Bioconcentration and Biotransformation of Amitriptyline in Gilt-Head

Jan 20, 2017 - Collectively these data indicate that, in addition to AMI, a broad suite of metabolites should be included in biomonitoring campaigns i...
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Bioconcentration and Biotransformation of Amitriptyline in Gilt-head Bream Haizea Ziarrusta, Leire Mijangos, Urtzi Izagirre, Merle M. Plassmann, Jonathan P. Benskin, Eneritz Anakabe, Maitane Olivares, and Olatz Zuloaga Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05831 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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Bioconcentration and Biotransformation of Amitriptyline

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in Gilt-head Bream

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Haizea Ziarrusta*,1,2 Leire Mijangos,1 Urtzi Izagirre,3 Merle M. Plassmann,2 Jonathan P.

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Benskin,2 Eneritz Anakabe,4 Maitane Olivares,1 Olatz Zuloaga1,3

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1. Department of Analytical Chemistry, University of the Basque Country (UPV/EHU), Leioa,

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Basque Country, Spain

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2. Department of Environmental Science and Analytical Chemistry (ACES), Stockholm

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University, Stockholm, Sweden

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3. Research Centre for Experimental Marine Biology and Biotechnology (PIE), University of the

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Basque Country (UPV/ EHU), Plentzia, Basque Country, Spain

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4. Department of Organic Chemistry, University of the Basque Country (UPV/EHU), Leioa,

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Basque Country, Spain

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*Corresponding author: Haizea Ziarrusta ([email protected])

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Department of Analytical Chemistry, Faculty of Science and Technology, University of the

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Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain.

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Tel: + 34 94 601 55 51; Fax: +34 94 601 35 00

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ABSTRACT

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Extensive global use of the serotonin-norepinephrine reuptake inhibitor Amitriptyline (AMI) for

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treatment of depression has led to its ubiquitous occurrence in the aquatic environment. To

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assess AMI bioconcentration factors, tissue distribution, and metabolite formation in fish, we

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exposed gilt-head bream (Spaurus aurata) to AMI in seawater for 7 days at two concentrations

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(0.2 µg/L and 10 µg/L). Day 7 proportional bioconcentration factors (BCFs) ranged from 6

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(10 µg/L dose, muscle) to 127 (0.2 µg/L dose, brain) and were consistently larger at the low

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dose level. The relative tissue distribution of AMI was consistent at both doses, with

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concentrations decreasing in the order brain ≈ gill > liver > plasma > bile >> muscle. Using a

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suspect screening workflow based on liquid chromatography–high resolution (Orbitrap) mass

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spectrometry we identified 33 AMI metabolites (both Phase I and Phase II), occurring mostly in

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bile, liver and plasma. Ten structures are reported for the first time. Remarkably, all 33

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metabolites retained the tricyclic ring structure common to tricyclic antidepressants, which

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may be toxicologically relevant. Collectively these data indicate that, in addition to AMI, a

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broad suite of metabolites should be included in biomonitoring campaigns in order to fully

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characterize exposure in aquatic wildlife.

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1. Introduction

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Globally, amitriptyline (AMI) is among the most extensively prescribed pharmaceuticals for the

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treatment of depression and psychiatric disorders (>10,000 kg in England alone for 2012).1 Like

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other tricyclic antidepressants (TCAs), the mechanism of action of AMI involves inhibition of

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norepinephrine and/or serotonin reuptake in the central nervous system, resulting in

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increased neurotransmitter concentrations in the brain.2,3 AMI enters waste streams mainly

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through human excretion.4 Despite considerable removal during wastewater treatment5 AMI is

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detectable at concentrations of up to 72 ng/L in surface water and 223 ng/L in wastewater

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treatment plant (WWTP) effluents.3,5-8

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The disposition of AMI in mammalian models has been well studied as part of human

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pharmaceutical safety assessments.9-11 However, few data exist for non-target organisms

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which may be exposed unintentionally in the environment. In aquatic species, AMI may disrupt

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the immune system and affect reproduction, development and growth at concentrations as

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low as 10 ng/L.12-14 AMI also has the potential to bioaccumulate15 (log Kow = 4.81, pKa = 9.8 16),

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and it has been observed in thicklip gray mullet (Chelon labrosus) liver from Spain8 and whole

