Environmental Toxicology and Chemistry, Vol. 32, No. 8, pp.

1810–1818, 2013
# 2013 SETAC
Printed in the USA

COMPARATIVE PHARMACEUTICAL METABOLISM BY RAINBOW TROUT

(ONCORHYNCHUS MYKISS) LIVER S9 FRACTIONS

KRISTIN A. CONNORS,*yzx BOWEN DU,yzk PATRICK N. FITZSIMMONS,# ALEX D. HOFFMAN,#

C. KEVIN CHAMBLISS,zkyy JOHN W. NICHOLS,# and BRYAN W. BROOKSyzxk
yDepartment of Environmental Science, Baylor University, Waco, Texas, USA
zCenter for Reservoir and Aquatic Systems Research, Baylor University, Waco, Texas, USA
xInstitute of Biomedical Studies, Baylor University, Waco, Texas, USA
kThe Institute of Ecological, Earth and Environmental Sciences, Baylor University, Waco, Texas, USA
#US Environmental Protection Agency, Duluth, Minnesota, USA
yyDepartment of Chemistry and Biochemistry, Baylor University, Waco, Texas, USA

(Submitted 10 December 2012; Returned for Revision 6 February 2013; Accepted 5 April 2013)

Abstract: The occurrence of pharmaceuticals in the environment presents a challenge of growing concern. In contrast to many industrial
compounds, pharmaceuticals undergo extensive testing prior to their introduction to the environment. In principle, therefore, it may be
possible to employ existing pharmacological safety data using biological “read-across” methods to support screening-level
bioaccumulation environmental risk assessment. However, few approaches and robust empirical data sets exist, particularly for
comparative pharmacokinetic applications. For many pharmaceuticals, the primary cytochrome P450 (CYP) enzymes responsible for their
metabolism have been identified in humans. The purpose of the present study was to employ a comparative approach to determine whether
rainbow trout biotransform pharmaceuticals known to be substrates for specific human CYPs. Seven compounds were selected based on
their primary metabolism in humans by CYP3A4, CYP2D6, or CYP2C9. Five additional test compounds are known to be substrates for
multiple CYPs. Metabolism by rainbow trout liver S9 fractions was evaluated using a substrate-depletion approach, which provided an
estimate of intrinsic hepatic clearance (CLIN VITRO,INT). An isotope dilution liquid chromatography–tandem mass spectrometry method
was employed for quantitation of parent chemical concentrations. Only 2 general CYP substrates demonstrated measurable levels of
substrate depletion. No significant biotransformation was observed for known substrates of human CYP2D6, CYP2C9, or CYP3A4. The
results of this study provide novel information for therapeutics that fish models are likely to metabolize based on existing mammalian data.
Further, these results suggest that pharmaceuticals may possess a greater tendency to bioaccumulate in fish than previously anticipated.
Environ Toxicol Chem 2013;32:1810–1818. # 2013 SETAC

Keywords: Bioaccumulation Contaminant of emerging concern Comparative pharmacology Risk assessment Alternative
model

