Aquatic Toxicology 109 (2012) 259–266

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil,

erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes
Emily M. Smith, Fathima I. Iftikar 1 , Sarah Higgins, Anam Irshad, Racquel Jandoc, Matthew Lee,
Joanna Y. Wilson ∗
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1

a r t i c l e i n f o a b s t r a c t

Article history: Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can
Received 9 June 2011 be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been
Received in revised form 22 August 2011 well established in fish yet these studies are important for several reasons. First, such studies will provide
Accepted 25 August 2011
functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnos-
tic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic
Keywords:
environment and adverse effects associated with drug–drug interactions, including CYP inhibition by
Fluoxetine
pharmaceuticals may be of concern. Using liver microsomes from untreated and ␤-naphthoflavone (BNF)-
Ciprofloxacin
Gemfibrozil
treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP
Erythromycin inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed
Cytochrome P450 zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected
Enzyme inhibition substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was
the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s
were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP
inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish.
These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the
patterns seen here are different than what would be expected based on CYP inhibition in mammals.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction inhibition of CYP activity. Yet, pharmaceutical metabolism and CYP

The presence of pharmaceuticals within the aquatic environ-
ment was first reported in the mid-1970s (Hignite and Azarnoff,
1977) and has since grown to be a major area of research in envi-
ronmental toxicology. Discharge from sewage treatment plants
has been identified as the primary source of pharmaceuticals to
the aquatic environment; it has been predicted that levels could
approach kilograms per year in the near future (Lindberg et al.,
2005).
In aquatic environments, pharmaceuticals are found in mixtures
and therefore, drug–drug interactions in aquatic species are of con-
cern. Cytochrome P450 enzymes (CYP) are important mediators
of drug–drug interactions in mammals because CYPs are primarily
responsible for drug metabolism. Drug–drug interactions are clin-
ically important in humans and mediated by both induction and
∗ Corresponding author. Tel.: +1 905 525 9140x20075; fax: +1 905 522 6066.
E-mail address: joanna.wilson@mcmaster.ca (J.Y. Wilson).
1
Present address: School of Biological Sciences, University of Auckland, New
Zealand.
inhibition has not been well studied in fish. Studies of CYP inhibition
by environmentally relevant pharmaceuticals in fish will provide
valuable information for assessing the risk of pharmaceuticals to
aquatic species.
CYPs from families 1 through 4 are well associated with
pharmaceutical metabolism and are highly expressed in mam-
malian hepatocytes. More specifically, CYP3A4, CYP2D6, CYP2C9
and CYP1A2 are responsible for 50%, 25%, 15% and 5% of human drug
metabolism, respectively (Hemeryck and Belpaire, 2002). These
specific CYP isoforms are among the major hepatic CYPs in mam-
mals. A review of the major CYP isoforms, their induction and
inhibition in mammals can be found in Martignoni et al. (2006).
Hepatic expression of multiple CYPs, including isoforms from
families 1–4, has been found in teleost fish (reviewed in Arellano-
Aguilar et al., 2009; Stegeman, 1993). Yet, novel CYP isoforms are
found in fish and assuming homologous function between mam-
malian and actinopterygian CYPs can be problematic (Scornaienchi
et al., 2010a,b; Smith and Wilson, 2010). The evolution of the CYP2
gene family is quite different between mammals and fish; subfam-
ilies are typically distinct across the vertebrates and neither CYP2C
nor CYP2D homologs exist in fish (Kirischian et al., 2011). Thus,

0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2011.08.022
260 E.M. Smith et al. / Aquatic Toxicology 109 (2012) 259–266
≥40% of human drugs must be metabolized by non-orthologous
CYPs in fish.
Laville et al. (2004) provided the first study of CYP inhibition by
pharmaceuticals using fish hepatocytes. Testing a broad range of
drug classes in vitro, 24 h exposure to fibrates and anti-depressants,
in particular, caused potent inhibition of ethoxyresorufin-O-
dealkylase (EROD), an indicator of CYP1 activity. Enrofloxacin
inhibited a number of monooxygenase reactions, both in vivo and
in vitro in sea bass, Dicentrarchus labrax (Vaccaro et al., 2003).