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mussels from San Francisco Bay17 at concentrations of 1.8 ng/g and 0.2 ng/g, respectively. A

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systematic investigation of AMI uptake and tissue distribution in aquatic species has not yet

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been conducted; however, a study involving brook trout (Salvelinus fontinalis) exposed to

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3.7 ng/L of AMI via diluted WWTP effluents observed up to 0.29 ng/g in liver, and non-

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detectable concentrations in muscle and brain.18 This result is surprising considering that

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observations in other species indicate a tendency for AMI to partition to brain followed by

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liver.9,10

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Mammalian studies indicate that AMI is extensively biotransformed in the liver by cytochrome

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P450 2D6 (CYP2D6) enzymes to a diverse range of potentially bioactive metabolites.2 In vitro

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incubations involving human liver microsomes observed up to 50 possible AMI transformation 3 ACS Paragon Plus Environment

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products,19,20 while an additional 12 glucuronide conjugates were observed in human urine.20

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Considerable inter-species variability in metabolism of AMI exists2,21,22 and to our knowledge

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there are no data on biotransformation of AMI in aquatic organisms. Consequently,

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environmental exposure assessments in aquatic wildlife which focus only on AMI, or a limited

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number of mammalian metabolites, may underestimate the true extent of exposure.

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The objective of the present work was to assess the distribution of AMI in fish in order to aid in

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assessing the risk(s) associated with occurrence of this substance in the aquatic environment.

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To achieve this goal, gilt-head bream (Spaurus aurata) were exposed to AMI in seawater for 7

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days at two concentrations (0.2 µg/L and 10 µg/L). In addition to tissue-specific

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bioconcentration factors, AMI-metabolites were characterized using a suspect screening

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strategy involving high resolution mass spectrometry (HRMS). To our knowledge this is the first

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time AMI uptake and biotransformation has been monitored in fish. These data provide

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valuable information for assessing exposure of aquatic organisms to AMI and its potentially

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bioactive metabolites.

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2. Materials and methods 2.1. Standards and Reagents

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Amitriptyline hydrochloride (AMI; 98 %), nortriptyline hydrochloride (NOR; 98 %), and the

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isotopically-labelled internal standards 2H3-amitriptyline hydrochloride (2H3-AMI) and 2H3-

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nortriptyline hydrochloride (2H3-NOR) were purchased from Sigma–Aldrich (St. Louis, MO,

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USA). Stock dosing solutions of AMI were prepared at 5000 mg/L in methanol (MeOH) and

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diluted down to 4.26 mg/L (high dose experiment) and 85.2 µg/L (low dose experiment) in

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Milli-Q water. The final concentration of MeOH in the dosing solution was 0.98) between them taking into account the concentrations observed

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at both dosing experiments, suggesting that AMI uptake occurred mainly through the gills and

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not the gut.28 Similar results are reported in the literature for uptake of several

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pharmaceuticals in wild fish exposed to wastewater.27 Though the uptake and tissue

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distribution of ionisable compounds is generally pH-dependent,29-32 AMI is a weak base (pKa

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9.8) with a relatively high hydrophobicity (log Kow of 4.81, log D 2.39 at pH 7.316), and

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therefore, it is always positively charged at physiological pH.

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With the exception of gill in the low-dose experiment, tissue BCFs were consistent over time

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(p > 0.05), suggesting that steady state was reached after the 7-days of exposure at both

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dosing levels (Figure 2). BCFs were higher in the low-dose experiment for brain, gill and liver,

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suggesting a concentration dependency on the accumulation of AMI in fish. This result is

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inconsistent with other studies investigating the effect of exposure concentration on

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pharmaceutical uptake.33 Nevertheless, an inverse relationship between concentration and

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BCF34 has been reported, possibly due to an increase in the reactive oxygen species (caused by

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higher contaminant concentrations), which result in increased AMI biotransformation.35 The

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same phenomena was not observed for muscle BCFs, but this could be due to the low

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concentrations observed (close to limit of detection, and hence, wider variability) in this tissue.