INTRODUCTION of pharmaceuticals in aquatic life therefore represents a critical

The occurrence of pharmaceuticals and personal care
products in the environment has prompted substantial research
activity and an increasing level of regulatory attention. In the
developed world, dozens of pharmaceuticals have been detected
in wastewater treatment plant effluent, surface water, and
groundwater at concentrations ranging from nanograms per liter
to low micrograms per liter [1]. Higher concentrations have been
found in some developing countries [2]. Bioaccumulation of
pharmaceuticals in fish and other aquatic organisms has attracted
additional attention, particularly in effluent-dominated sys-
tems [3–6]. For some compounds, tissue residues may increase
in aquatic organisms to several orders of magnitude higher than
surface water concentrations [7]. For example, in a recent study
by Brodin et al. [8], the concentration of the benzodiazepine
oxazepam was 6 times greater in fish tissue than in surface water.
In the laboratory, exposure to such environmentally relevant
concentrations of oxazepam resulted in similar tissue bioaccu-
mulation levels and subsequently behavioral alterations and
changes in feeding rate [8]. Understanding the bioaccumulation
All Supplemental Data may be found in the online version of this article.
* Address correspondence to Kristin_Connors@Baylor.edu.
Published online 18 April 2013 in Wiley Online Library
(wileyonlinelibrary.com).
DOI: 10.1002/etc.2240
1810
research need [9,10].
Pharmaceuticals are unusual among nonpesticidal environ-
mental contaminants because the drug-approval process provides a
wealth of information about their physicochemical and biological
properties. Therapeutic compounds are intentionally designed to
elicit specific physiological effects at low concentrations. If
therapeutic targets (e.g., receptors, enzymes) are sufficiently
conserved in nontarget species, adverse outcome pathways may be
predicted by mammalian pharmacodynamic information [11–14].
For example, Valenti and coworkers [15] studied the effect of
sertraline, a widely prescribed antidepressant, on adult fathead
minnows. Sertraline acts in humans by selectively inhibiting
serotonin reuptake. Previous work had shown that serotonin
reuptake transport (SERT) proteins in fish share substantial (65%–
75%) sequence homology with human SERT proteins [17]. Adult
male fathead minnows exposed to sertraline in water accumulated
chemical concentrations in plasma exceeding the human
therapeutic threshold [15]. These measured concentrations were
subsequently associated with functional changes in chemical
binding to SERT proteins and shelter-seeking, antianxiety
behavior. Thus, it may be possible to use mammalian pharmaco-
dynamic data to develop predictions for substances presenting
potential environmental hazards to aquatic life [9,12,16,17].
Unfortunately, the comparative pharmacokinetics of phar-
maceuticals in aquatic life are less well understood than the
Comparative pharmaceutical metabolism by rainbow trout
pharmacodynamic approaches described above [7,18]. Because
biotransformation has the potential to reduce chemical accumu-
lation within an organism, a question of particular concern is
whether aquatic biota can appreciably metabolize these
compounds. Using substrate-depletion methods developed by
the pharmaceutical industry, environmental toxicologists have
begun to characterize biotransformation rates in fish using in
vitro systems derived from liver tissue [19–21]. Though
measured rates of depletion provide no information on the
identity of metabolizing enzymes, the resulting information can
be extrapolated to estimate whole-body clearance. In vitro
systems, such as those that utilize the liver S9 fraction or liver
microsomes, are desirable because they can be rapidly
performed, are cost-effective, and thus can support screening-
level environmental risk-assessment efforts [22]. To date, most
of this work has been performed with pesticides and other
organic compounds; however, limited studies have been
conducted using pharmaceuticals [23–25]. These efforts have
shown that fish can biotransform several widely used drugs.
For many pharmaceuticals, the primary cytochrome P450
(CYP) enzymes responsible for their metabolism have been
identified in humans [26,27]. The CYP isoforms 3A4, 2C9, and
2D6 represent the major phase I enzymes involved in
pharmaceutical metabolism [28]. Research to identify orthologs
in fish to these and other CYP enzymes is ongoing. Based on
sequence information, several CYP1 and CYP3 genes in rainbow
trout (Oncorhynchus mykiss) have been shown to be orthologous
to human CYPs from the same gene families [29,30]. In contrast,
trout and other fish possess very few CYP2 family genes
orthologous to human CYP2 forms [31]. Gene sequences do not,
however, provide information on the functional behavior of fish
CYPs, including substrate specificity and inducibility. Thus, fish
may possess a CYP capable of catalyzing reactions typically
associated with a specific human CYP despite the absence of any
known orthologs to the human enzyme. It is of interest, therefore,
to determine whether fish and humans possess similar types of
metabolic activity as this may provide a basis for predicting fish
metabolism from human data. Because in vitro studies with fish
S9 can support screening-level bioaccumulation assessment of
organic contaminants [22,32,33], the primary objective of the
present study was to determine whether trout are capable of
metabolizing in vitro 7 pharmaceuticals identified as substrates
for specific CYP enzymes in humans. Additional studies were
performed with 5 therapeutics considered to be general CYP
substrates in mammals. A secondary objective was to compare
such in vitro pharmaceutical biotransformation data with
previous field observations of bioaccumulation in fish.
MATERIALS AND METHODS
Chemicals
Pharmaceuticals were selected for study based on whether
they are primarily metabolized in humans by CYP3A4, 2C9, or
2D6, or a general combination of CYPs (Table 1). Pharmaceuti-
cal substrates and isotopically labeled standards were purchased
from several providers and were of 98% purity or higher
(Supplemental Data, Table S1). The b form of reduced
nicotinamide adenine dinucleotide phosphate (b-NADPH,
>95% pure) was purchased from Oriental Yeast. Radiolabeled
benzo[a]pyrene (14C-BAP, >97% pure, 26.6 mCi/mmol; Sigma-
Aldrich) was diluted with unlabeled BAP to a specific activity of
5 mCi/mmol. All other reagents and cofactors were purchased
from Sigma-Aldrich and were reagent-grade or higher in quality.
Environ Toxicol Chem 32, 2013 1811
Fish cultures
Rainbow trout (Erwin strain) of mixed sex were obtained
from the Upper Midwest Environmental Sciences Center (La
Crosse, WI, USA) and reared until approximately 1.5 yr old
(average wt approximately 350 g) at the US Environmental
Protection Agency laboratory in Duluth, Minnesota. All fish
were in early stages of sexual maturation at the time of death and
had not previously undergone a spawning cycle. Fish were fed
Silver Cup trout chow (Nelson and Sons) and maintained on a
16 h light:8 h dark photoperiod at 11  1 8C. Water for fish
holding was drawn from Lake Superior and was treated before
use with ultraviolet light and sand filtration.
S9 fractions
Fish were killed with ethyl 3-aminobenzoate methanesulfo-
nate (MS222, 250 mg/L) buffered with 750 mg/L NaHCO3. In
an effort to reduce blood contamination of liver S9 preparations,
the hepatic vein was severed, and 10 mL to 20 mL of ice-cold
buffer (Hank’s balanced salt solution with 10 mM N-2-
hydroxyethylpiperazine-N0 -2-ethane-sulfonic acid and 3 mM