Gemfibrozil and fluoxetine have been reported to cause both inhi-
bition (Laville et al., 2004; Thibaut et al., 2006) and induction
of EROD activity (Thibaut and Porte, 2008). Inhibition of 7-
benzyloxy-4-trifluoromethyl-coumarin (BFC)-O-debenzyloxidase
has been reported by fluoxetine and gemfibrozil in carp liver micro-
somes (Thibaut et al., 2006). Erythromycin inhibited testosterone
6␤-hydroxylation in intestinal microsomes from channel catfish
(James et al., 2005). Collectively, these data suggest that pharma-
ceuticals that inhibit CYP mediated reactions in mammals are likely
teleost CYP inhibitors.
In this study, we assess the inhibition potential of four pharma-
ceuticals (erythromycin, ciprofloxacin, gemfibrozil and fluoxetine)
in rainbow trout liver microsomes and expressed zebrafish CYP1s
using a panel of fluorescent CYP assays. These compounds have
been selected because they are potent inhibitors of the domi-
nant CYPs responsible for pharmaceutical metabolism in mammals.
These selected compounds have been detected in sewage treat-
ment effluent and surface waters (Corcoran et al., 2010; Daughton
and Ternes, 1999; Hirsch et al., 1999; Kolpin et al., 2002; Metcalfe
et al., 2003) and are environmentally relevant pharmaceuticals.
This data will be useful for assessing the potential for drug–drug
interactions in aquatic species and for understanding important
differences between mammalian and fish CYP function.
2. Methods
2.1. Animals
Juvenile rainbow trout (Oncorhynchus mykiss) were purchased
from Humber Springs trout hatchery (Mono Mills, ON, Canada).
Trout were held at 12–15 ◦ C in flow-through dechlorinated city
water and fed floating trout pellets 3 times per week. All fish were
held for a minimum of two weeks before use. Feeding was sus-
pended 24 h before treatment.
2.2. In vivo CYP induction
␤-Naphthoflavone (BNF) was purchased from Sigma–Aldrich
(St. Louis, MO, USA). Trout were exposed to 50 mg kg−1 body
weight of BNF dissolved in corn oil and administered via i.p. injec-
tion, under anesthetic (100 mg L−1 methanesulfonate salt, MS-222).
Injection volume did not exceeding 10 ␮L g−1 total body weight and
control fish were injected with corn oil carrier alone. BNF is a proto-
typical CYP1A inducer and has been shown to induce many CYP
mediated reactions in rainbow trout liver at this dose (Smith and
Wilson, 2010). All fish were sacrificed 48-h after injection and liv-
ers were collected and flash-frozen in liquid nitrogen. Tissues were
stored at −80 ◦ C until use. Liver microsomes were prepared as in
Stegeman et al. (1995) and total protein content was determined
using a bicinchoninic acid (BCA) kit, according to manufacturer’s
protocols (Pierce protein research products, Rockford, IL, USA).
2.3. In vitro inhibition of catalytic activity
Ciprofloxacin, gemfibrozil and fluoxetine were dissolved in
DMSO and erythromycin was dissolved in methanol, to concen-
trated stock solutions (Sigma–Aldrich, St. Louis, MO). Stocks were
diluted in assay buffer just prior to use in assays. CYP inhibition
was determined using fluorescent-based catalytic activity assays
for eight substrates with inhibitor concentrations of 0, 10 and
100 ␮M. The specificity of these substrates is known for mam-
malian CYPs but not for non-mammalian CYPs. The substrates
were chosen based on the catalytic capacity of unexposed and BNF
induced trout liver for these substrates (Smith and Wilson, 2010).
Alkoxyresorufin-O-dealkylase (AROD) activities were determined
using ethoxyresorufin (ER), methoxyresorufin (MR), benzyloxyre-
sorufin (BR) and pentoxyresorufin (PR; Sigma–Aldrich, St. Louis,
MO) according to previously published methods (Hahn et al., 1993).
