Journal of Hazardous Materials 254–255 (2013) 242–251

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Effects of non-steroidal anti-inflammatory drugs on hormones and

genes of the hypothalamic-pituitary-gonad axis, and reproduction of
zebrafish
Kyunghee Ji a,b,c , Xiaoshan Liu a , Saeram Lee a , Sungeun Kang a , Younglim Kho d ,
John P. Giesy b , Kyungho Choi a,∗
a
School of Public Health, Seoul National University, Seoul 151-742, Republic of Korea
b
Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
c
Department of Occupational and Environmental Health, Yongin University, Yongin 449-714, Republic of Korea
d
School of Human & Environmental Sciences, Eulji University, Seongnam 461-713, Republic of Korea

h i g h l i g h t s

• Among five NSAIDs, ibuprofen and mefenamic acid increased E2 level in fish.
• The E2/T ratio and transcription of cyp19a gene were increased in both sexes.
• Transcriptional responses in gonadotropin hormone-related genes were sex-dependent.
• The average number of eggs spawned was significantly less upon exposure to ibuprofen.
• Parental exposure to ibuprofen resulted in delayed and lesser rates of hatching.

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

Article history: This study was conducted in two experiments, to identify non-steroidal anti-inflammatory drugs
Received 8 November 2012 (NSAIDs) with high endocrine disruption potentials, and to understand consequences of exposure to
Received in revised form 13 March 2013 such NSAIDs in fish. In the first experiment, the effects of five NSAIDs on hormones and gene tran-
Accepted 16 March 2013
scriptions of the hypothalamic-pituitary-gonad (HPG) axis were evaluated after 14 d exposure of adult
Available online 22 March 2013
zebrafish. Ibuprofen and mefenamic acids were identified to increase the concentrations of 17␤-estradiol
and testosterone in females significantly, while decreased those of testosterone among male fish. Sig-
Keywords:
nificant up-regulation of fshˇ, lhˇ, fshr and lhr were observed in females, whereas down-regulation was
Endocrine disruption
Hypothalamic-pituitary-gonad (HPG) axis
observed in males exposed to each NSAID. In the second experiment, ibuprofen was chosen as a model
Ibuprofen chemical. Adult zebrafish pairs were exposed to ibuprofen for 21 d, and the effects on reproduction and
NSAIDs development of offspring were examined. The egg production was significantly decreased at ≥1 ␮g/L
Reproduction ibuprofen, and parental exposure resulted in delayed hatching even when they were transferred to clean
Transgenerational effects water for hatching. The results demonstrated that ibuprofen could modulate hormone production and
related gene transcription of the HPG axis in a sex-dependent way, which could cause adverse effects on
reproduction and the development of offspring.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction frequently detected pharmaceuticals in treatment plants and sur-

Continuous use of non-steroidal anti-inflammatory drugs
(NSAIDs) leads to their persistent release into water, hence their
potential impact on water organisms is an important concern. Due
to their volume of consumption and incomplete removal during
the wastewater treatment processes, NSAIDs are among the most
∗ Corresponding author. Tel.: +82 2 880 2738; fax: +82 2 745 9104.
E-mail address: kyungho@snu.ac.kr (K. Choi).
face waters worldwide [1]. For example in sewage treatment plant
(STP) effluents, median concentrations of diclofenac, ibuprofen, and
mefenamic acid were 0.424, 3.086, and 0.133 ␮g/L, respectively.
In UK streams, median concentrations of these compounds were
<0.020, 0.826, and 0.062 ␮g/L, respectively [2]. In Germany, the
median concentrations of acetylsalicylic acid, diclofenac, ibupro-
fen, and naproxen were reported at 0.22, 0.81, 0.37, and 0.30 ␮g/L in
STP effluents, and <0.02, 0.15, 0.07, and 0.07 ␮g/L in rivers, respec-
tively [3]. In Korea, concentrations of acetylsalicylic acid, diclofenac,
ibuprofen, mefenamic acid, and naproxen were reported at as great