ethylenediamine tetraacetic acid [EDTA], pH 7.8) was passed
through the hepatic portal vein. Livers were excised, rinsed with
clearing buffer, minced in 2 volumes (w/v) of homogenization
buffer (50 mM Tris, 2 mM EDTA, 1 mM dithiothreitol,
150 mM KCl, and 250 mM sucrose; pH 7.8), and homogenized
using a Potter-Elvehjem mortar and pestle. Homogenates from 5
male (pool A) and 6 female (pool B) fish were pooled and
centrifuged at 13 000 g (4 8C) for 20 min. The S9 fraction was
carefully isolated, flash-frozen, and stored at 80 8C.
S9 characterization
Select phase I and phase II enzymatic activities were
characterized using well-known substrates. The activity of
CYP1A was described using 2 assays: BAP ring hydroxylation
(aryl hydrocarbon hydroxylase) and ethoxyresorufin O-deal-
kylation. A radiometric method described by Van Cantfort
et al. [34] was used to measure aryl hydrocarbon hydroxylase
activity. Each reaction consisted of 0.5 mg/mL S9 protein,
5 mM MgCl2, 2 mM NADPH, and 80 mM 14C-BAP in 50 mM
Tris buffer for a final incubation volume of 0.5 mL. Reactions
were initiated by adding BAP and terminated with 1 mL 0.15 M
KOH in 85% dimethyl sulfoxide. Unreacted BAP was removed
by extracting samples 3 times with hexane (5 mL per
extraction). A 750-mL aliquot of the resulting aqueous:dimethyl
sulfoxide phase was mixed with 20 mL UltimaGold scintillation
fluid (Perkin-Elmer), and polar BAP metabolites were quantified
by liquid scintillation counting (Packard 2550; Perkin-Elmer).
Heat-denatured S9 controls were employed to account for
radioactivity (e.g., by-products in the BAP) that partitioned to
the aqueous phase but were unrelated to enzymatic activity.
The ethoxyresorufin O-dealkylation assay was based on the
fluorescence method of Burk and Mayer [35]. Each reaction
consisted of 1 mg/mL S9 protein, 5 mM ethoxyresorufin, and
1 mM NADPH in 100 mM potassium phosphate buffer for a
final incubation volume of 200 mL. Reactions were initiated by
adding ethoxyresorufin and terminated with 600 mL methanol.
Samples were centrifuged at 3000 g to precipitate protein. The
metabolite resorufin was measured by fluorescence at excitation
and emission wavelengths of 550 nm and 580 nm, respectively,
on a microplate reader (BioTek Instruments) and quantified
using a standard curve.
Uridine diphosphate-glucuronosyltransferase (UGT) activity
was measured using p-nitrophenol as the substrate and uridine
1812 Environ Toxicol Chem 32, 2013
Table 1. Cytochrome P450 (CYP) enzyme substrates in humansa
1A2 2A6 2B6
Major CYP3A4 substrates
Diltiazem
Carbamazepine
Major human CYP2C9 substrates
Celecoxib
Sulfamethoxazole
Warfarin þ þ
Major human CYP2D6 substrates
Methylphenidate
Paroxetine
General human CYP substrates
Acetaminophen þ þ
Diclofenac þ þ
Diphenhydramine þ þ
Fluoxetine þ þ
Propranolol þþþþþ
a
Data from Lacy et al. [26], Lee et al. [27], and Akutsu et al. [59].
þþþþþ ¼ major CYP substrate; þ ¼ minor substrate.
50 -diphosphoglucuronic acid (UDPGA) as the glucuronosyl
donor [36]. Incubations consisted of 1 mg/mL S9 protein,
10 mM EDTA, 7 mM UDPGA, 100 mM p-nitrophenol, and
25 mg/mL alamethicin in 500 mM potassium phosphate buffer
with a final incubation volume of 200 mL. Reactions were
terminated with 600 mL of 3% trichloroacetic acid and 100 mL
of 5N sodium hydroxide. Samples were then centrifuged at
3000 g for 5 min to precipitate protein. The remaining substrate
was measured by fluorescence (400 nm) on a microplate reader
(BioTek Instruments) and quantified using a standard curve.
Samples containing denatured S9 or active S9 without UDPGA
were used as controls to correct for nonenzymatic losses.
Glutathione S-transferase activity was measured using the
method described by Habig et al. [37]. Reactions contained 2 mM
reduced glutathione (GSH) and 2 mM 1-chloro-2,4-dinitroben-
zene in 100 mM potassium phosphate buffer for a final incubation
volume of 1.4 mL. Absorbance (340 nm) was monitored for
5 min (Varian 300; Agilent Technologies) following the addition
of 0.1 mg/mL of S9 protein. Control samples without S9 protein
were analyzed to account for nonenzymatic changes in absor-
bance. Enzyme activity was calculated using a molar absorption
coefficient of 9.6 mM1 cm1.
All assays were conducted at trout physiological temperature
(11 8C) and pH (7.8) and under saturating substrate conditions.
Substrates were added to incubations using acetone as the carrier
solvent (1% v/v). The S9 protein concentration was measured
using Peterson’s modification of the Lowry method (Sigma
technical bulletin TP0300; Sigma-Aldrich).
In vitro metabolism assay
In vitro substrate-depletion experiments were conducted
using S9 fractions derived from liver tissue as outlined by
Johanning et al. [38], with slight modifications. The S9 fraction
was selected because of its ease of preparation and because it
contains both phase I and phase II metabolizing enzymes. For
compounds exhibiting measurable rates of clearance, prelimi-
nary experiments were run to assess the kinetics of depletion.
The goal of such studies was to establish conditions (protein
concentration, substrate concentration, incubation time) result-
ing in log-linear elimination.
Samples of S9 protein were preincubated with 25 mg/mL
alamethicin on ice for 15 min in 1-mL glass tubes. Cofactors
NADPH, UDPGA, adenosine 30 -phosphate 5-phosphosulfate,
K.A. Connors et al.
2C8 2C9 2C19 2D6 E21 3A4
þ þ þþþþþ
þ þþþþþ
þþþþþ þ
þþþþþ þ
þþþþþ þ þ
þþþþþ
þþþþþ
þ þ þ þ
þ þ þ þ þ
þ þþþþþ
þþþþþ þ þþþþþ þ þ
þ þþþþþ þ
and GSH (2 mM, 2 mM, 0.1 mM, and 5 mM final concen-
trations, respectively) dissolved in 100 mM potassium phos-
phate buffer (pH 7.8) were added and allowed to incubate with
S9 samples for 10 min in a shaking 11 8C water bath. Reactions
were initiated by adding 2 mL of test compound dissolved in
either methanol or acetonitrile. The resulting final concentration
of substrate was 1 mM, except for methylphenidate (0.5 mM),
propranolol (0.5 mM), paroxetine (0.5 mM), and pyrene
(0.38 mM). The final reaction volume was 200 mL, and the
final S9 concentration was 1 mg protein mL1, except for
diltiazem (2 mg mL1) and carbamazepine (2 mg mL1).
Paroxetine and diphenhydramine assays were performed with
S9 pool A; all other assays were conducted using S9 pool B.
For quality control, matrix blanks and heat-inactivated S9
controls were run with each assay. Reactions were terminated at
regular time intervals by adding 595 mL ice-cold acetonitrile,
followed by thorough vortexing. Each time point was collected
in duplicate. All samples were spiked with 5 mL of 25-ppm
isotopically labeled internal standard, vortexed, and centrifuged
at 3000 g at 4 8C for 6 min. Supernatants were collected via
pipette and analyzed by liquid chromatography–tandem mass
spectrometry (LC/MS-MS) or high-performance liquid chroma-
tography (HPLC) according to methods described in the
Instrumental analysis section, below.
Instrumental analysis
Pharmaceuticals were analyzed by LC/MS-MS on a Varian
model 410 autosampler, a ProStar model 212 binary pumping
system, and a model 1200L triple quadrupole mass analyzer.
Most aspects of LC/MS-MS analyses were based on methods
previously reported by our group [5,39]. In those studies, gradient
elution was utilized to monitor all target analytes in a single
chromatographic run. In the present study, only a single
compound needed to be analyzed in each sample. Accordingly,
an isocratic mobile-phase condition that resulted in compound
elution between 2 min and 10 min was identified for each
analyte. This enabled salts and other highly polar sample
constituents to be diverted from the MS during the first 2 min of
each run and reduced analysis time. Chromatography was
performed

