Chemosphere 81 (2010) 1189–1195

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Chemosphere
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The influence of gill and liver metabolism on the predicted bioconcentration

of three pharmaceuticals in fish
C.F. Gomez a, L. Constantine b, D.B. Huggett a,⇑
a
Department of Biological Sciences, University of North Texas, Denton, TX 76208, USA
b
Pfizer Global Research and Development, Groton, CT 06340, USA

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

Article history: The potential for xenobiotic compounds to bioconcentrate is typically expressed through the bioconcen-
Received 29 April 2010 tration factor (BCF), which has gained increased regulatory significance over the past decade. Due to the
Received in revised form 30 August 2010 expense of in vivo bioconcentration studies and the growing regulatory need to assess bioconcentration
Accepted 14 September 2010
potential, BCF is often calculated via single-compartment models, using KOW as the primary input. Recent
Available online 25 October 2010
efforts to refine BCF models have focused on physiological factors, including the ability of the organism to
eliminate the compound through metabolic transformation. This study looks at the ability of in vitro bio-
Keywords:
transformation assays using S9 fractions to provide an indication of metabolic potential. Given the impor-
Bioconcentration
In vitro tests
tance of the fish gill and liver in metabolic transformation, the metabolic loss of ibuprofen, norethindrone
Metabolic biotransformation and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus
Bioconcentration factor punctatus) gill and liver S9 fractions. Metabolic transformation rates (kM) were calculated and integrated
Risk assessment into a refined BCF model. A significant difference was noted between BCF solely based on KOW and BCF
including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted biocon-
centration estimates closer to in vivo values.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Background
The bioconcentration factor (BCF) is a quantitative expression of
a compound’s ability to partition into and remain in an organism
following aqueous exposure, increasing the potential for chronic
toxic effects and/or biomagnification in higher trophic levels.
Bioconcentration often represents a surrogate measure for ‘‘B” in
PBT, a regulatory classification for persistent, bioaccumulative
and toxic substances that has taken on greater importance with
the implementation of the European Union regulation on the Reg-
istration, Evaluation and Authorisation of Chemicals (REACH) and
the Canadian assessment of chemicals on the Domestic Substances
List (DSL). Of the thousands of chemicals currently in commerce,
most lack published data on their bioaccumulation potential. For
example, of the 11 300 organic chemicals under review on the
Canadian DSL, only 4% have publicly published BAF or BCF values
Abbreviations: BCF0, partition based BCF estimate (L kg1); BCFM, partition based
BCF estimate including kM (L kg1); CLi, intrinsic clearance in tissue (L d1 kg1);
CLm, intrinsic clearance rate of parent compound (mL h1 (mg protein)1); CLt,
tissue clearance (L d1 kg1); CO, cardiac output (mL min1 kg1); fu, free fraction
correction term; kM, metabolic transformation rate (d1); KOW, octanol to water
partition coefficient; PT, protein per g tissue (g g1); TF, fraction of blood flow
through tissue; TW, tissue weight (g tissue (kg body weight)1); Vd, volume of
distribution (L kg1).
⇑ Corresponding author. Tel.: +1 940 891 6956.
E-mail address: dbhuggett@unt.edu (D.B. Huggett).
(Arnot and Gobas, 2006). Thus, a method for accurately determin-
ing large quantities of BCF values is necessary. Multi-compartment
BCF models provide such an alternative method.
Historically, models utilize the octanol–water partition coeffi-
cient (KOW) as the primary determinant of BCF. Recent BCF model-
ing efforts have proposed a multi-compartment model based on
the principles of absorption, distribution, metabolism and elimina-
tion (ADME) (Arnot and Gobas, 2004; Cowan-Ellsberry et al., 2008;
Arnot et al., 2009). The model depicted as BCF = (k1 + kU)/
(k2 + kG + kM + kE), includes: uptake via gills (k1), dietary ingestion
(kU), elimination across gills (k2), growth (kG), metabolism (kM)
and egestion (kE) (Gobas, 1993).
By far, a majority of efforts have focused on the role of
metabolism on the process of bioconcentration (Arnot et al.,
2008; Cowan-Ellsberry et al., 2008; Dyer et al., 2009; Han et al.,
2009). Building on concepts developed within the human pharma-
ceutical sector, in vitro fish metabolism information may be able to
predict whole fish clearance, and thus, contribute to a more realis-
tic calculated BCF (Obach, 1999; Mohutsky et al., 2006; Cowan-
Ellsberry et al., 2008).
While human science has focused on ionizable pharmaceuticals,
most environmental work has focused on neutral, lipophilic chem-
icals. This study evaluates the ability of in vitro fish metabolism
data to determine whole fish intrinsic clearance, using ibuprofen
(a weak acid), norethindrone (a neutral compound) and proprano-
lol (a weak base) as the test compounds. At physiological pH,