0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhazmat.2013.03.036
 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251
as 0.269 ␮g/L, 0.793 ␮g/L, 3.528 ␮g/L, 1.390 ␮g/L, and 0.326 ␮g/L in
major rivers, respectively [4].
Both in vitro and in vivo studies have demonstrated that several
NSAIDs could modulate endocrine function. Significant reduction
in ovulation rates of female mouse and damaged fertility of male
mouse were reported following chronic administration of ibupro-
fen [5]. Among female Japanese medaka fish, exposure to 10 mg/L
diclofenac delayed hatching of the eggs and resulted in a greater
gonadosomatic index (GSI) [6]. Ibuprofen affected spawning behav-
ior of Japanese medaka [7], and delayed hatching [8]. Disruption of
endocrine system by exposure to NSAIDs might be partly explained
by alteration of aromatase activity which might subsequently influ-
ence sex hormone balance, but detailed mechanisms in aquatic
vertebrates are not well understood.
In vertebrates, reproduction is regulated by coordinated interac-
tions among hormones of the hypothalamic-pituitary-gonad (HPG)
axis [9]. Gonadotropin-releasing hormone (GnRH) released from
the hypothalamus stimulates secretion of gonadotropin hormones,
including follicle-stimulating hormone (FSH) and luteinizing hor-
mone (LH) from the pituitary. Gonadotropin hormones are then
transported to the gonads to induce steroidogenesis producing
sex steroid hormones, such as 17␤-estradiol (E2) and testos-
terone (T), which modulate the reproductive process [10,11].
Therefore, environmental contaminants that influence balance
of gonadotropin and sex hormones could affect reproduction
of the fish [12]. It has been previously shown that 8:2 fluo-
rotelomer alcohol [12] or 2,4-dichlorophenol [13] could disrupt
gonadotropins like FSH and LH, and sex steroid hormones, subse-
quently leading to decreased fecundity and reduced hatching rates
of offspring.
This study was conducted in two separate experiments, to
understand the endocrine disruption potentials of NSAIDs. In
the first experiment, five major NSAIDs, i.e., acetylsalicylic acid,
diclofenac, ibuprofen, mefenamic acid, and naproxen, were cho-
sen and measured for concentrations of sex steroid hormones and
expression of mRNA for 21 functionally relevant genes of the HPG
axis in zebrafish. In the second experiment, one of the most potent
compounds was selected and evaluated further for effects on repro-
duction and development of offspring.
2. Materials and methods
2.1. Test chemicals
Acetylsalicylic acid (CAS No. 50-78-2), diclofenac sodium salt
(CAS No. 15307-79-6), ibuprofen (CAS No. 15687-27-1), mefenamic
acid (CAS No. 61-68-7), and naproxen (CAS No. 22204-53-1) were
obtained from Sigma–Aldrich (St. Louis, MO, USA).
The actual concentrations of tested pharmaceuticals in expo-
sure medium were measured at the beginning of, and after the
48 h exposure, using high performance liquid chromatography tan-
dem mass spectrometry (LC–MS/MS). NSAIDs were separated on a
2.0 mm × 100 mm Unison UK-C18 column (Imtakt USA, Philadel-
phia, PA, USA). The injection volume was 5 ␮L and the flow rate
was 200 ␮L/min. The mobile phase consisted of a binary mix-
ture (A:B = 20/80, v/v); solvents A (5 mM ammonium acetate in
water) and B (methanol). Identification and quantification were
performed by a triple quadruple mass spectrometer with electro-
spray ionization in negative mode (API 4000 triple MS/MS system,
Applied Biosystems, Forster City, CA, USA) in the following condi-
tions: ion source voltage 4.5 kV and ESI temperature 400 ◦ C. The
mass analyzer was operated in the MRM mode: for acetylsali-
cylic acid (m/z 137 → 93, 65), diclofenac (m/z 294 → 250), ibuprofen
(m/z 205 → 161, 159), mefenamic acid (m/z 240 → 196, 192), and
naproxen (m/z 229 → 185, 170).
243
2.2. Fish culture and exposures
Adult zebrafish (Danio rerio) were acclimated in 30 L glass
tanks containing 20 L dechlorinated water for 2 weeks prior to the
experiment. The fish were maintained under a 16:8-h light:dark
photoperiod and fed with Artemia nauplii (<24 h after hatching)
twice daily. Water quality parameters of the culture water includ-
ing dissolved oxygen (YSI 5000; YSI Inc., OH, USA), pH (Orion 3-star
pH benchtop meter; Thermo Fisher Scientific, MA, USA), conduc-
tivity (NeoMet EST401C; iSTEK Inc., Seoul, Korea), and temperature
were monitored twice a week.
In the first set of experiment, four male and four female fish
(>3 months old) per group were exposed to control, solvent con-
trol (DMSO with a final concentration of 0.1% (v/v)), 10, 100 or
1000 ␮g/L of each NSAID under study. In each treatment, male and
female fish were separately placed in two 2 L beakers filled with
1.6 L of exposure medium (Fig. S1). For control and solvent control,
eight male and eight female fish were placed in the same way in
four 2 L beakers. The exposure duration was 14 d, during which the
fish were fed Artemia nauplii ad libitum twice a day. The exposure
medium was renewed with freshly prepared solution at every 2 d.
Mortality was recorded daily until the test termination, and dead
organisms were removed as soon as noted. After 14 d of exposure,
all surviving fish were sacrificed by immersion in an ice-water and
employed for further analysis.
In the second set of experiment, one of the most potent NSAID
identified among the 5 NSAIDs was chosen and its effects on repro-
duction and development of the next generation were evaluated.
Adult zebrafish were exposed to control, solvent control (MeOH
with a final concentration of 1:1000 (v/v) water), 0.1, 1, or 10 ␮g/L
ibuprofen for 21 d, following OECD test guideline 229 with minor
modification of sex ratio and water renewal [14]. Two replicates
were used for each control or treatment. Each replicate includes
four male and six female fish together in a spawning aquarium
(7.5 L tank with 6 L exposure medium). In every 2 d, exposure
medium was renewed, by carefully decanting old medium as much
as possible and adding freshly prepared test medium. Fish were fed
twice daily with freshly hatched Artemia nauplii. The number of
eggs spawned during the previous 24 h period were counted and
recorded daily. Exposure medium was routinely monitored for pH,
temperature, and dissolved oxygen. No mortalities were observed
at any treatment during the exposure period. All fish were euth-
anized in 2-phenoxyethanol (Sigma–Aldrich, St. Louis, MO, USA),
and total weight and snout-vent length were recorded for each fish.
Indices including condition factor (K), brain-somatic index (BSI),
hepatosomatic index (HSI), and GSI were calculated. For measure-
ment of hormones and gene transcription, 4 male and 4 female fish
were randomly sampled from two replicate tanks of each treat-
ment.
On the 18th day of fish exposure, fertilized eggs were collected
from each tank. Thirty eggs were randomly selected per each tank,
and separately placed in 48-well plate (Corning Life Sciences, CA,
USA) containing 1 mL exposure water (the same concentration) or
clean water (control) for 6 d under static conditions. Hatching rate,
time to hatch, and malformation were determined. The develop-
mental status of zebrafish was observed under a light microscope
Axioscop 2 (5× magnifications, Zeiss, Oberkochen, Germany).
2.3. Hormone measurement
After exposure, the tail of each zebrafish was transected, and
blood was collected from caudal vein in a glass capillary tube
treated with heparin following the method described elsewhere
[13] with a minor modification on buffer volume. Five microliter
of blood per each fish was centrifuged at 5000 × g for 20 min, and
the plasma was stored at −80 ◦ C. For the first experiment, plasma
244 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251