ER, MR, BR and PR were run at substrate concentrations of 2, 2,
3, and 7 ␮M, respectively. Benzyloxy-4-trifluoromethylcoumarin
(BFC), methoxy-4-trifluoromethylcoumarin (MFC), and benzy-
loxyquinoline (BQ; BD Biosciences, San Jose, CA), were used in
assays optimized for fish (Smith and Wilson, 2010), based on meth-
ods published for mammalian microsomes (Crespi et al., 1997;
Miller et al., 2000; Stresser et al., 2000). Assay conditions and lim-
its of detection (LODs) were as previously published (Smith and
Wilson, 2010) with the exception of BFC and BQ. Assays with BFC
and BQ were modified from Smith and Wilson (2010) with the addi-
tion of bovine serum albumin (1.1 mg mL−1 final concentration) and
lower final substrate concentrations (100 ␮M BQ; 500 ␮M BFC). All
reactions were run using kinetic assays at room temperature for
10 min in black 96-well plates and normalized for total protein con-
tent. Between 4 and 10 replicate liver microsome samples were
assayed for each substrate.
Pharmaceutical inhibition of ER, MR and BR metabolism was
investigated using heterologously expressed CYP1 proteins from
zebrafish (Danio rerio). Expressed CYP1A, 1B1, 1C1 and 1C2
(Scornaienchi et al., 2010a) were used in catalytic assays with
0, 1, 10 and 100 ␮M ciprofloxacin, gemfibrozil and fluoxetine;
compounds with inhibition were assessed for inhibition kinetics.
Reactions with expressed CYP proteins were normalized for total
CYP content, based on CO-reduction assays (Omura and Sato, 1964).
A minimum of triplicate measures were taken for each expressed
protein at each concentration of inhibitor.
2.4. Data and statistical analysis
Inhibition of CYP mediated reactions were determined using
one-way repeated measures (RM) Analysis of Variance (ANOVA) on
log-transformed enzyme activity (pmol mg−1 min−1 ) at 0, 10 and
100 ␮M inhibitor concentrations, except for MFC where one-way
ANOVA was used. If log-transformation did not normalize the data,
Friedman repeated measures Analysis of Variance on ranks was
performed (BFC – both control and BNF fish, BQ – BNF fish only). All
post hoc tests were completed using the Student–Newman–Keuls
method. Statistical tests were completed using Sigmaplot 11.0 (Sys-
tat Software Inc.).
Inhibition of EROD, MROD and BROD activity with expressed
zebrafish CYP1s were assessed using GraphPad Prism version 5.0
for Windows (GraphPad Software Inc., San Diego, CA). These assays
were run with single preparations of expressed proteins (CYP1A,
1B1, 1C1, 1C2) run in triplicate with 1 ␮M, 10 ␮M and 100 ␮M
inhibitor. The inhibitor concentration at which 50% activity is
achieved (IC50) was calculated using the non-linear enzyme inhi-
bition function (model: y = 100/(1 + 10(X-LogIC50) ) for normalized
response) within the GraphPad Software.
3. Results
The inhibition potential for four pharmaceuticals was deter-
mined for CYP mediated reactions in hepatic trout microsomes,
from undosed and BNF-treated animals. CYP mediated metabolism
 E.M. Smith et al. / Aquatic Toxicology 109 (2012) 259–266
Fig. 1. Inhibition of CYP mediated metabolism by fluoxetine. In vitro inhibition of
microsomal reactions with (A) un-treated and (B) BNF-exposed fish. Baseline data
represents the level of activity without fluoxetine added. Those bars marked with
an (*) are statistically different from baseline activities, n = 10.
was measured in fluorescent-based assays with ethoxyresorufin
(ER), methoxyresorufin (MR), benzyloxyresorufin (BR), pen-
toxyresorufin (PR), benzyloxy-4-trifluoromethylcoumarin (BFC),
methoxy-4-trifluoromethylcoumarin (MFC), or benzyloxyquino-
line (BQ). The inhibitory effects of each pharmaceutical for
CYP1 proteins was tested with bacterially expressed zebrafish
CYP 1 proteins (CYP1A, CYP1B1, CYP1C1 or CYP1C2) using the
resorufin-based substrates only.