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There are few examples of tissue-specific BCFs determined for AMI in the peer-reviewed

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literature.18 The limited existing data suggest that AMI BCFs are influenced by both inter-

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species differences in uptake as well as exposure conditions. For example, a BCF=78 was

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obtained for liver in book trout,18 which is in the same order of magnitude to the liver BCFs

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calculated in the present work (21 at high dose, 48 at low dose). In contrast, much higher

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values (BCF= 6028) were reported in mussels exposed to AMI via a river receiving wastewater

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effluent.36 Additionally, it is germane to note that the BCFs in Figure 2 only take into account

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not metabolized AMI. Therefore, the BCFs may underestimate the total burden of

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psychoactive drug in a given tissue, since they do not account for the presence of bioactive

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metabolites, which could have an additive effect together with AMI.

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3.2. Metabolite Identification

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An overview of the biotransformation pathway with the annotated structures for AMI

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metabolites in gilt-head bream is provided in Figure 3. Structural assignments based on

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accurate mass and fragment ion data are summarized in Table S2. No reference standards

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were available for the detected metabolites, thus we reached a confidence level 2b – 3

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according to Schymanski et al.,37 leading to the assignments of probable structures or tentative

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candidates. In all cases, the difference between measured and theoretical masses was

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< 3.3 ppm. Nevertheless, due to the possibility of multiple isomers, structural assignments

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were often ambiguous and relied on expert judgement. A total of 33 AMI transformation

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products were observed, made up of both Phase I and Phase II metabolites. To the best of our

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knowledge, 10 of these metabolites (M12, M13, M15, M16, M20, M21, M22, M23, M27 and

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M28) are reported for the first time. Detailed information about the identification of individual

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structures is compiled in the SI and summarized herein.

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NOR (M1) was observed in all tissues and biofluids in the present work, but it was never the

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major metabolite. This was surprising considering that N-demethylation of AMI to NOR (M1) is

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the main metabolic route in human liver microsomes.19 Similarily, N, N- didemethylation 19,20

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which was reported in human liver microsomes, was not observed in the present work. This

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could be attributable to inter-species variability or to differences between in vivo and in vitro

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biotransformation pathways. Despite these differences, oxidation remained a major metabolic

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route of AMI in gilt-head bream, consistent with observations in humans.2 Up to 5 AMI

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monohydroxylated metabolites of AMI and NOR (M2-M6) and an AMI N-oxidation metabolite

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(M7) were observed (Figure 3). These structures are consistent with those observed from in

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vitro experiments involving AMI.19,20 Notably, M2 and M3 were estimated to be the most

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abundant of all metabolites observed in the present work, based on a relative comparison of

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peak areas.

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M2/M3 (m/z 294.1857) and M4/M5 (m/z 280.1701) were identified as monohydroxylated

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metabolites in the endocyclic ethylene group of AMI or NOR, respectively, according to the

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observed fragmentation (see Figure S1a for M2/M3 and discussion in the SI). Hydroxylation in

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the endocyclic ethylene group of the central ring of AMI is well described in the literature.2 On

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the other hand, M6 (m/z 294.1857) was identified as a monohydroxylated metabolite of AMI

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with the hydroxyl group in the exocyclic ethylene group of AMI (see Figure S1b) and M7 (m/z

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294.1857) as a N-oxidation metabolite of AMI (commonly known as amitriptylinoxide) (see SI).

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M8 (m/z 276.1752) likely forms from dehydration of M2/M3 according to the similar

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fragmentation pattern observed (see SI). Similarly, M9 (m/z 262.1595) is likely formed from

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dehydration of hydroxy-metabolites of NOR (i.e., M4/M5). Both M8 and M9 were identified

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during in vitro experiments using human liver microsomes.20

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M10 and M11 were identified as keto-derivatives of AMI in the central ring (m/z 292.1701)

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(see discussion in SI), similar to the metabolites observed in in vitro experiments.19,20 M12 had

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the same exact mass as M10/11 (m/z 292.1701) and was likely formed by dehydrogenation

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plus methoxylation of NOR (see Figures S2a and S2b). M13 (m/z 306.1857) showed an

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equivalent structure to M12 but with the tertiary amine head group as in AMI (see Figure S3c).