using a 15 cm  2.1 mm Extend-C18 column (5mm,
80 A; Agilent Technologies) connectedto a 12.5 mm  2.1 mm
Extend-C18 guard cartridge (5 mm, 80 A; Agilent Technologies).
Analyte-dependent mobile-phase conditions, MS parameters,
Comparative pharmaceutical metabolism by rainbow trout Environ Toxicol Chem 32, 2013 1813

and method detection limits are available in Supplemental Data, Table 2. Rainbow trout liver S9 characterization parameters
Table S1. A minimum of 6 standards, ranging in concentration Pool A Pool B
from just below each analyte’s method detection limit to (mean  SD, (mean  SD,
500 ng mL1, were used to construct linear calibration curves (r2 n ¼ 3) n ¼ 3)
 0.998) for each analyte. Instrument calibration was monitored
over time via analysis of continuing calibration verification Protein recovery (mg/g liver) 26.3  0.6 26.0  0.0
AHH (pmol/min/mg protein) 48.5  1.2 45.0  1.1
samples with an acceptability criterion of  20%. EROD (pmol/min/mg protein) 11.3  1.7 10.6  1.7
Pyrene was analyzed by HPLC on a Beckman 126 HPLC UGT (pmol/min/mg protein) 1075  197.5 1032  47.1
system (Beckman Instruments) with a multiwavelength fluores- GST (nmol/min/mg protein) 507  26 559  13
cence detector (Waters). Chromatography was performed using a
SD ¼ standard deviation; AHH ¼ aryl hydrocarbon hydroxylase; EROD
reverse-phase Hypersil Green polyaromatic hydrocarbon column ¼ ethoxyresorufin O-dealkylation; UGT ¼ uridine diphosphate-glucurono-
(5 mm, 100  2.1 mm; Thermo Fisher Scientific). Solvent A syltransferase; GST ¼ glutathione S-transferase.
consisted of 90% water (Millipore) and 10% acetonitrile. Solvent
B was made of 10% water and 90% acetonitrile. The solvents
were run isocratically at a flow rate of 0.5 mL/min. Proportions of
solvents A (15%) and B (85%) were chosen to optimize run time norethindrone, and propranolol by trout and catfish [23,24] as
and peak symmetry. Identification and quantification were based well as fluoxetine biotransformation by rainbow trout, goldfish,
on reproducible retention times of standards and samples. zebrafish, and killifish [25]. Collectively, these observations
Quantification was performed at excitation and emission wave- show that fish are capable of metabolizing several widely
lengths of 237 nm and 385 nm, respectively. prescribed drugs and provide some initial information related to
the functional conservation of this activity in fish and humans.
Data analysis In the present study, we employed a more targeted approach
Measured concentrations of test compounds were log- to investigate the comparative biotransformation of pharma-
transformed and plotted against reaction time. In each case, ceuticals in trout and humans. Twelve compounds were chosen
there was no apparent loss of compound from heat-denatured based on their extensive hepatic metabolism and CYP-isoform
controls. Data for active and heat-denatured samples (n ¼ 2 per selectivity in humans (Table 1). Substrate-depletion experiments
time point) were evaluated using linear regression. Slopes from were then conducted to determine whether trout possess
each regression were compared for significant differences comparable “CYP-like” activity (CYP3A4, 2D6, etc.). Impor-
(p < 0.05) using a Student’s t test [40]. If a significant tantly, the resulting data do not indicate whether this expressed
difference was observed, the depletion rate constant (k; per activity is performed by the same or similar enzymes. Instead,
hour) was calculated from the slope of the line according to the value of this information depends on the existence of
functional patterns that can be used to predict reactions in 1
k ¼ 2:3  Slope species from activities measured in a second. It is also important
to note that all S9 assays in the present study were performed at
Rate constants were divided by the S9 protein concentration to the physiologically relevant temperature of 11 8C in order to
calculate intrinsic hepatic clearance and extrapolated to describe describe the baseline ability of uninduced rainbow trout to
intrinsic hepatic clearance per gram of liver (ClIN VITRO, INT, biotransform selected pharmaceuticals in an environmental
milliliters per hour per gram of liver tissue) [41]. exposure.
The activity of trout S9 fractions was initially characterized
using well-known substrates for CYP1A, UGT, and glutathione
RESULTS AND DISCUSSION
S-transferase (Table 2). As an additional confirmation of S9
Determining the potential for a chemical to bioaccumulate activity, we performed a set of substrate-depletion experiments
within aquatic ecosystems represents an important component of with pyrene, a well-characterized polycyclic aromatic hydrocar-
environmental risk assessments. Due to the cost and labor bon (Figure 1). The observed intrinsic clearance rate for pyrene
intensity of performing in vivo bioaccumulation studies,
bioaccumulation is often estimated using modeling ap-
proaches [42,43]. However, such estimates are inherently 0.0
log pyrene concentration (uM)

affected if an organism possesses the ability to biotransform

or metabolize a chemical. In fact, a recent kinetic modeling -0.5
exercise demonstrated the ability for metabolic rates to alter fish
bioaccumulation [32]. Even low levels of microsomal intrinsic -1.0
clearance can alter bioaccumulation for compounds with high
octanol–water partition coefficient (log KOW >5) [32,33]. Thus, -1.5
incorporating in vitro metabolic transformation information,
such as S9 substrate-depletion assays, promises to improve the -2.0
accuracy of bioaccumulation models employed during environ-
mental risk assessments [22]. In the present study, S9 substrate- -2.5
depletion methods were employed to derive novel in vitro
metabolism information that can inform comparative bioaccu- -3.0
mulation predictions during screen-leveling environmental 0 10 20 30
assessments of pharmaceuticals. Time (min)
Three recent studies have applied a substrate-depletion Figure 1. Biotransformation of pyrene by trout liver S9 fraction. Closed
approach to investigate pharmaceutical metabolism in fish. circles denote heat-inactive S9; open circles denote active S9 (n ¼2 at each
These efforts demonstrated the biotransformation of ibuprofen, time point).
1814 Environ Toxicol Chem 32, 2013
(472 mL/h/g liver; Table 3) was in good agreement with
previously determined values (P. Fitzsimmons, unpublished
observations).
Liver S9 fractions from trout exhibited no measurable activity
toward the prototypical human CYP3A4 substrates diltiazem
and carbamazepine (Figure 2). Some CYP3A-like isoforms have
been identified in several fish species, including trout [44] and
zebrafish [31]. Moreover, several studies have described the
metabolism by fish of mammalian CYP3A substrates, including
testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluor-
omethylcoumarin [19,45,46]. In a recent study by Alderton
et al. [47], larval zebrafish were shown to hydroxylate
testosterone, although the predominant metabolism pathway
appeared to be glucuronidation. Metabolic clearance of
nortethindrone, a CYP3A4 substrate in humans, was described
in both trout and catfish [23]. The results of the present study
suggest that there are differences in CYP3A substrate specificity
between trout and mammals that can lead to an apparent absence
of CYP3A4-like activity in trout, depending on the compound of
interest. Alternatively, trout and other fish species may
metabolize some, but not all, mammalian CYP3A substrates
utilizing enzymes that belong to a different CYP family (e.g.,
CYP1A).
Trout S9 fractions also exhibited a lack of activity toward the
prototypical human CYP2C9 substrates celecoxib, sulfameth-
oxazole, and warfarin (Figure 2). Metabolism of ibuprofen,
which occurs through CYP2C9 and UGT enzymes in humans,
was previously described in trout and catfish liver and gill S9
fractions [23,24]. Both phase I and phase II ibuprofen
metabolites were observed in liver S9 studies [24]. However,
experiments with selective inducers and inhibitors of CYP
activity indicated that this metabolism occurs through a CYP1A
homolog [24]. The results of the present study, combined with
those of previous efforts, suggest that CYP2C9-like activity is
low or absent in trout, except when metabolism is catalyzed by a
non–CYP2 family enzyme.
In the present study, methylphenidate, a typical human
CYP2D6 substrate, displayed a slow trend toward depletion;
however, the slope of the log-linear depletion curve was not
significantly different (p > 0.05) from that of heat-inactivated
controls (Figure 2). Also, no significant depletion was observed
for the human CYP2D6 substrate paroxetine (Figure 2). By
Table 3. In vitro intrinsic clearance (CLIN VITRO, INT) rate of pharmaceuticals by rainbow trout liver S9 fractions and human hepatocytes and microsomes
Trout S9 CLIN VITRO, INT
Substrate (mL/h/g liver)
Major CYP3A4 substrates
Diltiazem NM
Carbamazepine NM
Major human CYP2C9 substrates
Celecoxib NM
Sulfamethoxazole NM
Warfarin NM
Major human CYP2D6 substrates
Methylphenidate NM
Paroxetine NM
General human CYP substrates
Acetaminophen NM
Diclofenac 9.5
Diphenhydramine NM
Fluoxetine NM
Propranolol 137
Pyrene 472
CYP ¼ cytochrome P450; NM ¼ no metabolism observed; LOQ ¼ limit of quantitation.
K.A. Connors et al.
contrast, dextromethorphan, a typical human CYP2D6 substrate,
was shown previously to be metabolized by larval zebrafish [47].
The CYP2 family of enzymes differs widely among species,
and several genomic studies have described the diversity of
CYP2 isoforms in fish [29,31,48]. Limited homology between
human and fish CYP2 isoforms exists [31]. Presently, however,
only CYP2R1 and CYP2U1 have been described as con-
served [31,48]. Furthermore, CYP2 catalytic activity in fish has
been reported in some [45,49], but not all [46,50], studies. Given
these inconsistencies, it cannot be presumed that fish possess
similar CYP2 biotransformation capabilities as mammals.
Several drugs considered to be broad CYP enzyme substrates
(acetaminophen, diphenhydramine, fluoxetine) also displayed
no significant depletion by rainbow trout S9 fractions (Figure 2).
In a recent study, Smith et al. [25] described fluoxetine
metabolism by liver microsomes from several fish species.
Due to high intraspecific variability, differences among species
could not be demonstrated. Generally, however, fluoxetine
metabolism by trout microsomes was slow or undetectable
unless the animals were induced by preexposure to
carbamazepine [25]. Additionally, the predominant fluoxetine
biotransformation product in mammals, norfluoxetine, was often
not detected. These findings are thus consistent with our results
and highlight the potential pitfalls of presuming the presence of
common metabolic pathways across species. It is important to
note that the Smith et al. [25] experiments were conducted at
25 8C despite several study fish species having been reared and
held at lower temperatures. Though the authors described fish
microsomes as appearing to be not “as sensitive to temperature
differences as mammalian microsomal proteins [25], it is
possible that differences in experimental temperature between
Smith et al.’s work and the present studies may have influenced
CYP activity.
Contrary to observations for acetaminophen, diphenhydra-
mine, and fluoxetine, significant (p < 0.05) metabolism was
observed for diclofenac and propranolol (Figure 2). Both of these
pharmaceuticals were shown previously to be metabolized by
fish. Alderton et al. [47] observed diclofenac metabolism in
larval zebrafish resulting in hydroxylated metabolites. Addition-
ally, phase I and phase II metabolites of diclofenac have been
described in biotransformation experiments with juvenile
rainbow trout [51,52]. In the present study, diclofenac displayed
Human hepatocyte Human microsome
CLIN (mL/h/g liver)
VITRO, INT CLIN (mL/h/g liver)
VITRO, INT References
48.96, 72, 64.8, 93.6, 93.6 93, 117, <21 [60–63]
NM, 3.312, 14.4 1.47 [60,61,64,65]
<LOQ 6.6, <21 [62,63]
10.08 150 [64,65]
NM, 7.2 24.3, 10.8 [63–65]
115.2, 316.8, 338.4 534, 426, 144 [60,62,64,65]
5.976, 14.4, 43.2 28.8, <21, 23.4 [60,62–65]
7.2 [60]
38.16, 56.16, 72, 108, 136.8 66, 39 [60,63–65]
Comparative pharmaceutical metabolism by rainbow trout Environ Toxicol Chem 32, 2013 1815