0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2010.09.043
1190 C.F. Gomez et al. / Chemosphere 81 (2010) 1189–1195
ibuprofen and propranolol reside in their ionizable forms with
log KOW of 1.0 and 0.78, respectively (Cleuvers, 2003; Johnson
et al., 2007). Conversely, norethindrone has a reported log KOW of
2.99 at physiological pH (Saha et al., 2000; Lee et al., 2007). Pre-
dicted BCF of norethindrone based on KOW may be unduly influ-
enced by the neutral state of the compound compared to other
readily metabolized compounds that reside in their ionized form
at neutral pHs.
Test compounds were chosen for their known metabolic poten-
tial as they are readily metabolized in human and mammalian li-
ver. In the human and rat liver, CYP2C9 and CYP2C8 contribute
to the majority of Phase I metabolism of ibuprofen (Jacqz-Aigrain
and Anderson, 2006). Ibuprofen also undergoes glucuronidation
by multiple UGT isoforms (Ritter, 2000). In human liver, norethin-
drone is primarily metabolized by CYP3A4 (Korhonen et al., 2008).
CYP2D6 is thought to catalyze a large portion of propranolol bio-
transformation in humans (Masubuchi et al., 1994).
Test compounds were not only chosen for their metabolic po-
tential, but also for their reported presence in the aquatic environ-
ment at trace levels. All three compounds have been observed in
either wastewater effluent or surface water with ibuprofen noted
in wastewater effluent at up to 4.2 lg L1 (Farre et al., 2001; Öllers
et al., 2001; Ternes, 2001; Andreozzi et al., 2003; Roberts and Tho-
mas, 2006; Fernandez et al., 2007; Nebot et al., 2007; Huggett et al.,
2003). Understanding the ability of aquatic species to metabolize
pharmaceuticals and other emerging contaminants not only aides
in refining BCF prediction, but also in advancing the overall risk
assessment of the compound. Biotransformation of a compound
generally leads to the formation of more polar metabolites that
can be eliminated more rapidly than the parent compound, reduc-
ing the potential for a compound to bioaccumulate in fatty tissue,
but also reducing the general opportunity for the compound to del-
eteriously affect the organism. However, biotransformation may
also increase toxicity through the formation of reactive metabo-
lites. As fish do not contain the same CYP isoforms as mammals,
and are specifically known not to have CYP2C or 2D homologs
(Buhler and Wang-Buhler, 1998), it cannot be assumed that fish
biotransformation will be equivalent to that of mammals. The lack
of direct homologs is particularly noted with regards to the test
compounds ibuprofen and propranolol. Thus, understanding the
metabolism of the test compounds should provide further insight
beyond bioconcentration as to the overall toxicological profile of
the compound in the aquatic environment.
2. Materials and methods
2.1. Materials
All chemicals used in incubation assays were purchased from
Sigma–Aldrich Corp. (St. Louis, MO). Homogenization buffer
(50 mM potassium phosphate, 0.15 M potassium chloride, 0.2 M
sucrose) materials were obtained from Fisher Chemical (Fairlawn,
NJ). The Bradford Protein Assay was procured from Bio-Rad Labora-
tories (Hercules, CA). Uninduced, male Sprague–Dawley rat liver S9
and uninduced, male CD-1 mouse liver S9 were purchased from
Moltox (Boone, NC). (±)-Ibuprofen, d3-ibuprofen, norethindrone
and d6-norethindrone were sourced from Toronto Research Chem-
icals (North York, ON, Canada). (±)-Propranolol was supplied by
TOCRIS Bioscience (Ellisville, MO). d7-propranolol was purchased
from CDN Isotopes Inc. (Pointe-Claire, Quebec, Canada).
2.2. Fish cultures
Gill and liver tissue were harvested from anesthetized (e.g. MS-