with 250 ␮L enzyme-linked immunosorbent assay (ELISA) buffer concentrations, and visualized the differences between different
was used for quantification of sex hormones. For the second exper- levels of exposure.
iment, plasma sample (5 ␮L for female and 3 ␮L for male fish)
with 400 ␮L UltraPure water was extracted twice with 2 mL diethyl
ether at 2000 × g for 10 min. The solvent used to extract hormones 3. Results
was evaporated under a stream of nitrogen, and the residues were
dissolved in 120 ␮L ELISA buffer. E2 (Cat No. 582251) and T (Cat 3.1. Experiment 1: effects of five NSAIDs on hormone production
No. 582701) were quantified by use of ELISA kit (Cayman Chem- and gene transcription
ical Company, Ann Arbor, MI, USA), following the manufacturer’s
instructions. Measured concentrations at the beginning of the exposure
were generally similar to the nominal concentrations for all tested
NSAIDs (Table S3). After the 48 h exposure, differences between
2.4. Real-time polymerase chain reaction (PCR) assay
nominal and measured concentrations were still less than 20%
for all tested NSAIDs except for acetylsalicylic acid (Table S3). For
Brain and gonad were collected from each fish and preserved
simplicity, the nominal concentrations were used for statistical
in 250 ␮L RNAlater reagent (QIAGEN, Korea Ltd., Seoul, Korea) at
analysis and presentation of the results throughout the present
−20 ◦ C until analysis. Transcriptions of 22 genes were measured
study.
as well as one housekeeping gene (ˇ-actin) which is reported to
During the 14 d exposure period, mortalities were observed
be stably expressed following chemical treatment [15] (Table S1).
at the maximum concentration of diclofenac, mefenamic acid,
Full names of the determined genes are shown in Table S2. Total
and naproxen (Fig. S2). The concentration 1000 ␮g/L caused
RNA was isolated from the sample by use of the RNeasy mini-
significant mortality and therefore was not used for measure-
kit (QIAGEN). Two micrograms of total RNA for each sample were
ment of hormone production and gene transcription for all test
used for reverse transcription by use of the iScriptTM cDNA Synthe-
NSAIDs.
sis Kit (BIORAD, Hercules, CA, USA). The ABI 7300 Fast Real-Time
Concentrations of E2 in blood plasma were significantly greater
PCR System (Applied Biosystems, Foster City, CA, USA) was used to
in both male and female fish after exposure to ibuprofen or mefe-
perform quantitative real time PCR. PCR reaction mixtures (20 ␮L)
namic acid (10 and 100 ␮g/L) (Fig. 1A). Concentrations of plasma T
contained 1.8 ␮L (0.9 ␮M) of forward and reverse primers each,
were also significantly greater in female fish, while T was decreased
3 ␮L of cDNA sample, 3.4 ␮L of RNase-free water (QIAGEN), and
among the males (Fig. 1B). The E2/T ratio was significantly greater
10 ␮L of 2× SYBR GreenTM PCR master mix (Applied Biosystems).
in female fish after exposure to acetylsalicylic acid, ibuprofen,
Samples were denatured at 95 ◦ C for 10 min, followed by 40 cycles
mefenamic acid, or naproxen. Such effects were more pronounced
of denaturation for 15 s at 95 ◦ C, annealing together with exten-
among male fish by the exposure to ibuprofen or mefenamic acid
sion for 1 min at 60 ◦ C. For each sample a dissociation step was
(Fig. 1C).
performed at 95 ◦ C for 15 s, 60 ◦ C for 15 s, and 95 ◦ C for 15 s at the
The sex- and organ-specific profiles of gene transcription are
end of the amplification phase to ensure amplification of a single
summarized for each NSAID (Table 1 and Fig. S3). The effects
product. Efficiency of each quantitative real time PCR assay was
of NSAIDs exposure on gene transcription were generally sex-
assessed by construction of standard curves with serially diluted
dependent. Among female fish, significant up-regulation of cyp19b,
cDNA standards. For quantification of PCR results, the threshold
gnrh2, gnrh3, gnrhr1, gnrhr2, and gnrhr4 was observed after expo-
cycle (Ct) was determined for each reaction. Ct values for each gene
sure to ibuprofen or mefenamic acid. Transcription of er˛, er2ˇ,
of interest were normalized to the endogenous control gene, ˇ-
and ar in brain of female fish were significantly up-regulated by
actin, by use of the Ct method [16]. ˇ-actin was chosen as the
exposure to ibuprofen or mefenamic acid, but were significantly
most stable gene among the six candidate housekeeping genes, i.e.,
down-regulated in male fish by all tested NSAIDs. Similar sex-
ˇ-actin, tubulin alpha 1 (tuba1), glyceraldehydes-3-phosphate dehy-
dependent differences were observed for fshˇ and lhˇ in brain and
drogenase (gapdh), elongation factor 1-alpha (elfa), 18s ribosomal RNA
fshr and lhr in gonad. Up-regulation was observed in these genes
(18S rRNA), and ribosomal protein L8 (rpl8), in both brain and gonads
among females, while down-regulation was observed in males.
of male and female fish, using geNorm analysis (results not shown).
In ovary, significant up-regulation of fshr, lhr, hmgra, and star
Normalized values were used to calculate the degree of induction or
mRNA was observed in females exposed to 10 or 100 ␮g/L ibuprofen
inhibition expressed as a “fold difference” compared to normalized
or mefenamic acid. Transcriptions of 17ˇhsd and cyp19a genes were
control values.
significantly up-regulated in females exposed to 100 ␮g/L acetyl-
salicylic acid, 10 or 100 ␮g/L ibuprofen, or 100 ␮g/L mefenamic
2.5. Statistical analysis acid. In testis, however transcription of fshr, lhr, 3ˇhsd, cyp17, or
17ˇhsd was significantly down-regulated in males exposed to the
Normality and homogeneity of variances of observations were test NSAIDs.
analyzed by the Kolmogorov–Smirnov test and Levene’s test,
respectively. One-way analysis of variance (ANOVA) with Dun-
nett’s test was performed by use of SPSS 15.0 for Windows® (SPSS, 3.2. Experiment 2: effects of ibuprofen on F0 and F1 fish
Chicago, IL, USA) to determine significant differences between
control and exposure groups. The value p < 0.05 was used as Significant decrease of GSI was observed in males at 10 ␮g/L
the criterion for statistical significance. All data are expressed as and females at >0.1 ␮g/L ibuprofen (Fig. 2). However, the exposure
mean ± standard deviation. range tested in the present study did not result in significant effects
Correlations between gene transcripts were investigated by use on K, BSI, and HSI (Fig. 2).
of Spearman correlation analysis using SAS (Version 9.2, Cary, NC, Concentrations of plasma E2 were significantly increased in both
USA). Principal component analysis was conducted to transform a male and female fish at ≥1 ␮g/L ibuprofen (Fig. 3A). Significant
number of possibly correlated gene variables into a smaller number decrease of T was observed in males at ≥1 ␮g/L and significant
of uncorrelated variables called “principal components” using SAS. increase of T was observed in females at 10 ␮g/L ibuprofen (Fig. 3B).
With the first and second principal components, we assessed the The E2/T ratio was significantly increased at ≥1 ␮g/L ibuprofen in
relationship between gene transcriptions and sex steroid hormone males (Fig. 3C).
Table 1
Transcriptional response profiles of genes in HPG axis in female and male zebrafish (Danio rerio) after the exposure to acetylsalicylic acid, diclofenac, ibuprofen, mefenamic acid, or naproxen.a