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M14 (m/z 278.2544) corresponded to an epoxidation of M9 (see SI). Zhou and co-workers also

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described epoxy metabolites as intermediate products20 and Liu et al. observed epoxy

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metabolites during an investigation into the degradation of cyclobenzaprine.38

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Five dioxidated metabolites of AMI (M15-M19; m/z 310.18066) and two of NOR (M20 and

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M21; m/z 296.1650) were also identified in all the biological matrices except in muscle and

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brain. M15 and M16 metabolites showed a similar fragmentation pattern to one another, with

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one hydroxylation on the endocyclic ethylene group and a N-oxydation. M17-M19 were

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identified as dihydroxymetabolites in the endocyclic ethylene group of AMI (see Figure S3a),

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which were also reported during in vitro experiments.20 M20 and M21 were identified as

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dihydroxymetabolites of NOR in the endocyclic ethylene group (see Figure S3b). Compared to

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Zhou and coworkers,20 we did not observe dihydroxy metabolites with one hydroxyl group in

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the ethylene bridge and the other one in the aromatic ring. This may be due to interspecies

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differences or due to in vitro20 versus in vivo test systems.

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M22 (m/z 308.1650) showed a similar fragmentation pattern to M10/M11. This metabolite

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contains a keto group in the endocyclic ethylene group and the second oxidation was

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determined as a N-oxidation due to the lack of a second loss of water and the unmodified side

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chain.

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M23 (m/z 269.1541) could come from either AMI or NOR after the loss of (-C2H6N) or (-CH3N),

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respectively, followed by dihydroxylation. However, due to poor fragmentation, we could not

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locate these two hydroxyl groups in this metabolite.

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Up to ten glucuronide derivatives were observed mainly in bile (M24-M33), of which seven

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(M24-M26, M29, M30, M32 and M33) were previously reported by Zhou and co-workers in

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human urine20. Although mentioned in the literature,2,19,20 N-glucuronidation of AMI was not

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observed. This discrepancy may be attributed to the different glucuronidation process in

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mammalian metabolism21 or due to metabolic differences arising from the use of in-vivo versus

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in-vitro tests. In some cases we could determine the position of the glucuronide group but we

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could not always determine where the conjugation (N- or O-glucuronidation) occurred, as is

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also stated in the literature.39 M24-M26 (m/z 470.2178) was attributed to the glucuronidation

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of M2/M3 based on the same fragment ions. This is consistent with Zhou et al.20 who also

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found three glucuronide derivatives with the hydroxyl group at that position. M27 and M28 13 ACS Paragon Plus Environment

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(m/z 470.2178, see Figure S4 for fragmentation) were considered as N-glucuronides of

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methoxy-metabolite of NOR in the endocyclic ethylene group. While monohydroxy-

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metabolites of AMI in the aromatic ring were not observed in the present work, M29 (m/z

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470.2178) was attributed to a glucuronide of a hydroxyl metabolite of AMI in an aromatic ring.

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M30 (m/z 486.2127) was attributed to the glucuronidation of M17-M19. In the case of M31

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(m/z 486.2127) we could not define the structure of this glucuronide metabolite. Finally,

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glucuronides M32-M33 (m/z 456.2022) were only observed in bile and showed a similar

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fragmentation in accordance with the fragmentation of M4-M5 monohydroxylates of NOR.

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3.3. Changes in metabolite profiles with time and in different tissues

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AMI metabolites were found primarily in bile (29), liver (21) and plasma (19) (see Table S2).

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This is not surprising considering that liver is the main site of xenobiotic metabolism, and

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metabolites formed in the liver are likely to undergo either systemic circulation (i.e. via

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plasma) or elimination (via bile). Bile receives metabolites produced in both the liver and gut,

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and thereby most metabolites were observed in bile.

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Although it is only an approach, in order to compare the abundances of the different

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metabolites in the different tissue/biofluids, we considered the same sensitivity for all the

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metabolites. The relative abundances of metabolites (calculated as the ratio of the individual

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peak area with respect to the sum of all peak areas) in different tissues and biofluids over the

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course of the exposure period are shown in Figure 4. All peak areas were corrected using

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sample size and internal standard (2H3-NOR). While metabolite profiles displayed considerable

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differences between tissues/biofluids, the relative profile within a given tissue/biolfluid

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remained fairly consistent over the duration of the experiment.

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AMI was estimated to be the most abundant compound in comparison to its metabolites in gill

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(30% of the total chromatographic peak area) and brain (40% of the total chromatographic

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concentrations of AMI in this tissue are expected. In the case of brain the high abundance of

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AMI is also expected due to passive diffusion across the blood-brain barrier is likely to favour

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AMI as opposed to the more polar Phase I and II metabolites.10 Next to AMI, M2 (24 %) and

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M13 (11 %) were the most abundant compounds detected in brain.