222) mature rainbow trout (Oncorhynchus mykiss) and channel cat-
fish (Ictalurus punctatus). Fish were procured from Greers Ferry
National Fish Hatchery (Heber Springs, AR). Fish were maintained
in dechlorinated tap water at the appropriate temperature (16 °C
for rainbow trout and room temperature (25 °C) for channel cat-
fish) in Frigid Units Living Streams under fluorescent lights with a
16 h light/8 h dark photoperiod. Fish were fed ground Purina Trout
Chow. All studies were conducted in accordance with University of
North Texas animal use protocols.
2.3. S9 fractions
Tissues were rinsed thoroughly with a homogenization buffer
and immediately placed on ice (Kelly et al., 2000). Gill, liver
and whole fish wet weights were collected prior to further pro-
cessing. Gill tissue was then composited, minced and mixed with
a homogenization buffer in a 2:1 buffer to tissue ratio (v:w). Liver
tissues were homogenized with a 4:1 buffer to tissue ratio. Tissue
from five to ten fish were homogenized together to reduce inter-
fish variability. Additionally, multiple fish batches were prepared
to evaluate batch variability. Four to ten batches of fish were uti-
lized to evaluate loss of parent (i.e., n = 4–10) within each tissue
type. Tissues were homogenized on ice using an electric homog-
enizer. The homogenate was placed in chilled centrifuge tubes
and centrifuged at 9000g for 20 min at 4 °C. The S9 supernatant
was carefully separated from the pellet via pipette and stored
at 80 °C.
Uninduced, male Sprague–Dawley rat liver S9 and uninduced,
male CD-1 mouse liver S9 were purchased from Moltox (Boone,
NC). Mammalian S9 was solely used in ibuprofen incubations.
2.4. Incubation of S9 fractions
S9 fractions were diluted to 2 mg protein mL1 with 0.01 M
phosphate buffer (pH 7.4). The Bradford Protein Assay was used
to determine the protein content of each sample. A NADPH regen-
eration system containing 7 mM isocitrate and isocitrate dehydro-
genase (0.5 units of activity mL1) was then added to 600 lL S9 in
microcentrifuge tubes. Test compounds were dissolved in ethanol
to create 100 stock solutions. To reduce any reaction interference
from the ethanol, only 6.5 lL was added to the S9 samples to bring
the reaction vessel concentration to 10 lM test compound. Sam-
ples were run in duplicate or triplicate. S9 matrix controls and sol-
vent controls were run with each assay to assure that loss of parent
was not due to binding effects. Samples were placed in a temper-
ature controlled shaker (15 °C for trout, 25 °C for catfish and
37 °C for mammalian) to equilibrate for 10 min. To initiate the
reactions, 6.5 lL of 50 mM NADPH in phosphate buffer was added
to each sample. S9 and solvent controls did not receive NADPH.
Samples were returned to the shaker and incubated for 90–
120 min. Aliquots of 100 lL were removed at the appropriate time
points for each test material. Aliquots were placed in 100 lL cold
methanol and vortexed to stop biological activity. Deuterated
internal standard was then added. Samples were centrifuged at
2500g for 10 min to pellet the denatured protein. The supernatant
was carefully removed via pipette and stored at 4 °C until GC/MS or
LC–MS/MS analysis.
2.5. Instrumental analysis
2.5.1. GC/MS
Ibuprofen samples were dried under a gentle stream of nitro-
gen. The derivatizing agent boron trifluoride (BF3)-methanol and
acetonitrile were added. Samples were incubated at 85 °C for 2 h.
Approximately 200 lL of milli-Q water (MQ) was added and the
derivatized ibuprofen was back extracted into 1:1 hexane:ethyl
acetate. Samples were then dried again under nitrogen gas and
 C.F. Gomez et al. / Chemosphere 81 (2010) 1189–1195 1191