3.1. Inhibition of CYP activity in trout liver microsomes
All compounds tested showed inhibition of a number of sub-
strates. Of these, fluoxetine showed the broadest inhibition across
substrates (Fig. 1). Fluoxetine (100 ␮M) was capable of inhibiting
the metabolism of all substrates catalyzed by both untreated and
BNF induced trout liver microsomes. The largest inhibitions by flu-
oxetine were seen at 100 ␮M fluoxetine with BFC (untreated liver
microsomes, 84% reduction in activity), MROD and BROD (BNF-
treated microsomes, both 82% inhibition) and MFC (control and BNF
microsomes, 80–81% inhibition; Fig. 1). The lower concentration
of fluoxetine (10 ␮M) caused significant inhibition (35–58% inhi-
bition) of all but MFC and BQ metabolism (Fig. 1). In general, the
BNF-treated microsomes showed similar or higher percent inhi-
bition compared to control microsomes at the same fluoxetine
concentration; BFC and BQ were both exceptions to this pattern.
In reactions with BFC, higher inhibition was seen in untreated fish
at both fluoxetine concentrations. Similarly, BQ metabolism was
261
Fig. 2. EROD inhibition by pharmaceuticals in un-treated and BNF exposed trout.
EROD activity is shown without inhibitor (baseline) and in the presence of
ciprofloxacin (cipro), erythromycin (erythro), and gemfibrozil (gem). Those bars
marked with an (*) are statistically different from baseline activities, n = 10.
inhibited 45% by 100 ␮M fluoxetine in untreated fish assays and
only 14% in BNF-treated microsomes (Fig. 1).
Ciprofloxacin, erythromycin and gemfibrozil showed more
modest inhibition, both in terms of degree of inhibition and number
of substrates inhibited (Figs. 2–4). BFC metabolism was inhibited by
all inhibitors tested, regardless whether untreated or BNF induced
trout microsomes were used to catalyze the reaction (Figs. 1 and 4).
In control fish, EROD was inhibited by 100 ␮M, but not 10 ␮M,
ciprofloxacin and gemfibrozil (40% and 45% reduction in activity,
respectively; Fig. 2). EROD assays with BNF-induced trout were
more susceptible to inhibition; EROD was inhibited with 100 ␮M
ciprofloxacin and erythromycin and both 10 and 100 ␮M gemfi-
brozil (Fig. 2).
Contrary to BCF metabolism, which was widely inhibited, BQ
metabolism was only inhibited by 100 ␮M ciprofloxacin, gem-
fibrozil and fluoxetine (Figs. 1 and 4). These reductions in BQ
metabolism were evident in both control and BNF-fish; in all cases,
assays with microsomes from control fish showed 2–3 times the
level of inhibition compared to BNF-treated fish (Figs. 1 and 4).
No inhibitor showed an affect on BQ metabolism at 10 ␮M
concentration; erythromycin did not inhibit BQ metabolism at
either concentration tested (Fig. 4). BR metabolism, at least
Fig. 3. BFC inhibition by pharmaceuticals in un-treated and BNF exposed trout. BFC
activity is shown without inhibitor (baseline) and in the presence of ciprofloxacin
(cipro), erythromycin (erythro), and gemfibrozil (gem). Those bars marked with an
(*) are statistically different from baseline activities, n = 10.
262 E.M. Smith et al. / Aquatic Toxicology 109 (2012) 259–266
Fig. 4. BQ inhibition by pharmaceuticals in un-treated and BNF exposed trout. BQ
activity is shown without inhibitor (baseline) and in the presence of ciprofloxacin
(cipro), erythromycin (erythro), and gemfibrozil (gem). Those bars marked with an
(*) are statistically different from baseline activities, n = 10.
in liver microsomes from BNF treated trout, was inhibited by
ciprofloxacin, erythromycin and gemfibrozil at both concentra-
tions; MR metabolism in trout liver microsomes from BNF treated
trout, was inhibited at the high concentration only (Supplementary
Table 1).