Sex Tissue Gene Acetylsalicylic acid Diclofenac Ibuprofen Mefenamic acid Naproxen

10 ␮g/L 100 ␮g/L 10 ␮g/L 100 ␮g/L 10 ␮g/L 100 ␮g/L 10 ␮g/L 100 ␮g/L 10 ␮g/L 100 ␮g/L

Female Brain gnrh2 1.60 ± 0.53 1.94 ± 0.68 1.41 ± 0.44 1.16 ± 0.54 3.50 ± 0.81*
3.28 ± 1.05*
3.85 ± *
0.0 8 4.24 ± 2.41*
1.96 ± 1.14 2.03 ± 0.91
gnrh3 1.06 ± 0.43 1.85 ± 0.41 1.05 ± 0.46 1.21 ± 0.31 5.54 ± 0.84* 6.45 ± 2.21* 7.02 ± 0.59* 8.07 ± 0.37* 1.23 ± 0.81 1.04 ± 0.32
gnrhr1 0.80 ± 0.27 1.12 ± 0.26 0.84 ± 0.43 0.87 ± 0.28 1.80 ± 0.65 2.36 ± 1.09* 1.96 ± 0.54 1.75 ± 0.49 0.66 ± 0.33 0.84 ± 0.16
gnrhr2 1.18 ± 0.12 2.01 ± 0.32 1.27 ± 0.44 1.44 ± 0.41 2.18 ± 0.27* 2.76 ± 0.32* 3.26 ± 0.72* 4.23 ± 1.21* 0.99 ± 0.72 1.79 ± 0.23
gnrhr4 0.86 ± 0.50 1.22 ± 0.25 1.11 ± 0.49 1.05 ± 0.32 2.17 ± 0.72* 1.80 ± 0.52 2.04 ± 0.20* 2.27 ± 0.76* 2.05 ± 0.54* 0.89 ± 0.38
fshˇ 2.04 ± 0.45* 3.02 ± 0.21* 3.10 ± 0.15 2.78 ± 0.84* 1.92 ± 0.23* 2.76 ± 0.40* 2.23 ± 0.45* 3.21 ± 0.38* 1.85 ± 0.26 2.49 ± 0.79*
lhˇ 2.06 ± 0.79 2.82 ± 0.56* 1.59 ± 0.40 2.05 ± 0.26 3.18 ± 1.05* 3.09 ± 0.07* 3.75 ± 1.27* 5.50 ± 1.04* 2.51 ± 1.09 2.43 ± 0.60
cyp19b 1.87 ± 0.60 1.90 ± 0.12 1.16 ± 0.62 1.25 ± 0.20 5.36 ± 1.88* 6.35 ± 1.82* 5.28 ± 2.20* 6.86 ± 0.68* 1.74 ± 0.44 1.86 ± 0.31
er˛ 0.56 ± 0.05 0.71 ± 0.04 0.75 ± 0.22 1.28 ± 0.17 2.11 ± 0.45* 2.85 ± 0.30* 2.49 ± 0.22* 3.40 ± 0.56* 1.24 ± 0.36 1.23 ± 0.19

K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251

er2ˇ 0.74 ± 0.18 1.23 ± 0.31 1.19 ± 0.49 1.56 ± 0.32 1.54 ± 0.21 2.31 ± 0.25* 2.53 ± 0.85* 2.65 ± 0.27* 1.09 ± 0.68 1.58 ± 0.10
ar 0.79 ± 0.40 1.78 ± 0.48 1.12 ± 0.46 1.40 ± 0.64 2.08 ± 1.09 2.74 ± 0.55* 2.13 ± 0.93 3.43 ± 0.46* 0.74 ± 0.40 0.97 ± 0.07