reconstituted to 100 lL with hexane. Samples were analyzed using
an Agilent 6890N Network GC System coupled with an Agilent
5973 inert Mass Selective Detector and an Agilent autosampler
(Agilent Technologies, Inc.). Samples were injected at 260 °C in
pulsed splitless mode onto an Econo-Cap™ EC™-5 (30 m 
0.25 mm  0.25 lm) column. Injection pressure was held constant
at 9.5 psi and with a flow of 18.5 mL min1. Initial temperature of
40 °C was held for 3 min, increased 10 °C min1 until 200 °C, fur-
ther increased 15 °C min1 until 300 °C and then held for 5 min.
Analytes were detected and quantified in selective ion monitoring
(SIM) mode with the quantitative ions 161 m/z for ibuprofen and
164 m/z for d6-ibuprofen. A seven-point calibration curve was used
for quantification.
2.5.2. LC–MS/MS analysis
Norethindrone and propranolol samples were dried under
nitrogen and reconstituted in 100 lL 1:1 methanol:MQ. LC–MS/
MS was carried out using a Waters 2695 Separations Module and
autosampler (Waters). Chromatography was performed on a Sun-
fire C18 (2.1 mm  50 mm, 3.5 lm) column (Waters). Analyte
identification was performed using a Micromass Quattro Ultima
mass spectrometry system with an electrospray interface run in
positive mode (Micromass). The flow rate for all applications was
0.2 mL min1 with an injection volume of 10 lL.
The mobile phase for norethindrone analysis consisted of MQ
with 0.1% formic acid (solvent A) and methanol with 0.1% formic
acid (Solvent B1). Initial mobile phase conditions of 70% solvent
A:30% solvent B1 were held for 1 min and then solvent B1 was in-
creased to 70% over 3 min. Solvent B1 was further increased to 95%
over 30 s and then decreased to 70% over 2 min. Initial mobile
phase conditions were returned over 30 s and held for 3 min. For
propranolol, the mobile phase comprised of solvent A and 2:1 ace-
tonitrile:methanol with 0.1% formic acid (solvent B2). Initial mo-
bile phase of 90% solvent A:10% solvent B2 was held for 30 s.
Solvent B2 linearly increased to 90% over 9.5 min. Initial conditions
were then linearly resumed over the course of 2 min.
Individual tune files were created for each analyte. Norethin-
drone settings consisted of 4.0 kV capillary voltage, 60 V cone volt-
age and 30 eV collision energy. For propranolol, capillary voltage
was 4.0 kV with cone voltage at 45 V and collision energy at
17 eV. Protonated ions were dissociated with argon for norethin-
drone (m/z 298.5 > 108.2), d6-norethindrone (m/z 304.1 > 113.8),
propranolol (m/z 260.1 > 183.4) and d7-propranolol (m/z
267.1 > 190.1) with detection in multiple reaction monitoring
(MRM) mode. Six-point calibration curves of analyte standards
were used for quantification.
2.6. Ethoxyresorufin-O-deethylase (EROD) activity
To ensure S9 fractions were metabolically active, the dealkyla-
tion of ethoxyresorufin to resorufin was measured. S9 fractions
were diluted to 2 mg protein mL1 with 0.01 M phosphate buffer
(pH 7.4). Ethoxyresorufin (5 lM) was added. NADPH (10 lM)
was added to initiate the reaction. Samples were covered in alumi-
num foil and placed in a shaker within a dark incubator (15 °C for
rainbow trout fractions, 25 °C for channel catfish fractions). S9 ma-
trix controls and blanks did not receive NADPH. Aliquots were re-
moved at 0, 20, 40 and 60 min time points and placed in four
volumes of cold methanol in black 96-well plates to stop the reac-
tion. Samples were immediately read for fluorescence (excitation
530 nm, emission 590 nm) on a Synergy 2 Multi-Mode Microplate
Reader (BioTek). A five-point resorufin standard curve (10–
50 lg mL1) was used for quantification. All work was conducted
in low light conditions.
2.7. In vitro metabolic transformation rates of the gill and liver
In vitro metabolic transformation rates of the gill and liver were
determined using the bioconcentration model described by Cow-
an-Ellsberry et al. (2008). The model extrapolates in vitro test data
to determine a whole body metabolic transformation rate using
key fish physiological information (Table 1). This extrapolation is
based on concepts commonly applied in mammalian pharmacoki-
netics (Obach, 1999). To evaluate loss of parent compound, first or-
der kinetics was assumed for each reaction. Remaining parent
compound concentrations were plotted versus time. Data points
were fit to an exponential curve with the following integrated
equation where k is the first order loss constant and t is time.
lmoles lost ¼ intercept  ekt ð1Þ
Intrinsic clearance rate of parent compound (CLm) was deter-
mined from in vitro S9 data per mg protein in the assay vessel. Mul-
tiplication of tissue weight and total protein content determined
intrinsic clearance in tissue (CLi), which was extrapolated to tissue
clearance (CLt) by including cardiac output, fraction of blood flow
through tissue and a free fraction correction term. Division by
the volume of distribution then produced the metabolic transfor-
mation rate (kM). BCF was then calculated, incorporating kM (BCFM)