Surprisingly, ciprofloxacin, gemfibrozil and fluoxetine signifi-
cantly elevated catalytic activity compared to baseline for several
substrates. PROD, in particular, showed significantly higher rates
of metabolism in hepatic microsomes from BNF treated fish with
the addition of all three pharmaceuticals (Supplementary Table
1). Erythromycin increased the metabolism of MFC in assays with
microsomes from BNF treated fish (Supplementary Table 1).
3.2. Inhibition of AROD activity in expressed CYP1s
Three substrates (ER, MR and BR) were selected to test the
inhibition potential of fluoxetine, gemfibrozil, ciprofloxacin and
Fig. 5. Inhibition of EROD activity mediated by heterologously expressed zebrafish CYP1s. Data are shown for (A) CYP1A, (B) CYP1B1, (C) CYP1C1, and (D) CYP1C2 as log
[Inhibitor, ␮M] versus % Activity. Pharmaceutical inhibitors were ciprofloxacin (CIPRO), erythromycin (ERYTH), fluoxetine (FLX), and gemfibrozil (GEM).
erythromycin for heterologously expressed zebrafish CYP1A, 1B1,
1C1 and 1C2; these substrates were metabolized well by zebrafish
CYP1s (Scornaienchi et al., 2010a,b). Contrary to inhibition of
trout liver microsomes, erythromycin showed potent inhibition
of expressed CYP1s (Figs. 5–7). Erythromycin (100 ␮M) inhib-
ited CYP 1A, 1B1, 1C1 and 1C2-mediated EROD by 75%, 79%, 57%
and 41%, respectively (Fig. 5). CYP1A-mediated EROD activity was
inhibited by all compounds except gemfibrozil (Fig. 5a), although
ciprofloxacin reduced activity by only 10% at the high concentra-
tion. The highest concentration (100 ␮M) of fluoxetine showed
strong inhibition of CYP1A (60% reduction in activity, Fig. 5a).
The addition of ciprofloxacin to CYP1C1 and 1C2-mediated EROD
assays caused a concentration-dependent increase in activity that
was similar for both proteins (13–35% increase; data not shown).
IC50s for ciprofloxacin and gemfibrozil inhibition of EROD activ-
ity were relatively high compared to fluoxetine and erythromycin
(Table 1).
Inhibition of the MROD assay was seen for most inhibitors,
except gemfibrozil, with the CYP1 proteins (Fig. 6a and b). Gem-
fibrozil was only able to inhibit CYP1C1 and 1C2-mediated MROD
at 100 ␮M (Fig. 6c and d). CYP 1C1 and 1C2-mediated MROD
showed amongst the highest levels of inhibition; 94% inhibition
was seen with 100 ␮M erythromycin for both proteins (Fig. 6c and
d). IC50s for erythromycin inhibition of MROD activity were con-
sistently lower than the other inhibitors for all expressed CYP1s
(Table 1).
Inhibition of CYP1-mediated BROD activity was fairly limited.
Only CYP1C1-mediated BROD showed a reduction in activity with
all compounds (Fig. 7c). 100 ␮M erythromycin was the most effec-
tive inhibitor for CYP1A and 1B1-mediated BROD activity (75%
and 95% reduction, respectively); fluoxetine was the most effec-
tive inhibitor for CYP1C1 and 1C2-mediated BROD (42% and 63%
reduction, respectively; Fig. 7). Ciprofloxacin caused increased
metabolism in CYP 1A, 1B1 and 1C2-mediated BROD (data not
shown). Although erythromycin caused inhibition for CYP 1A, 1B1
and 1C1-mediated BROD (Fig. 7), CYP1C2 activity was increased to
over twice the baseline metabolism with erythromycin (1 ␮M and
10 ␮M), the highest activation seen across all substrates tested with
expressed CYP1s (data not shown).
 E.M. Smith et al. / Aquatic Toxicology 109 (2012) 259–266 263

Fig. 6. Inhibition of MROD activity mediated by heterologously expressed zebrafish CYP1s. Data are shown for (A) CYP1A, (B) CYP1B1, (C) CYP1C1, and (D) CYP1C2 as log
[Inhibitor, ␮M] versus % Activity. Pharmaceutical inhibitors were ciprofloxacin (CIPRO), erythromycin (ERYTH), fluoxetine (FLX), and gemfibrozil (GEM).