Gonad fshr 0.97 ± 0.29 2.15 ± 0.29 1.23 ± 0.28 2.30 ± 0.32 4.82 ± 1.05* 5.16 ± 0.75* 6.03 ± 1.45* 5.82 ± 1.26* 0.86 ± 0.35 1.43 ± 0.33
lhr 2.65 ± 1.51 2.82 ± 0.32 1.31 ± 0.41 2.49 ± 0.13 20.29 ± 2.39* 25.36 ± 3.52* 9.27 ± 1.23* 14.00 ± 2.03* 2.63 ± 1.02 2.46 ± 0.22
hmgra 2.20 ± 1.08 2.67 ± 0.93 0.71 ± 0.50 1.49 ± 0.59 7.37 ± 1.02* 7.90 ± 0.85* 2.95 ± 0.29* 5.89 ± 2.05* 1.77 ± 0.70 2.27 ± 0.55
hmgrb 1.58 ± 0.80 0.96 ± 0.40 1.26 ± 0.25 0.86 ± 0.17 1.23 ± 0.38 1.30 ± 0.37 1.49 ± 0.38 1.09 ± 0.43 1.01 ± 0.10 0.71 ± 0.14
star 1.52 ± 0.35 1.97 ± 0.17 0.49 ± 0.04 2.16 ± 0.32 4.56 ± 0.55* 4.47 ± 0.77* 2.85 ± 1.03* 5.77 ± 1.45* 1.72 ± 0.70 3.35 ± 0.39
cyp11a 0.52 ± 0.14 0.87 ± 0.38 1.00 ± 0.64 0.97 ± 0.39 1.08 ± 0.51 0.98 ± 0.07 0.77 ± 0.16 1.04 ± 0.17 0.48 ± 0.21 0.99 ± 0.39
3ˇhsd 0.59 ± 0.32 0.93 ± 0.45 1.67 ± 0.71 1.76 ± 0.39 1.56 ± 0.50 1.25 ± 0.18 1.01 ± 0.58 1.30 ± 0.40 0.92 ± 0.17 1.00 ± 0.23
cyp17 0.62 ± 0.47 1.33 ± 0.56 1.11 ± 0.38 1.69 ± 0.38 1.55 ± 0.68 1.13 ± 0.13 0.89 ± 0.43 0.98 ± 0.39 1.08 ± 0.81 1.41 ± 0.61
17ˇhsd 0.55 ± 0.11 3.03 ± 1.06* 0.91 ± 0.05 1.95 ± 0.06 2.80 ± 1.33* 3.38 ± 0.48* 2.46 ± 0.93* 3.81 ± 0.99* 0.83 ± 0.51 2.25 ± 0.55
cyp19a 2.66 ± 0.74 3.35 ± 0.51* 1.25 ± 1.10 1.70 ± 0.19 5.98 ± 0.61* 8.51 ± 1.96* 2.56 ± 0.46* 5.66 ± 1.85* 2.69 ± 0.74 2.93 ± 0.95

Male Brain gnrh2 1.75 ± 1.04 1.59 ± 0.47 1.68 ± 1.01 1.75 ± 0.87 1.19 ± 0.98 2.08 ± 0.73 2.26 ± 1.16 2.30 ± 0.23 1.92 ± 1.34 1.94 ± 0.71
gnrh3 1.46 ± 1.01 1.46 ± 0.45 0.81 ± 0.26 2.21 ± 0.38 2.15 ± 0.33 4.48 ± 0.33 2.36 ± 0.98* 3.75 ± 1.21* 1.41 ± 0.35 1.17 ± 0.37
gnrhr1 1.28 ± 0.54 1.03 ± 0.09 1.15 ± 0.89 1.38 ± 0.49 1.18 ± 0.64 1.24 ± 0.31 1.70 ± 0.36 1.82 ± 0.64 0.96 ± 0.49 1.10 ± 0.33
gnrhr2 1.49 ± 1.35 1.73 ± 0.58 1.59 ± 0.81 2.82 ± 0.16* 0.94 ± 0.68 2.29 ± 0.54 1.69 ± 0.58 2.25 ± 0.45 1.98 ± 1.15 2.03 ± 0.61
gnrhr4 1.29 ± 0.77 1.63 ± 0.48 0.99 ± 0.76 1.63 ± 0.46 0.71 ± 0.37 1.37 ± 0.62 1.30 ± 0.60 1.30 ± 0.64 1.67 ± 0.42 1.25 ± 0.22
fshˇ 0.48 ± 0.07* 0.33 ± 0.13* 0.90 ± 0.26 0.84 ± 0.15 0.49 ± 0.12* 0.39 ± 0.06* 0.44 ± 0.18* 0.27 ± 0.08* 0.81 ± 0.12 0.65 ± 0.06*
lhˇ 0.40 ± 0.11* 0.34 ± 0.06* 0.69 ± 0.16 0.56 ± 0.25* 0.29 ± 0.08* 0.34 ± 0.11* 0.39 ± 0.20* 0.25 ± 0.13* 0.94 ± 0.31 0.77 ± 0.13
cyp19b 0.76 ± 0.64 1.27 ± 0.55 1.57 ± 1.15 1.16 ± 0.28 1.30 ± 0.20 1.56 ± 0.41 2.04 ± 0.28 1.68 ± 0.78 1.60 ± 0.63 1.33 ± 0.35
er˛ 0.81 ± 0.37 0.37 ± 0.15* 0.43 ± 0.05 0.33 ± 0.17* 0.46 ± 0.12* 0.39 ± 0.10* 0.40 ± 0.07* 0.43 ± 0.10* 0.83 ± 0.21 0.78 ± 0.25
er2ˇ 0.58 ± 0.27 0.41 ± 0.08* 0.59 ± 0.36 0.43 ± 0.05* 1.04 ± 0.37 0.82 ± 0.13 0.84 ± 0.18 0.71 ± 0.10 0.64 ± 0.20 0.54 ± 0.06*
ar 0.57 ± 0.27* 0.32 ± 0.02* 0.37 ± 0.05 0.31 ± 0.09* 0.39 ± 0.11* 0.32 ± 0.04* 1.09 ± 0.23 0.41 ± 0.06* 0.54 ± 0.24* 0.42 ± 0.04*

