less specificity than in mammals. However, some studies with early vertebrates56 and loricariids have described that these fish express high deethylation of EC and lack EROD activity,48 similar to the present observations with A. rostratus. In the latest reported study, a CYP1A-like protein was inmunodetected, and ECOD activity was inducible by a promutagen polycyclic aromatic hydrocarbon (DMBA) but not with the model CYP1A inducer βNF.48 No induction studies could be carried out with these deep-sea species, but further research on CYPs characterization using other molecular tools warrant consideration. The specific action of diclofenac and fluoxetine on ECOD activity, although species dependent, supports the inclusion of this measure when screening for drug interactions in fish. In addition to species-specific differences in basal EROD and BFCOD activities and their respective responses to the selective inhibitors αNF and KETO, the interaction of PPCPs with these two metabolic pathways was also highly species dependent, with T. scabrous being the most sensitive. Another issue of particular interest is the lack of response to the azole fungicide KETO on BFCOD activity in M. moro. KETO is regarded as a selective CYP3A4 inhibitor in mammals and many fish species.57,58 However, as revealed in this study, some deep-sea species seem less responsive to this fungicide. We can hypothesize that in M. moro either the substrate BFC can be metabolized by other CYPs (as already revealed in some fish studies) or KETO does not interact with the CYP3A isoforms from this species. The results of this study highlight the interaction of PPCPs with the xenobiotic and endogenous system of deep-sea fish. Although water-borne and sediment exposures to these chemicals are unlikely to be of toxicological significance at present, (1) their increasing worldwide production and use, (2) their lipophilic properties and potential transfer potential through the food chain, (3) the high trophic level of some deep-sea fish, and (4) the low IC50 values reported for some enzymatic activities, all alert against the consequences that chronic exposures could produce. As a result of our

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AUTHOR INFORMATION

Corresponding Author

*Fax 34 932309555; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financed by Spain’s Ministry of Science and Innovation (ref CTM2007-66316-C02-02/MAR-PROMETEO and CTM2010-16611-DEPURAMAR). D. Romeo is acknowledged for his help in sample processing. Prof. D. Lloris is acknowledged for providing the T. scabrus photograph.

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REFERENCES

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Interaction of Emerging Contaminants with the Cytochrome P450

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