0.2 0.2 0.2

0.0 0.0 0.0
-0.2 -0.2
-0.2
-0.4 -0.4
-0.6 -0.6 -0.4
-0.8 -0.8 -0.6
-1.0 -1.0
-0.8
-1.2 -1.2
-1.0
-1.4
A -1.4
B C
-1.6 -1.6 -1.2
0 50 100 150 0 50 100 150 0 50 100 150
0.2 0.2 0.2
0.0 0.0 0.0
-0.2 -0.2 -0.2
log pharmaceutical concentration (uM)

-0.4 -0.4 -0.4

-0.6 -0.6 -0.6
-0.8 -0.8 -0.8
-1.0 -1.0 -1.0
D E F
-1.2 -1.2 -1.2
0 50 100 150 0 50 100 150 0 50 100 150
0.2 0.2 0.2
0.0 0.0 0.0
-0.2 -0.2 -0.2
-0.4 -0.4 -0.4
-0.6 -0.6 -0.6
-0.8 -0.8 -0.8
-1.0 -1.0 -1.0
G H I
-1.2 -1.2 -1.2
0 50 100 150 0 50 100 150 0 50 100 150
0.2 0.2 0.2
0.0 0.0 0.0
-0.2
-0.2 -0.2
-0.4
-0.4 -0.4 -0.6
-0.6 -0.6 -0.8
-1.0
-0.8 -0.8
-1.2
-1.0 -1.0
J K -1.4
L
-1.2 -1.2 -1.6
0 50 100 150 0 50 100 150 0 20 40 60

Time (min)
Figure 2. Biotransformation of select pharmaceuticals by trout liver S9 fraction. Major human CYP3A4 substrates (A) diltiazem and (B) carbamazepine; major
human CYP2C9 substrates (C) celecoxib, (D) sulfamethoxazole, and (E) warfarin; major human CYP2D6 substrates (F) methylphenidate and (G) paroxetine; and
general human CYP substrates (H) acetaminophen, (I) diclofenac, (J) diphenhydramine, (K) fluoxetine, and (L) propranolol. Closed circles denote heat-inactive
S9; open circles denote active S9 (n ¼ 2 at each time point).