Gonad fshr 0.27 ± 0.24* 0.19 ± 0.09* 0.48 ± 0.11 0.39 ± 0.13 0.50 ± 0.27 0.30 ± 0.03* 0.31 ± 0.25* 0.18 ± 0.02* 0.97 ± 0.87 0.44 ± 0.21
lhr 0.40 ± 0.15* 0.27 ± 0.08* 1.11 ± 0.59 0.35 ± 0.10* 0.97 ± 0.33 0.24 ± 0.06* 1.29 ± 0.18 0.30 ± 0.07* 0.12 ± 0.07* 0.10 ± 0.02*
hmgra 0.74 ± 0.39 0.55 ± 0.16 1.25 ± 0.28 0.59 ± 0.22 0.62 ± 0.47 0.67 ± 0.18 0.54 ± 0.21 0.49 ± 0.11 1.33 ± 0.50 0.86 ± 0.33
hmgrb 0.65 ± 0.23 0.80 ± 0.23 0.83 ± 0.32 0.82 ± 0.15 0.53 ± 0.39 0.98 ± 0.39 0.57 ± 0.21 0.75 ± 0.26 1.78 ± 1.59 0.81 ± 0.12
star 0.89 ± 0.74 0.57 ± 0.23 2.13 ± 0.47 1.20 ± 0.40 0.48 ± 0.34 0.78 ± 0.31 0.47 ± 0.41 0.69 ± 0.29 1.66 ± 1.06 0.68 ± 0.24
cyp11a 0.56 ± 0.39 0.67 ± 0.26 0.86 ± 0.32 0.88 ± 0.26 0.59 ± 0.40 1.28 ± 0.21 0.68 ± 0.25 1.24 ± 0.58 1.01 ± 0.79 0.77 ± 0.25
3ˇhsd 0.19 ± 0.11* 0.18 ± 0.04* 0.42 ± 0.13 0.32 ± 0.19* 0.59 ± 0.51 0.18 ± 0.06* 0.54 ± 0.24 0.18 ± 0.03* 0.51 ± 0.36* 0.55 ± 0.23
cyp17 0.25 ± 0.23* 0.18 ± 0.08* 0.89 ± 0.12 0.60 ± 0.17 0.69 ± 0.21 0.52 ± 0.08 0.41 ± 0.21* 0.49 ± 0.24* 0.62 ± 0.53 0.54 ± 0.32
17ˇhsd 0.18 ± 0.14* 0.13 ± 0.04* 0.88 ± 0.39 0.63 ± 0.32 1.13 ± 0.20 0.53 ± 0.05* 0.42 ± 0.12* 0.28 ± 0.07* 0.17 ± 0.20* 0.32 ± 0.11*
cyp19a 0.70 ± 0.28 2.46 ± 0.32* 1.35 ± 0.53 1.61 ± 0.42 2.37 ± 0.59* 2.83 ± 1.17* 1.71 ± 0.31 2.22 ± 0.33* 1.51 ± 0.17 1.83 ± 0.41
a
mRNA expression is expressed as the fold change compared to the corresponding DMSO control mRNA expression. The results are shown as mean ± standard deviation of three replicate samples. Asterisk indicates significant
difference from corresponding DMSO control (p < 0.05).

245
246 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251

Male zebrafish Female zebrafish

A 8 8

relative to DMSO control

17β-estradiol fold-change
*
6 * 6
* *
* *
4 4
* *
2 2

0 0
0 10 100 10 100 10 100 10 100 10 100 0 10 100 10 100 10 100 10 100 10 100
ASA DCF IBP MFA NPX ASA DCF IBP MFA NPX
Concentration ( μg/L) Concentration ( μg/L)
B 3 3
relative to DMSO control
Testosterone fold-change

2 2 *
* *

1 * 1
*
* * *

0 0
0 10 100 10 100 10 100 10 100 10 100 0 10 100 10 100 10 100 10 100 10 100
ASA DCF IBP MFA NPX ASA DCF IBP MFA NPX

Concentration ( μg/L) Concentration ( μg/L)

C 15 15
relative to DMSO control

*
E2/T ratio fold-change

12 12
*
9 9
*
*
6 6
* * * * *
3 * 3 * *

0
*
0
0 10 100 10 100 10 100 10 100 10 100 0 10 100 10 100 10 100 10 100 10 100
ASA DCF IBP MFA NPX ASA DCF IBP MFA NPX
Concentration ( μg/L) Concentration ( μg/L)

Fig. 1. (A) Blood 17␤-estradiol (E2), (B) testosterone (T), and (C) E2/T ratio in male and female zebrafish (Danio rerio) by the exposure to 0, 10, or 100 ␮g/L acetylsalicylic acid
(ASA), diclofenac (DCF), ibuprofen (IBP), mefenamic acid (MFA), or naproxen (NPX) for 14 d. The results are shown as mean ± standard deviation of three replicate samples.
Asterisk indicates significant difference from control (p < 0.05).

Exposure to ibuprofen affected transcription of genes of the
HPG axis (Fig. 4). In male fish, exposure to ibuprofen induced up-
regulation of gnrh3, gnrhr2, and cyp19b in brain (Fig. 4A) and cyp11a,
3ˇhsd, and cyp19a in testis (Fig. 4B). However, the transcriptions of
fshˇ, lhˇ and ar in brain (Fig. 4A) and lhr and 17ˇhsd in testis (Fig. 4B)
were significantly down-regulated in male zebrafish. In females,
significant up-regulation of brain gnrh2, gnrh3, gnrhr2, gnrhr4, lhˇ,
and cyp19b mRNAs (Fig. 4C) and ovary fshr, lhr, hmgra, star, 17ˇhsd,
and cyp19a mRNAs (Fig. 4D) were observed.
The relationship between gene transcriptions and sex steroid
hormone concentrations in male and female fish was evaluated.
Since several genes among the 21 target genes are highly corre-
lated with each other (r > 0.5, p < 0.01, Table S4), PCA was used to
reduce the number of independent variables to fewer factors, i.e.,
PCs. The first PC (PC1) explains 43.4% of the total variance for male
and 50.3% for female, and the second PC (PC2) explains additional
9.9% for male and 11.2% for female of the total variances (Fig. 5).
PC1 was highly influenced by variables such as cyp19a, cyp19b,
gnrh3, gnrhr2, fshˇ, lhˇ, lhr, cyp11a, 3ˇhsd, and 17ˇhsd, and was
significantly correlated with the concentrations of E2 (ˇ = −0.275,
p < 0.0001) and T (ˇ = 0.251, p = 0.0001) in male fish (Table S5). In
females, PC1 was significantly correlated with the concentration
of E2 (ˇ = 0.229, p = 0.0002) and T (ˇ = 0.235, p = <0.0001) (Table
S5).
The average number of eggs spawned was significantly less
at ≥1 ␮g/L ibuprofen (Fig. 6A). Continuous exposure to 10 ␮g/L
ibuprofen significantly reduced the rate of hatching (Fig. 6B). In
the F1 generation, over 89% of the control embryos hatched suc-
cessfully at 4 dpf. Parental exposure to ≥1 ␮g/L ibuprofen resulted
in significant delay in hatching, even when they were trans-
ferred to clean culture water (Fig. 6C). Continuous exposure to
ibuprofen through F1 generation increased malformation rates
compared to those which did not receive an exposure after fer-
tilization (Fig. 6D). Phenotypic malformation, e.g., cardiac edema,
was observed in embryos subsequently exposed to 10 ␮g/L ibupro-
fen, and these effects were significantly greater compared to those
observed in embryos without ibuprofen exposure (Fig. 6D and
E).
 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251 247