Table 1
Physiological and physical characteristics used to extrapolate in vitro metabolic transformation within the BCF model. Mean values are reported. Tissue batch specific values were
used in the calculation of individual kM and BCFM. Values were determined during the course of the study or in other unpublished laboratory studies.

Trout gill Trout liver Catfish gill Catfish liver Rat liver Mouse liver
Body weight (kg) 0.204 0.204 0.157 0.157 0.289a 0.028a
Tissue weight (g (g bw)1) 12.2 15.1 6.9 19.4 37.8a 37.9a
Protein level (g protein (g tissue)1) 0.042 0.078 0.024 0.094 0.045b 0.045b
Cardiac output (mL min1 kg1) 16.9 16.9 26.2 26.2 263b 484b
Fraction of blood flow through tissue 1.0 0.20 1.0 0.20 0.20b 0.25b
Body compositionc
Lipid 0.10 0.10 0.10 0.10 0.1 0.1
Non-lipid organic 0.20 0.20 0.20 0.20 0.2 0.2
Water 0.70 0.70 0.70 0.70 0.7 0.7
Blood compositionc
Blood lipid 0.01 0.01 0.01 0.01 0.01 0.01
Non-lipid organic 0.15 0.15 0.15 0.15 0.15 0.15
Water 0.84 0.84 0.84 0.84 0.84 0.84
Temperature of environment 15 °C 15 °C 25 °C 25 °C 37 °C 37 °C
a
Information to calculate characteristic provided by supplier (Moltox).
b
Information collected from literature (Davies and Morris, 1993; Gearhart et al., 1994; Houston, 1994; Janssen et al., 2002).
c
Obtained from Cowan-Ellsberry et al. (2008). Values held constant to reduce variability from factors not related to metabolism.
1192 C.F. Gomez et al. / Chemosphere 81 (2010) 1189–1195
and with kM equal to zero (BCF0). BCF calculations also included
predicted k1, k2, kE and kG values based upon KOW, total fish weight
and fish lipid content.
K 1 ¼ 1=ðð0:01 þ ð1=K OW ÞÞ  W 0:4 Þ
K 2 ¼ k1 =ðV lb  K OW Þ
K E ¼ ð0:125  0:02  W 0:15  e0:06T Þ=ðð5  108  K OW Þ þ 2Þ
K G ¼ 0:0251  W 0:2 ; ifT > 12  C
KOW at physiological pH of 7.4 was used for each compound (1.0
for ibuprofen, 2.99 for norethindrone and 0.78 for propranolol)
(Saha et al., 2000; Cleuvers, 2003; Johnson et al., 2007; Lee et al.,
2007). Biotransformation reaction vessels were also buffered to
pH 7.4.
2.8. Statistical analysis
Statistical analysis was conducted using GraphPad Prism 5
(GraphPad Software, Inc.). Loss of parent data was found to be
homoscedastic within each S9 fraction type (i.e., catfish liver S9,
etc.). Two-way repeated measures ANOVA confirmed statistical
significance of loss of parent compound over time while Bonferroni
post tests were used to evaluate the differences between batches at
each sample time point. CLm and BCF values were analyzed either
Fig. 1. Mean loss of parent compound over 90 min study time period. Error bars indicate standard error.
via an unpaired t-test or one-way ANOVA with Bonferroni’s multi-
ple comparison post test, depending upon the number of items
compared. All analyses were conducted at a = 0.05.
ð2Þ
3. Results
ð3Þ 3.1. EROD activity
ð4Þ EROD assays confirmed metabolic activity in the multiple tissue
fractions for quality control purposes. Resorufin concentration in-
ð5Þ creased over time in each test article, signifying CYP1A activity.
Although EROD activity was relatively low, results are on par with
other uninduced EROD activity levels in fish reported in the litera-
ture (Whyte et al., 2000). Resorufin was observed for trout liver
and gill S9 at 376.4 ± 23.4 and 363.7 ± 17.9 pmol mg protein1
after 60 min, which equated to mean 32.7% and 13.3% increases
in resorufin from time zero. For catfish liver and gill S9, resorufin
concentrations were 269.5 ± 37.3 and 268.2 ± 15.8 pmol mg pro-
tein1 after 60 min, which equated to mean 16.3% and 27.7%
increases.
3.2. Intrinsic clearance rate of parent compound
Loss of parent compound was observed in all trials, indicating
metabolic activity in the S9 fractions. However, loss of parent com-
pound occurred at varying rates among species and tissue type
(Fig. 1). CLm was calculated from loss of parent using analytically
 C.F. Gomez et al. / Chemosphere 81 (2010) 1189–1195
Fig. 2. Mean and standard error of CLm of S9 fractions. Indicates significant
difference from S9 of the same species.
confirmed initial parent concentrations (Fig. 2). In general, liver
fractions provided higher clearance rates than gill fractions. For
norethindrone and propranolol, a significant difference was ob-
served between liver and gill CLm within each species (p < 0.01).