a modest rate of intrinsic clearance (9.5 mL/h/g liver; Table 3).
Because liver S9 fractions contain both phase I and II
metabolizing enzymes, it is not possible to attribute the observed
diclofenac depletion to any specific enzymatic processes.
Propranolol is a substrate for CYP1A2 and CYP2D6 in
humans (Table 1). Given the demonstrated CYP1A-like activity
of trout S9 fractions (Table 2), it is not surprising that we
observed a comparatively large intrinsic clearance rate for this
compound. Gomez et al. [23] employed a substrate-depletion
approach to measure propranolol metabolism in trout and
catfish, gill and liver S9 fractions. The intrinsic clearance rate for
trout liver determined by these authors was 0.3 mL/hr/mg
protein to 0.5 mL/hr/mg protein (due to a unit conversion error,
the value of 180 mL/hr/mg protein given in Figure 2 and Table 3
of Gomez et al. [23] was incorrect; D.B. Huggett, University of
North Texas, Denton, TX, USA, personal communication). This
rate is considerably lower than that measured in the present study
(2.74 mL/h/mg protein or 137 mL/h/g liver; Table 3). Differ-
ences in intrinsic clearance rate may result from the use of
different trout subspecies, S9 fraction protein concentrations,
and starting substrate concentrations. For comparison, Gomez
et al. [23] measured propranolol depletion using a starting
substrate concentration of 10 mM, which is 20 times higher than
levels used in the present study.
During the drug-development process, intrinsic clearance
rates are often measured using microsomes and hepatocytes
obtained from human livers. In general, microsomes are used to
characterize phase I metabolism while hepatocytes possess both
phase I and phase II metabolic activity. A literature review was
performed to obtain human clearance rates for drugs examined in
the present effort, measured using a substrate-depletion
approach (Table 3). To provide a basis for comparing these
values to our calculated trout clearance rates, we converted all
literature values to milliliters per hour per gram of liver using the
extrapolation factors 120  106 hepatocyte cells/g liver and
50 mg microsomal protein/g liver [41,53].
1816 Environ Toxicol Chem 32, 2013
The compounds in Table 3 possessing the highest rates of
clearance in humans are diltiazem, diclofenac, and propranolol.
Of these, only diclofenac and propranolol exhibited measurable
metabolism in trout. As indicated previously, both compounds
are substrates for more than 1 human CYP, including CYP1A2.
In contrast, diltiazem is primarily a substrate for human CYP3A4.
In the present study, 10 common pharmaceuticals demon-
strated no significant, measurable metabolism in naive rainbow
trout liver S9 fractions. The lack of depletion observed in our
studies suggests that these pharmaceuticals may be accumulated
by rainbow trout. Furthermore, given the importance of
CYP3A4, 2C9, and 2D6 for metabolism of pharmaceuticals in
humans, the low or nonexistent levels of comparable activity in
trout liver S9 suggest that fish are likely to accumulate substrates
for these enzymes, provided that they possess other attributes
(e.g., hydrophobic character) required to elicit this behavior.
Though the present study focused on the enzymatic activity of
trout liver S9, fish gills also may be capable of metabolizing
pharmaceuticals [23,24]. Clearly, the possibility for tissue-
specific expression of CYP isozymes in fish warrants additional
investigation. Furthermore, the results reported in the present
study describe the baseline ability of uninduced rainbow trout to
biotransform selected pharmaceuticals. Additional in vivo
metabolism studies are necessary to understand comparative
pharmaceutical metabolism and clearance by fish.
Several recent studies have focused on quantifying pharma-
ceutical residue concentrations in fish tissues in both field studies
and laboratory exposures. Interestingly, many of the pharma-
ceuticals that displayed no significant metabolism in this study
have been detected in fish collected from the field. For example,
following the first report that fluoxetine, sertraline, and their
metabolites accumulate in fish liver, brain, and muscle
tissues [5], fluoxetine and other selective serotonin reuptake
inhibitors including paroxetine have been reported to accumulate
at nanogram per gram levels in fish from rivers in Canada [54],
the United States [39], and Sweden [55]. In the US
Environmental Protection Agency’s national pilot study of
pharmaceuticals and personal care products in fish tissue,
diphenhydramine, diltiazem, and carbamazepine were detected
in fish muscle tissue at concentrations ranging from 0.13 ng/g to
3.1 ng/g [5]. Diltiazem and carbamazepine have been observed
to bioaccumulate in fish from effluent-dominated rivers of the
United States [4,39] but not in Germany [56]. In a laboratory
study, waterborne exposures of 1 mg/L to 500 mg/L diclofenac
resulted in measurable concentrations in the gill, kidney, and
liver of rainbow trout [57]. Diclofenac has also been quantified in
the plasma of rainbow trout exposed to treated sewage
effluents [55,58]. Collectively, such reports, together with the
results of the present study, highlight the need to better
understand pharmaceutical metabolism in fish models and its
impact on bioaccumulation and toxicity in biomedical and
ecological applications. This suggestion is consistent with the
findings of a recent international workgroup that identified
pharmaceutical bioaccumulation as one of the top research needs
to understand the risks of pharmaceuticals and personal care
products in the environment [10].
SUPPLEMENTAL DATA
Table S1. (105 KB PDF).
Acknowledgment—This research was supported by a Glasscock Fund for
Excellence in Environmental Science grant to K.A. Connors and a Texas Sea
Grant to B.W. Brooks and C.K. Chambliss. The Center for Reservoir and
K.A. Connors et al.
Aquatic Systems Research, the Institute of Biomedical Studies, and the
Department of Environmental Science at Baylor University provided
additional support. We thank J. Berninger for useful discussions.
REFERENCES
1. Monterio SC, Boxall ABA. 2010. Occurrence and fate of human
pharmaceuticals in the environment. Rev Environ Contam Toxicol
202:53–154.
2. Larsson DGJ, de Pedro C, Paxeus N. 2007. Effluent from drug
manufactures contains extremely high levels of pharmaceuticals. J
Hazard Mater 148:751–755.
3. Brooks BW, Riley TM, Taylor RD. 2006. Water quality of effluent-
dominated ecosystems: Ecotoxicological, hydrological, and manage-
ment considerations. Hydrobiologia 556:365–379.
4. Ramirez AJ, Mottaleb MA, Brooks BW, Chambliss CK. 2007. Analysis
of pharmaceuticals in fish using liquid chromatography–tandem mass
spectrometry. Anal Chem 79:3155–3163.
5. Ramirez AJ, Brain RA, Usenko S, Mottaleb MA, O’Donnell JG, Stahl
LL, Wathen JB, Snyder BD, Pitt JL, Perez-Hurtado P, Dobbins LL,