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AMI in liver and plasma accounted for close to 20 % of the sum peak areas for all compounds,

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highlighting the metabolic role of liver and the distribution role of plasma. M2 (30 %), was the

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most abundant metabolite in liver whereas M10 (33 %), the dehydrogenation product of M2,

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was mainly found in plasma. In contrast, AMI accounted for a very small (2%) proportion of the

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sum peak areas in bile. Here, M24 and M26, glucuronide metabolites of M2/M3, were the

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most abundant metabolites, accounting for approximately 30 % of the total peak area each.

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3.4. Implications to Environmental Exposure Assessment

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Assessing risks associated with the occurrence of pharmaceuticals in the aquatic environment

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is hampered by the occurrence of numerous and potentially bioactive transformation

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products. These substances may not be the same as those elucidated as part of initial

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pharmaceutical safety assessments, on account of diverse reaction pathways occurring in the

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environment but also inter-species differences in metabolism. In order to fully characterize the

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extent of exposure to pharmaceuticals in aquatic wildlife, a complete understanding of the

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numerous and potentially bioactive structures, and their tissue distribution, is required. In the

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present work, brain contained the highest burden of AMI and only 10 metabolites, while liver

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contained only ~20% of AMI and 21 metabolites. Consequently, standard exposure

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assessments in aquatic wildlife focusing only on AMI in muscle or liver tissue would severely

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underestimate the concentration of AMI in the target organ (brain) and completely overlook

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the concentrations of potentially bioactive metabolites in the entire animal. In fact, recent

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data suggest that hydroxylated and N-oxidated metabolites (including NOR) are active in

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mammals, and may even be more bioactive than parent compounds.2,40-42 While this highlights

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the need to include transformation products in environmental exposure assessments, this

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clearly represents a challenge, since authentic analytical standards are unavailable for many of

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these substances. In the absence of analytical standards, a suspect screening analytical

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approach, such as that employed here, can be an effective means of qualitative identification

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of novel and potentially bioactive metabolites. Important structures identified through this

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approach can then be confirmed and semi-quantified using an a posteriori approach at such

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time that a standard becomes available.

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4. Supporting Information

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Reagents information; LC-QqQ-MS/MS and LC-q-Orbitrap analysis details; Table S1, Phase I and

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Phase II reactions; Table S2, details for AMI and all annotated metabolites; Metabolite

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identification decription; Figures S1-S4, MS2 spectra and fragmentation of some metabolites

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(M2-M3, M6, M10-M11, M12, M13, M17-M18-M19, M20 and M27-M28).

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5. Acknowledgements

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This work was financially supported by the Ministry of Economy and Competitiveness through

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the project CTM2014-56628-C3-1-R. H. Ziarrusta is grateful to the Spanish Ministry and L.

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Mijangos to the Basque Government for their pre-doctoral fellowships.

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Figure 1. Concentration of AMI in the different tissues (as ng/g) and fluids (as ng/mL) plus the confidence intervals (as two times the standard deviation, n=3, for a 95 % of confidence level) detected for fishes exposed to 10 µg/L (a) and 0.2 µg/L (b) of AMI. Concentrations of NOR (c) in fish exposed to 10 µg/L of AMI. Figure 1 179x300mm (96 x 96 DPI)

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Figure 2: Evolution of BCF values and confidence interval (expressed as two times the standard deviation, n=3, 95 % of confidence level) in the different tissues: (a) brain, (b) gill, (c) liver and (b) muscle during the uptake. Note: even if the steady-state was not reached in some cases, BCFs were calculated as the ratio between AMI concentration in each fish tissue and the average water concentration during the uptake. Asterisk indicates significant differences (p < 0.05) between the values in high and low dosing levels. Figure 2 300x179mm (96 x 96 DPI)

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Figure 3: Biotransformation pathway of AMI in Gilt-head bream. Inset shows MS/MS fragmentation and major m/z ions. The circles indicate that bond can take place at any position. Figure 3 349x203mm (300 x 300 DPI)

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Figure 4. Relative abundances of AMI and its metabolites (calculated as the proportion of the sum peak areas) in biological tissues (muscle, brain, gill and liver) and fluids (plasma and bile) along the AMI exposure experiment (day 2, 4 and 7). Figure 4 300x179mm (96 x 96 DPI)

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