Male zebrafish Female zebrafish Male zebrafish Female zebrafish

A 14 14 A 5000 5000

17β-estradiol (pg/mL)
12 12
4000 4000 * *
Condition factor

10 10
8 8
3000 3000

6 6 2000 * 2000
4 4
*
1000 1000
2 2
0 0
0 0
Ctrl SC 0.1 1 10 Ctrl SC 0.1 1 10
B 2.0 2.0
IBP ( μg/L) IBP ( μg/L)
B 2000 2000
Brainsomatic index

1.5 1.5

Testosterone (pg/mL)
*
1500 1500
1.0 1.0

1000 1000
0.5 0.5 *
500 * 500
0.0 0.0

C 0 0
2.0 2.0 Ctrl SC 0.1 1 10 Ctrl SC 0.1 1 10
IBP ( μg/L) IBP ( μg/L)
Hepatosomatic index

C
1.5 1.5
E2/T ratio relative to control
8 4

1.0 1.0
6 * * 3

0.5 0.5
4 2

0.0 0.0 2 1

D 2.0 20 0 0
Ctrl SC 0.1 1 10 Ctrl SC 0.1 1 10
Gonadosomatic index

1.5 15 * * IBP ( μg/L) IBP ( μg/L)

Fig. 3. Effects of ibuprofen on (A) 17␤-estradiol (E2) hormone concentration,
1.0 * 10 * (B) testosterone (T) concentration, and (C) E2/T ratio. The results are shown as
mean ± standard deviation (n = 4 for each sex). Asterisk indicates significant differ-
ence from control (p < 0.05).
0.5 5

neuro-modulater to regulate reproductive behaviors [17,18].

0.0 0 Among the five NSAIDs, ibuprofen or mefenamic acid exposure
Ctrl SC 0.1 1 10 Ctrl SC 0.1 1
IBP ( µg/L) IBP ( µg/L)
Fig. 2. (A) Condition factor, (B) brainsomatic index, (C) hepatosomatic index, and
(D) gonadosomatic index in zebrafish (Danio rerio) after exposure to control (Ctrl),
solvent control (SC), 0.1, 1, and 10 ␮g/L ibuprofen (IBP) for 21 d. The results
are shown as mean ± standard deviation (n = 8 for male, n = 12 for female fish).
Asterisk indicates significant difference between exposure and control group. Con-
dition factor = weight (g)/snout-vent length (cm)3 × 100, brainsomatic index = brain
weight × 100/body weight, hepatosomatic index = liver weight × 100/body weight.
Gonadosomatic index = gonad weight × 100/body weight.
4. Discussion
The present study demonstrates that NSAIDs including ibupro-
fen caused reproductive dysfunction, altered plasma sex hormone
levels as well as gene transcription in the HPG axis in zebrafish.
In fish, GnRH is the central hormone which regulates the syn-
thesis and release of gonadotropin hormone and also acts as a
10 resulted in significantly greater transcription of gnrh2, gnrh3,
gnrhr1, gnrhr2, and gnrhr4 genes in brain of female zebrafish.
Therefore modulation of GnRHs by exposure to ibuprofen
and mefenamic acid could subsequently disrupt production of
gonadotropin hormones.
Gonadotropin hormones are secreted by the pituitary and act
through binding to gonadal receptors such as FSHR and LHR to
induce steroidogenesis and gametogenesis [19,20]. In female fish,
vitellogenesis is primarily under control of FSH, while maturation
of oocytes is primarily controlled by LH [21]. In this study, tran-
scriptions of fshˇ, lhˇ, fshr, and lhr genes in females increased after
the exposure to NSAIDs at environmentally relevant concentration
which could subsequently accelerate gametogenesis and matura-
tion of oocytes. The greater abundances of transcripts of fshr and lhr
in gonads from female fish exposed to NSAIDs might be a response
to greater fshˇ and lhˇ released from the pituitary. In male fish,
FSH and LH are the most important pituitary hormones regulat-
ing fish spermatogenesis [22]. FSH plays a regulatory role during
248 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251

Fig. 4. Gene expression profiles in zebrafish (Danio rerio) after exposure to ibuprofen (IBP; 0, 0.1, 1, and 10 ␮g/L) for 21 d. Responses in (A) male brain, (B) male gonad, (C)
female brain, and (D) female gonad are summarized. The results are shown as mean ± standard deviation (n = 4 for each sex). Gene expressions were expressed as fold change
relative to control. Asterisk indicates significant difference between exposure groups and control group (p < 0.05).