Brooks BW, Chambliss CK. 2009. Occurrence of pharmaceuticals and
personal care products in fish: Results of a national pilot study in the
United States. Environ Toxicol Chem 28:2587–2597.
6. Brooks BW, Chambliss CK, Stanley JK, Ramirez A, Banks KE, Johnson
RD, Russell JL. 2005. Determination of select antidepressants in fish
from an effluent-dominated stream. Environ Toxicol Chem 24:464–469.
7. Daughton CG, Brooks BW. 2011. Active pharmaceutical ingredients
and aquatic organisms. In Bayer WN, Meador JP, eds, Environmental
Contaminants in Biota: Interpreting Tissue Concentrations, 2nd ed.
Taylor and Francis, Boca Raton, FL, USA, pp 287–347.
8. Brodin T, Fick J, Jonsson M, Klaminder J. 2013. Dilute concentrations of
a psychiatric drug alter behavior of fish from natural populations. Science
339:814–815.
9. Brooks BW, Huggett D, Boxall A. 2009. Pharmaceuticals and personal
care products in the environment: Research needs for the next decade.
Environ Toxicol Chem 28:2469–2472.
10. Boxall ABA, Rudd M, Brooks BW, Caldwell D, Choi K, Hickmann S,
Innes E, Ostapyk K, Staveley J, Verslycke T, Ankley GT, Beazley K,
Belanger S, Berninger JP, Carriquiriborde P, Coors A, DeLeo P, Dyer S,
Ericson J, Gagne F, Giesy JP, Gouin T, Hallstrom L, Karlsson M,
Larsson DGJ, Lazorchak J, Mastrococco F, McLaughlin A, McMaster
M, Meyerhoff R, Moore R, Parrott J, Snape J, Murray-Smith R, Servos
M, Sibley PK, Straub JO, Szabo N, Tetrault G, Topp E, Trudeau VL, van
der Kraak G. 2012. Pharmaceuticals and personal care products in the
environment: What are the big questions? Environ Health Perspect
120:1221–1229.
11. Brooks BW, Foran CM, Richards SM, Weston J, Turner PK, Stanley JK,
Solomon KR, Slattery M, La Point TW. 2003. Aquatic ecotoxicology of
fluoxetine. Toxicol Lett 142:169–183.
12. Huggett DB, Cook JC, Ericson JF, Williams RT. 2003. A theoretical
model for utilizing mammalian pharmacology and safety data to
prioritize potential impacts of human pharmaceuticals to fish. Hum Ecol
Risk Assess 9:1789–1799.
13. Ankley GT, Brooks BW, Huggett DB, Sumpter JP. 2007. Repeating
history: Pharmaceuticals in the environment. Environ Sci Technol
41:8211–8217.
14. Owen SF, Giltrow E, Huggett DB, Hutchinson TH, Saye J, Winter MJ,
Sumpter JP. 2007. Comparative physiology, pharmacology and
toxicology of b-blockers: Mammals versus fish. Aquat Toxicol 82:
145–162.
15. Valenti TW, Gould GG, Berninger JP, Connors KA, Keele NB, Prosser
KN, Brooks BW. 2012. Human therapeutic plasma levels of the selective
serotonin reuptake inhibitor (SSRI) sertraline decrease serotonin
reuptake transporter binding and shelter-seeking behavior in adult
male fathead minnows. Environ Sci Technol 46:2427–2435.
16. Gunnarsson L, Jauhiainen A, Kristiansson E, Nerman O, Larsson DGJ.
2008. Evolutionary conservation of human drug targets in organisms
used for environmental risk assessment. Environ Sci Technol 42:5807–
5813.
17. Berninger JP, Brooks BW. 2010. Leveraging mammalian pharmaceuti-
cal toxicology and pharmacology data to predict chronic fish responses
to pharmaceuticals. Toxicol Lett 193:69–78.
18. Brooks BW, Brain RA, Huggett DB, Ankley GT. 2009. Risk assessment
considerations for veterinary medicines in aquatic ecosystems. In
Henderson KL, Coats JR, eds, Veterinary Pharmaceuticals in the
Environment. ACS Symposium Series 1018. Oxford University Press,
New York, NY, USA pp 205–223.
Comparative pharmaceutical metabolism by rainbow trout
19. Han X, Nabb DL, Yang C, Snajdr SI, Mingoia RT. 2009. Liver
microsomes and S9 from rainbow trout (Oncorhynchus mykiss):
Comparison of basal-level enzyme activities with rat and determination
of xenobiotic intrinsic clearance in support of bioaccumulation
assessment. Environ Toxicol Chem 28:481–488.
20. Nichols JW, Schultz IR, Fitzsimmons PN. 2006. In vitro–in vivo
extrapolation of quantitative hepatic biotransformation data for fish. I. A
review of methods, and strategies for incorporating intrinsic clearance
estimates into chemical kinetic models. Aquat Toxicol 78:74–90.
21. Mingoia RT, Glover KP, Nabb DL, Yang C, Snajdr SI, Han X. 2010.
Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss):
A validation study to support their application in bioaccumulation
assessment. Environ Sci Technol 44:3052–3058.
22. Nichols JW, Erhardt S, Dyer S, James M, Moore M, Plotzke K, Segner
H, Schultz I, Thomas K, Vasiluk L, Weisbrod A. 2007. Use of in vitro
absorption, distribution, metabolism, and excretion (ADME) data in
bioaccumulation assessments for fish. Human and Ecological Risk
Assessment 13:1164–1191.
23. Gomez CF, Constantine L, Huggett DB. 2010. The influence of gill and
liver metabolism on the predicted bioconcentration of three pharma-
ceuticals in fish. Chemosphere 81:1189–1195.
24. Gomez CF, Constantine L, Moen M, Vaz A, Wang W, Huggett DB.
2011. Ibuprofen metabolism in the liver and gill of rainbow trout,
Oncorhynchus mykiss. Bull Environ Contam Toxicol 86:247–251.
25. Smith EM, Chu S, Paterson G, Metcalfe CD, Wilson JY. 2010. Cross-
species comparison of fluoxetine metabolism with fish liver microsomes.
Chemosphere 79:26–32.
26. Lacy CF, Armstrong LL, Goldman MP, Lance LL. 2007. Drug
Information Handbook: A Comprehensive Resource for All Clinicians
and Healthcare Professionals, 15th ed. Lexi-Comp, Hudson, OH, USA.
27. Lee MD, Ayanoglu E, Gong L. 2006. Drug-induced changes in P450
enzyme expression at the gene expression level: A new dimension to the
analysis of drug–drug interactions. Xenobiotica 36:1013–1080.
28. Brunton L, Chabner B, Knollman B. 2011. Goodman & Gilman’s The
Pharmacological Basis of Therapeutics, 12th ed. McGraw-Hill, New
York, NY, USA.
29. Buhler DR, Wang-Buhler J. 1998. Rainbow trout cytochrome P450s:
Purification, molecular aspects, metabolic activity, induction and role
in environmental monitoring. Comp Biochem Physiol Part C 121:
107–137.
30. Schlenk D, Celander M, Gallagher EP, George S, James M, Kullman
SW, van den Hurk P, Willet K. 2008. Biotransformation in fishes. In Di
Giulio RT, Hinton DE, eds, The Toxicology of Fishes. CRC Press, Boca
Raton, FL, USA, pp 153–234.
31. Goldstone JV, McArthur AG, Kubota A, Zanette J, Parente T, Jonsson
ME, Nelson DR, Stegeman JJ. 2010. Identification and developmental
expression of the full complement of cytochrome P450 genes in
zebrafish. BMC Genomics 11:643.
32. Nichols JW, Fitzsimmons PN, Burkhard LP. 2007. In vitro–in vivo
extrapolation of quantitative hepatic biotransformation data for fish. II.
Modeled effects on chemical bioaccumulation. Environ Toxicol Chem
26:1304–1319.
33. Cowan-Ellsberry CE, Dyer SD, Erhardt S, Bernhard M, Roe AL, Dowty
ME, Weisbrod AV. 2008. Approach for extrapolating in vitro
metabolism data to refine bioconcentration factor estimates. Chemo-
sphere 70:1804–1817.
34. Van Cantfort J, De Graeve J, Gielen JE. 1986. Radioactive assay for aryl
hydrocarbon hydroxylase. Improved method and biological importance.