Fig. 7. Inhibition of BROD activity mediated by heterologously expressed zebrafish CYP1s. Data are shown for (A) CYP1A, (B) CYP1B1, (C) CYP1C1, and (D) CYP1C2 as log
[Inhibitor, ␮M] versus % Activity. Pharmaceutical inhibitors were ciprofloxacin (CIPRO), erythromycin (ERYTH), fluoxetine (FLX), and gemfibrozil (GEM).

Table 1
IC50s (␮M) for inhibition of CYP1 mediated metabolism of resorufin-based substrates. IC50s were calculated through nonlinear enzyme inhibition regression plots. CYP1
proteins were heterologously expressed zebrafish proteins (see Section 2 for details).

EROD MROD BROD

CYP1A CYP1B1 CYP1C1 CYP1C2 CYP1A CYP1B1 CYP1C1 CYP1C2 CYP1A CYP1B1 CYP1C1 CYP1C2

Fluoxetine 75 475 1000 180 115 300 30 140 200 280 125 70
Ciprofloxacin 850 >1000 n/a n/a 900 680 >1000 410 n/a n/a 180 n/a
Erythromycin 40 40 90 145 5 25 15 25 60 50 275 n/a
Gemfibrozil >1000 >2000 n/a n/a >1000 96 350 330 n/a n/a 170 n/a

n/a, reactions do not fit inhibition model and therefore IC50 could not be calculated.
264 E.M. Smith et al. / Aquatic Toxicology 109 (2012) 259–266
4. Discussion
The inhibitors chosen represent environmentally relevant
pharmaceuticals, which have specificity for the major drug metab-
olizing CYPs in human, CYP1A2, CYP2D6, CYP2C9 and CYP3A4.
Ciprofloxacin is a selective inhibitor of CYP1A2 (Fuhr et al., 1992;
Granfors et al., 2004). Gemfibrozil is a modest inhibitor of CYP1A2
in humans (Wen et al., 2001). Fish do not have a CYP1A2 ortholog
and MR, a CYP1A2 substrate in mammals, is catalyzed by several
CYP1s in fish including the single CYP1A protein (Scornaienchi et al.,
2010b; Smith and Wilson, 2010), which metabolizes ER and MR
equally (Gooneratne et al., 1997).
Human CYP3A4 is strongly inhibited by erythromycin (Okudaira
et al., 2007; Yamazaki and Shimada, 1998) and fluoxetine (Jeppesen
et al., 1996). BFC and BQ are CYP3A substrates in mammals and
in most fish studies, such activity is attributed to CYP3A activity
(Hegelund et al., 2004; Kashiwada et al., 2005). The assumption
that these substrates are selectively catalyzed by CYP3A in fish may
be premature; BFC metabolism was higher with CYP1s than with
CYP3A65 in zebrafish (Scornaienchi et al., 2010a,b). Interestingly,
fish CYP3A isoforms are more closely related to CYP3B and CYP3C
genes (Yan and Cai, 2010), suggesting that CYP3A genes are not
orthologous across vertebrates.
Gemfibrozil is a potent inhibitor of CYP2C9 and a modest
inhibitor of CYP1A2 and 2C19 in humans (Wen et al., 2001). Fluox-
etine effectively inhibits human CYP2D6 and 2C19 (Jeppesen et al.,
1996). There are no CYP2C or CYP2D orthologs in fish, although
many CYP2 genes are present in fish genomes (Kirischian et al.,
2011). The actinopterygian CYP(s) responsible for metabolism of
CYP2C9 and CYP2D6 mammalian substrates is unknown.