However, a difference between species was not observed as the
CLm of trout and catfish liver and the CLm of trout and catfish gill
were not significantly different. Ibuprofen metabolism did not fol-
low the trend of higher liver clearance rates. The difference be-
tween liver and gill fractions was not considered significantly
different. While gill clearance rates for ibuprofen were in the same
range as those observed for norethindrone and propranolol, ibu-
profen liver clearance rates were significantly less than those of
norethindrone and propranolol (p < 0.0001). Ibuprofen metabolism
was also tested in uninduced Sprague–Dawley rat and CD-1 mouse
1193
S9 to provide further perspective on the lower intrinsic clearance
rates found in fish liver S9. CLm of rat and mouse were not signifi-
cantly different than those of fish. Thus, ibuprofen appears to have
a lower clearance rate across species than propranolol and
norethindrone.
The method proved reliable in providing reproducible intrinsic
clearance rates between different batches of S9. A significant dif-
ference was not observed between the CLm of the different fish
batches within each S9 type. Thus, homogenization of multiple fish
reduced the variability observed in other studies evaluating the
metabolic potential of individual fish (Crawford and Oleksiak,
2007; Smith et al., 2010). Additionally, the fish used during this
study were procured at different times over the course of 2 years.
Variability from the assorted fish procurement batches was not
seen.
3.3. Metabolic transformation rate
As described in Section 2.8, the metabolic transformation rate
(kM, d1) was extrapolated from loss of parent (Table 2). Fish tissue
fractions with higher CLm had correspondingly higher kM as the
physiological parameters entered for trout and catfish were not
greatly different. However, the increased liver to body weight,
TW and CO of the rat enhanced its calculated kM.
3.4. BCF
Not all tissue fractions consistently contributed to a signifi-
cantly reduced BCF. Predicted BCF0 for norethindrone, propranolol
and ibuprofen were 97.3 ± 0.05, 0.60 ± 0.00002 and 1.0 ± 0.0001
respectively. BCFM including trout liver kM were highly signifi-
cantly less than the original predicted BCF0 for all three compounds
(p < 0.001). Catfish liver BCFM was also significantly less than BCF0
for all compounds (p < 0.01). Trout gill metabolism significantly
lowered BCFM for ibuprofen and propranolol (p < 0.01); however,
its impact on norethindrone was considered not significant. Catfish
gill did not significantly reduce BCF in any of the compounds.
Mean gill and liver kM were combined additively to obtain
kM(total), representing total fish metabolic transformation. Trout
BCFM(total) was significantly smaller than BCFM based on liver alone
(BCFM(liver)) for propranolol (0.53 ± 0.005) and ibuprofen
(0.93 ± 0.02) (p < 0.001). Trout BCFM(total) was not significantly less
than trout BCFM(liver) for norethindrone (83.5 ± 1.6) due to minimal
gill clearance. Significant differences were not observed between
catfish BCFM(total) and BCFM(liver) for norethindrone (76.2 ± 0.9), pro-
pranolol (0.53 ± 0.004) or ibuprofen (0.95 ± 0.006). Lastly, in
continuing with mammalian comparisons for ibuprofen, rat liver
BCFM (0.88 ± 0.02) was significantly less than trout and catfish
BCFM(total) (p < 0.001). However, mouse BCFM (0.97 ± 0.004) was
not significantly different.
4. Discussion and conclusions
This study aimed at finding a simple method for determining kM
in vitro. As intrinsic clearance rates were scaled to metabolic trans-
formation rates, the physiological parameters (Table 1) used in the
model influenced calculated kM values in different ways. For exam-
ple, mean TW differed between liver and gill with liver accounting
for a larger proportion of body weight than gill. The larger liver TW
increased kM, and thus correspondingly decreased BCFM. The same
is true for tissue protein levels (PL). Liver had larger PL than gill,
which increased kM. TW and PL appropriately influence kM as lar-
ger, more protein dense tissues would be expected to contribute
more greatly to overall metabolism. Differences in cardiac output
between the two fish species was not great enough to result in sig-
1194 C.F. Gomez et al. / Chemosphere 81 (2010) 1189–1195