Biochem Biophys Res Commun 79:505–512.
35. Burke MD, Mayer RT. 1974. Ethoxyresorufin: Direct fluorimetric assay
of a microsomal O-dealkylation which is preferentially inducible by 3-
methylcholanthrene. Drug Metab Dispos 2:583–588.
36. Hanninen O. 1968. On the metabolic regulation in the glucuronic acid
pathway in the rat tissues. Ann Acad Sci Fenn 142:1–96.
37. Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione S-transferases.
The first enzymatic step in mecapturic acid formation. J Biol Chem
249:7130–7139.
38. Johanning K, Hancock G, Escher B, Adekola A, Bernhard M, Cowan-
Ellsberry C, Domoradzki J, Dyer S, Eickhoff C, Embry M, Erhardt S,
Fitzsimmons P, Halder M, Hill J, Holden D, Johnson R, Rutishauser S,
Segner H, Schultz I, Nichols J. 2012. Assessment of metabolic stability
using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction.
Current Protocols in Toxicology 14:10.11–10.28.
39. Du B, Perez-Hurtado P, Brooks BW, Chambliss CK. 2012. Evaluation
of an isotope dilution liquid chromatography tandem mass spectrometry
method for pharmaceuticals in fish. J Chromatogr A 1253:177–183.
40. Zar JH. 1984. Biostatistical Analysis, 2nd ed. Prentice Hall, Englewood
Cliffs, NJ, USA.
Environ Toxicol Chem 32, 2013 1817
41. Riley RJ, McGinnity DF, Austin RP. 2005. A unified model for
predicting human hepatic, metabolic clearance from in vitro intrinsic
clearance data in hepatocytes and microsomes. Drug Metab Dispos
33:1304–1311.
42. Weisbrod AV, Burkhard LP, Arnot JA, Mekenyan O, Howard PH,
Russom C, Boethling R, Sakuratani Y, Traas T, Bridges T, Lutz C,
Bonnell M, Woodburn K, Pakerton T. 2007. Workgroup report: Review
of fish bioaccumulation databases used to identify persistent, bio-
accumulative, toxic substances. Environ Health Perspect 115:255–261.
43. Veith GD, DeFoe DL, Bergstedt BV. 1979. Measuring and estimating
the bioconcentration factor of chemicals in fish. J Fish Res Board Can
36:1040–1048.
44. Celander M, Buhler DR, Forlin L, Goksoyr A, Miranda CL, Woodin BR,
Stegeman JJ. 1996. Immunochemical relationships of cytochrome
P4503A-like proteins in teleost fish. Fish Physiol Biochem 15:323–332.
45. Nabb DL, Mingoia RT, Yang C, Han X. 2006. Comparison of basal level
metabolic enzyme activities of freshly isolated hepatocytes from
rainbow trout (Oncorhynchus mykiss) and rat. Aquat Toxicol 80:52–59.
46. Smith EM, Wilson JY. 2010. Assessment of cytochrome P450
fluorometric substrates with rainbow trout and killifish exposed to
dexamethasone, pregnenolone-16alpha-carbonitrile, rifampicin and
beta-naphthoflavone. Aquat Toxicol 97:324–333.
47. Alderton W, Berghmans S, Butler P, Chassaing H, Fleming A, Golder Z,
Richards F, Gardner I. 2010. Accumulation and metabolism of drugs and
CYP probe substrates in zebrafish larvae. Xenobiotica 40:547–557.
48. Nelson DR. 2003. Comparison of P450s from human and fugu: 420
million years of vertebrate P450 evolution. Arch Biochem Biophys
409:18–24.
49. Matsuo AYO, Gallagher EP, Trute M, Stapleton PL, Levado R, Schlenk
D. 2008. Characterization of phase I biotransformation enzymes in coho
salmon (Oncorhynchus kisutch). Comp Biochem Physiol Part C 147:78–
84.
50. Gonzalez JF, Reimschuessel R, Shaikh B, Kane AS. 2009. Kinetics of
hepatic phase I and II biotransformation reactions in eight finfish species.
Mar Environ Res 67:183–188.
51. Lahti M, Brozinski J, Jylha A, Kronberg L, Oikari A. 2011. Uptake from
water, biotransformation, and biliary excretion of pharmaceuticals by
rainbow trout. Environ Toxicol Chem 30:1403–1411.
52. Mehinto AC, Hill EM, Tyler CR. 2010. Uptake and biological effects of
environmentally relevant concentrations of the nonsteroidal anti-
inflammatory pharmaceutical diclofenac in rainbow trout (Oncorhyn-
chus mykiss). Environ Sci Technol 4:2176–2182.
53. Iwatsubo T, Hirota N, Ooie T, Suzuki H, Shimada N, Chiba K, Ishizaki
T, Green CE, Tyson CA, Sugiyama Y. 1997. Prediction of in vivo drug
metabolism in the human liver from in vitro metabolism data.
Pharmacol Ther 73:147–171.
54. Chu S, Metcalfe CD. 2007. Analysis of paroxetine, fluoxetine and
norfluoxetine in fish tissues using pressurized liquid extraction, mixed
mode solid phase extraction cleanup and liquid chromatography-tandem
mass spectrometry. J Chromatogr A 1132:112–118.
55. Fick J, Lindberg RH, Parkkonen J, Arvidsson B, Tysklind M, Larsson
DGJ. 2010. Therapeutic levels of levonorgestrel detected in blood
plasma of fish: Results from screening rainbow trout exposed to treated
sewage effluents. Environ Sci Technol 44:2661–2666.
56. Subedi B, Du B, Chambliss CK, Koschorreck J, Rudel H, Quack M,
Brooks BW, Usenko S. 2012. Occurrence of pharmaceuticals and
personal care products in German fish tissue: A national study. Environ
Sci Technol 46:9047–9054.
57. Schwaiger J, Ferling H, Mallow U, Wintermayr H, Negele RD. 2004.
Toxic effects of the non-steriodal anti-inflammatory drug diclofenac.
Part I: Histopathological alterations and bioaccumulation in rainbow
trout. Aquat Toxicol 68:141–150.
58. Brown JN, Paxeus N, Forlin L, Joakim Larsson DG. 2007. Variations in
bioconcentration of human pharmaceuticals from sewage effluents into
fish blood plasma. Environ Toxicol Pharmacol 24:267–274.
59. Akutsu T, Kobayashi K, Sakurada K, Ikegaya H, Furihata T, Chiba K.
2007. Identification of human cytochrome P450 isozymes involved in
diphenhydramine N-demethylation. Drug Metab Dispos 35:72–78.
60. McGinnity DF, Soars MG, Urbanowicz RA, Riley RJ. 2004. Evaluation
of fresh and cryopreserved hepatocytes as in vitro drug metabolism tools
for the prediction of metabolic clearance. Drug Metab Dispos 32:1247–
1253.
61. Chao P, Uss AS, Cheng K. 2010. Use of intrinsic clearance for prediction
of human hepatic clearance. Expert Opinion on Drug Metabolism &
Toxicology 6:189–198.
62. Beaumont K, Gardner I, Chapman K, Hall M, Rowland M. 2011.
Toward an integrated human clearance prediction strategy that
minimizes animal use. J Pharm Sci 100:4518–4535.
1818 Environ Toxicol Chem 32, 2013
63. Sohlenius-Sternbeck A, Afzelius L, Prusis P, Neelssen J, Hoogstraate J,
Johansson J, Floby E, Bengtsson A, Gissberg O, Sternbeck J, Peterson C.
2010. Evaluation of the human prediction of clearance from hepatocyte
and microsome intrinsic clearance for 52 drug compounds. Xenobiotica
40:637–649.
64. Bauman JN, Kelly JM, Tripathy S, Zhao SX, Lam WW, Kalgutkar AS,
Obach RS. 2009. Can in vitro metabolism-dependent covalent binding
data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis
K.A. Connors et al.
using human hepatocytes and liver S-9 fraction. Chem Res Toxicol
22:332–340.
65. Obach RS, Kalgutkar AS, Soglia JR, Zhao SX. 2008. Can in vitro
metabolism-dependent covalent binding data in liver microsomes
distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18
drugs with consideration of intrinsic clearance and daily dose. Chem Res
Toxicol 21:1814–1822.