The inhibition potential for pharmaceuticals was determined for
CYP mediated reactions in hepatic trout microsomes, a widely used
aquatic toxicology model species that was large enough to allow the
analyses on individual fish. CYP mediated metabolism was mea-
sured in fluorescent-based assays with substrates that have been
used previously with trout hepatic microsomes (Smith and Wilson,
2010) such that reasonable catalytic activity was expected for all
substrates and we were determining inhibition for multiple hepatic
CYPs. The inhibitory effects of pharmaceuticals for CYP1 proteins
was tested with bacterially expressed zebrafish CYP 1 proteins
(CYP1A, CYP1B1, CYP1C1 or CYP1C2) using the resorufin-based sub-
strates only; catalytic activity was expected for most CYP1 proteins
with these substrates (Scornaienchi et al., 2010a,b). Collectively,
this data will identify if CYP inhibition by pharmaceuticals is widely
expected, suggest which microsomal reactions are CYP1 mediated,
and determine whether the selectivity of mammalian inhibitors are
conserved in fish.
4.1. Inhibition of EROD and MROD
To our knowledge, no studies have previously determined the
inhibition potential of ciprofloxacin in fish. Ciprofloxacin is a potent
CYP1A2 inhibitor in mammals (Fuhr et al., 1992; Granfors et al.,
2004). In comparison to other inhibitors, ciprofloxacin did not show
preferential inhibition of ER or MR; this was evident in assays
with microsomes and expressed zebrafish CYP1A. In microsomes,
EROD activity was reduced by over 60% by 100 ␮M ciprofloxacin
from both BNF-treated and untreated (control) trout and yet MROD
activity was inhibited by 100 ␮M ciprofloxacin in BNF treated trout
only. Inhibition of a number of CYP-mediated reactions, including
EROD, have been reported for enrofloxacin, the parent compound
of ciprofloxacin (Vaccaro et al., 2003, 2007). In vivo treatment with
enrofloxacin and in vitro exposures caused reduced EROD activity
in sea bass liver microsomes (Vaccaro et al., 2003).
Thibaut et al. (Thibaut and Porte, 2008; Thibaut et al., 2006)
reported inhibition of EROD activity with 1 mM gemfibrozil. In the
present study, lower concentrations of gemfibrozil elicit inhibi-
tion of EROD, with BNF-trout microsomes susceptible at the lowest
concentration (10 ␮M). Yet, EROD activity mediated by zebrafish
CYP1s was not hugely affected by gemfibrozil. Likewise, IC50s for
ciprofloxacin inhibition of zebrafish CYP1s, based on EROD and
MROD activity, were in excess of 850 and 410 ␮M, respectively,
in contrast to the large inhibition of EROD activity in rainbow trout
microsomal reactions. The reason for the incongruence between
the data for microsomes and expressed proteins is not clear.
Certainly, species differences may be one plausible reason; the
microsomes were from rainbow trout and the expressed proteins
were from zebrafish.
4.2. Inhibition of BFC and BQ metabolism
All four inhibitors, at both concentrations, were successful at
reducing the metabolism of BFC, in both undosed and BNF-treated
fish. BFC inhibition by gemfibrozil has been previously reported
in carp liver microsomes (Thibaut et al., 2006), although at much
higher concentrations (1 mM) than used in this study. BFC is a
mammalian CYP3A substrate, metabolized selectively by CYP3A4
in humans (Stresser et al., 2000). BQ, another mammalian CYP3A
substrate, was inhibited by the high concentration of ciprofloxacin,
gemfibrozil and fluoxetine, but not erythromycin. Previous data
suggested that different hepatic CYPs were responsible for the
metabolism of BCF and BQ in fish (Scornaienchi et al., 2010b; Smith
and Wilson, 2010); a notion supported here because erythromycin
was not capable of inhibiting the metabolism of both substrates.
These microsomal data provide evidence that erythromycin
inhibits fish CYPs, but to a minimal degree compared to other
tested compounds. The one substrate significantly inhibited at
both concentrations in untreated and BNF treated trout micro-
somes was BFC. In a study with catfish intestinal microsomes,
erythromycin was less potent compared to other inhibitors, like
ketoconazole (James et al., 2005). Erythromycin metabolism in
sea bass hepatic microsomes has been reported in the range
of 1000 pmol min−1 mg−1 protein (Vaccaro et al., 2003, 2007). If
hepatic CYPs in rainbow trout are capable of metabolizing ery-
thromycin, erythromycin may be competitively inhibiting the
metabolism of BCF. The metabolism of erythromycin in microsomal
preparations could significantly reduce erythromycin concentra-
tions in the reaction and explain the significant inhibition of CYP1
proteins, which have no CYP3A, but minimal inhibition of EROD or
MROD activity in microsomes where CYP1 and CYP3A isoforms are
co-expressed.