nificant differences in kM. However, a 10-fold increase in cardiac

output, as seen in the rat, did significantly increase kM (p < 0.01).

0.97 ± 0.004
Mouse liver

46.72 ± 7.1
Differences in metabolism and CLm were observed between tis-

1.1 ± 0.17
sue types throughout the study. For norethindrone and proprano-
lol, gill CLm was smaller than that of liver. Other studies have

S9
a
suggested that gill metabolic activity is less than that of the liver.

89.7 ± 16.0
2.02 ± 0.35
0.88 ± 0.02
Arachidonic acid metabolism in scup (Stenotomus chrysops) gill
Rat liver
microsomes was found to be 10- to 30-fold less than that of scup
liver microsomes (Schlezinger et al., 1998). Metabolic activity in
S9
a
rainbow trout gill epithelial cells was observed at between seven-

0.99 ± 0.003
and 60-fold less than isolated hepatocytes, depending on the sub-
74.5 ± 22.1
0.17 ± 0.05
Catfish gill

strate (7-ethoxycoumarin, aniline and testosterone) (Leguen et al.,

2000). Others have reported similar metabolic differences (Lind-
S9
6

strom-Seppa et al., 1981; Pesonen and Andersson, 1991).

Although differences in CLm were noted between tissues, differ-
Catfish liver

0.96 ± 0.005
0.82 ± 0.10

ences in intrinsic clearance rates were not found between species.

41.0 ± 6.0

In contrast, significant differences were seen in BCFM values be-

tween species in some cases. The biological and ecological rele-
S9
8

vance of these differences in BCFM must be put into perspective.

Trout gill S9

0.97 ± 0.005

For instance, BCFM for propranolol was highly significantly differ-

48.9 ± 18.1
0.69 ± 0.12

ent between trout and catfish for both liver and gill (p < 0.001).
Nonetheless, these differences were less than a 0.1 change in BCF
6

and are most likely not biologically or ecologically relevant to

either fish species.
1.0 ± 0.0001
46.7 ± 12.3
0.56 ± 0.12
0.97 ± 0.02
Trout liver
Ibuprofen

Overall, the inclusion of gill and liver kM decreased norethin-

Comparison of intrinsic clearance rate (CLm), extrapolated metabolic transformation rate (kM), BCFM and BCF0. Mean and standard error reported.

drone BCFs by 14.2% in trout and 21.7% in catfish. Propranolol BCFs

S9

decreased by 11.7% in both fish species. Ibuprofen BCFs decreased

7

by 7.0%, 5.0%, 12.0% and 3.0% for trout, catfish, rat and mouse
0.60 ± 0.002
61.2 ± 15.9
0.17 ± 0.04
Catfish gill

respectively. The inclusion of kM brought predicted BCFs closer to

observed in vivo BCFs for ibuprofen and norethindrone. In vivo
BCF values for ibuprofen in catfish range from 0.2 to 0.4 (Nallani
S9
5

et al., submitted for publication). In vivo BCF values for norethin-

drone in trout range from 6.7–13.5 to 4.5–24.5 in catfish (Nallani,
149.1 ± 12.5
Catfish liver

0.54 ± 0.003
2.45 ± 0.12

unpublished data). In vivo data is not available for propranolol.

Although the test compounds are not expected to bioconcen-
trate due to their small KOW, they demonstrate the metabolic activ-
S9
6

ity of fish S9 fractions and the potential for fish to clear xenobiotics.
460.6 ± 16.8
Trout gill S9

0.59 ± 0.004

Furthermore, they serve to advance method development of

0.50 ± 0.13

in vitro metabolism assays. The application of in vitro metabolism

assays in an integrated ADME bioconcentration model has the po-
7

tential to derive more realistic BCF values while dramatically

0.60 ± 0.00002

reducing study time, cost and the number of animals required

180.9 ± 13.8

0.55 ± 0.003
Propranolol

1.75 ± 0.07
Trout liver

(OECD, 1996; de Wolf et al., 2007; Weisbrod et al., 2007).

S9
6

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