4.3. Specificity of CYP inhibition
Our data does not support the finding that fish CYPs are
inhibited similarly to mammals. Fluoxetine, a CYP2D6 and 3A4
inhibitor in mammals, had the broadest inhibition, affecting all
substrates tested with rainbow trout microsomes. Fluoxetine has
been reported to inhibit EROD activity in fish hepatocytes and liver
microsomes (Laville et al., 2004; Thibaut et al., 2006). In our study,
fluoxetine significantly inhibited EROD activity in trout micro-
somes and was particularly potent against the zebrafish CYP1A
protein. Yet, strong inhibition was seen with zebrafish CYP1C1-
mediated MROD activity and CYP1C2-mediated BROD activity.
Collectively, this data suggests fluoxetine is an inhibitor of multiple
hepatic CYPs in fish including CYP1s.
Like fluoxetine, the other inhibitors did not have the specificity
expected. Ciprofloxacin is a potent CYP1A2 inhibitor in mam-
mals yet was neither a preferential inhibitor of zebrafish CYP1s
nor a potent inhibitor of EROD or BROD activity in trout micro-
somes. Erythromycin inhibits mammalian CYP3A4 but was capable
of nearly completely abolishing CYP1C mediated MROD activity;
 E.M. Smith et al. / Aquatic Toxicology 109 (2012) 259–266
something no other inhibitor was capable of, even ciprofloxacin
the mammalian CYP1 inhibitor. Gemfibrozil is a potent inhibitor of
mammalian CYP2C9 and a modest inhibitor of CYP1A2, yet it was
most potent in inhibiting BFC metabolism and BROD activity. Col-
lectively, mammalian CYP inhibitors appear to inhibit fish CYPs but
do not share isoform specificity.
4.4. Activation of CYP activity
Surprisingly, several CYP mediated reactions were increased
with the addition of pharmaceuticals. PROD, in particular, showed
significantly higher rates of metabolism in hepatic microsomes
from BNF treated fish with the addition of pharmaceuticals yet all
pharmaceuticals increased at least one CYP mediated reaction. Acti-
vation of CYP reactions has been reported with mammalian CYP3A4
(Ludwig et al., 1999; Niwa et al., 2003) yet, to our knowledge, CYP
activation has not been reported in fish. Until the CYP isoform pri-
marily responsible for hepatic PROD activity is identified, studies
of how pharmaceuticals activate PROD are not possible. There are
several reported mechanisms by which CYP mediated metabolism
can be activated in mammals.
4.5. Implications of CYP inhibition
With the exception of fluoxetine and general BFC metabolism,
the lowest inhibitor concentration, 10 ␮M, did not significantly
inhibit the majority of reactions in un-treated fish. The higher con-
centration chosen (100 ␮M) is likely orders of magnitude higher
than what would be found in the environment. However, many
pharmaceuticals have been found to bioaccumulate in the tissues
of wild fish (Brooks et al., 2005; Nakamura et al., 2008). Additive
and synergistic effects are something that should be considered
particularly because these data suggest broad CYP inhibition by
the tested compounds. The environmental relevance of inhibition
by these compounds is therefore not straightforward. Clearly, our
data suggests that inhibition of CYP mediated reactions is possible
with environmentally relevant pharmaceuticals but the concentra-
tions found in the environment may be too low to cause significant
drug–drug interactions.
Acknowledgements
This work was supported by funding from the Canadian Water
Network, the Ontario Ministry of the Environment Best in Sci-
ence program, and NSERC discovery (grant #328204) program. E.S
was partially supported by funding provided by the Department
of Biology, McMaster University. The authors wish to thank Dr.
Malin Celander and Johanna Gräns for advice on modifying the BCF
assays. We thank Mr. James Harskamp for preparation of the CYP1
expressed proteins used in this study.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.aquatox.2011.08.022.
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