early stages of spermatogenesis, and LH is mainly involved in later
stages of maturation, e.g., regulating spermiation [22,23]. Down-
regulation of fshˇ and lhˇ in brain and fshr and lhr in testis in
the male zebrafish exposed to NSAIDs including ibuprofen suggests
possible delay in spermatogenesis as well as maturation.
Measurement of sex steroid hormones has been suggested
to be one of the most integrative and functional endpoints for
reproduction, and corresponds well with alteration of steroido-
genic gene transcriptions in zebrafish [13]. In the present study,
significant increase of E2 and decrease of T levels in male fish
were accompanied by down-regulation of 3ˇhsd, 17ˇhsd and
cyp17 genes and up-regulation of cyp19a gene, following expo-
sure to acetylsalicylic acid, ibuprofen or mefenamic acid. CYP17
plays a key role in the conversion of 17␣-hydroxyprogesterone to
 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251
Fig. 5. Plot of first two factors of principal component analysis of gene transcrip-
tion along the hypothalamic-pituitary-gonad axis. Clusters A–E represent control,
solvent control, 0.1 ␮g/L ibuprofen, 1 ␮g/L ibuprofen, and 10 ␮g/L ibuprofen group,
respectively. (A) Male, (B) female.
androstenedione in fish gonads. Inhibitory effects of CYP17 by
ibuprofen and diclofenac have been reported in vitro in testicular
mitochondrial fractions of carp [24]. CYP19 catalyzes a conversion
of androgen to estrogen, and therefore changes in aromatase activ-
ity can influence the concentration and balance of sex hormones
in zebrafish [25,26]. In fish brain, cyp19b gene is known to be con-
trolled by a positive auto-regulatory feedback loop which is driven
by E2 [27]. Greater transcription of cyp19b in male and female
zebrafish brain exposed to ibuprofen and mefenamic acid might
be a response to a greater concentration of circulatory E2. The ratio
of E2/T is indicative of endocrine disruption and has been used as
a sensitive biomarker of abnormal sex hormones in fish [28,29].
Greater ratio of E2/T in fish exposed to NSAIDs suggest that NSAIDs
exposure could disrupt balance of sex hormones in fish, and could
result in adverse effects on gametogenesis, sexual development or
reproduction of fish [28,30].
The pharmacological target of NSAIDs is cyclooxygenase (COX),
which catalyses production of prostaglandins (PGs) [31]. In verte-
brates including fish, there are two COX isozymes, COX-1 and COX-
2. COX-2 is active in ovaries during follicular development, and
its inhibition is thought to reduce not only PGs synthesis, but also
249
aromatase expression thereby inhibiting synthesis of estrogen [32].
The estrogenic responses that were observed upon exposure to
NSAIDs in the present study and other studies [4,6–8], however, do
not correspond well with such anti-ovulatory properties of NSAIDs.
In fact, several published works provide evidences that NSAIDs lack
inhibition of COX activities in fish. Green sunfish (Lepomis cyanellus)
were treated with a number of COX inhibitors including ibuprofen,
and no effect on COX enzyme was found by treatment of ibuprofen
[33]. Following exposure to indomethacin up to 100 ␮g/L for 16 d,
the COX activity in ovary and whole body homogenates of zebrafish
were not altered [34]. Following ibuprofen exposure, no changes
in COX enzyme activity were reported in either gill or kidney tis-
sue in rainbow trout [35]. Our observation of no change in gonadal
ptgs2 mRNA (cox mRNA) expression also supports the existing body
of evidences. Therefore one explanation for increased estrogenic-
ity by ibuprofen is that up-regulation of transcription of star and
17ˇhsd might stimulate basal synthesis of T, subsequently leading
to greater basal concentrations of E2 due to aromatization of T.
Exposure to NSAIDs resulted in sex-specific effects on expres-
sion of sex steroid hormone receptor in male and female zebrafish.
Increase of er˛ and er2ˇ transcription in female zebrafish follow-
ing exposure to ibuprofen and mefenamic acid suggests estrogenic
potentials of these pharmaceuticals. Activation of ER signaling
might be due to greater synthesis of E2 which is stimulated by FSH
and LH. In contrast, lesser transcriptions of er˛, er2ˇ, and ar in males
exposed to NSAIDs might be compensation to greater production
of E2 as a negative feedback.
In the second experiment, the altered plasma levels of E2
and T accompanied by significantly less production of eggs were
observed in fish exposed to ≥1 ␮g/L ibuprofen. Changes in hor-
mone levels and genes transcriptions of the HPG axis often link
to changes in reproduction, e.g., fecundity [36,37] or rate of
hatching [13]. The results of reproduction of eggs were in good
agreement with those reported in previous study of ibuprofen:
exposure to 100 ␮g/L ibuprofen resulted in fewer spawning event
[7]. Significant decrease on the weight of gonad was observed in
environmentally relevant concentrations of ibuprofen, suggesting
that ibuprofen has potential to inhibit the normal growth of gonad
and reproduction as a xenoestrogen. A lesser GSI value accom-
panied by an inhibition of egg production has been frequently
reported in fish exposed to estrogenic compounds [38].
Delayed and lesser rates of hatching, and increased malforma-
tion rates following parental exposure suggest the possibility of
trans-generational effects of ibuprofen exposure. Han et al. also
reported similar delayed hatching was observed among the F1
O. latipes following parental exposure to ≥0.1 ␮g/L ibuprofen [8].
Delayed hatchability, that was observed in the present study in the
absence of direct ibuprofen exposure among the offspring, may
be explained by impaired gamete quality by the parental expo-
sure, or by consequence of parental transfer of ibuprofen to the
gametes. Significantly increased malformation rates and worse
hatchability among the F1 embryos under continuous ibuprofen
exposure reflect consequences of exposure during embryonic stage.
Those embryos which showed cardiac edema died before hatching.
Similar observations were reported for well-known endocrine dis-
rupting chemicals: following prenatal exposure to sublethal con-
centrations of nonylphenol [39] and endosulfan [40], adverse mor-
phological changes, e.g., spinal malformation or defective hearts,
and subsequent mortality were reported among the F1 larvae.
In summary, our results clearly showed that exposure to NSAIDs
could increase the estrogenicity in fish, although the detailed mech-
anisms of sex dependent responses by exposure to NSAIDs remain
unknown. To the best of our knowledge, this study is the first report
which links the transcriptions of genes of HPG axis to hormonal
changes in fish by exposure to NSAIDs. It should be noted that
such changes could happen even at the environmentally relevant
250 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251

Fig. 6. Reproductive endpoints in F0 fish and the toxicity endpoints in offspring after maternal exposure to ibuprofen (IBP). (A) Number of eggs/breeding tank/day, (B)
hatchability (%), (C) time to hatch (d), (D) malformation rate (%), and (E) phenotypic changes in F1 embryo at 80 h post fertilization (left: control embryo, middle: embryo
continuously exposed to 10 ␮g/L IBP with cardiac edema, right: embryo continuously exposed to 0.1 ␮g/L IBP with cardiac edema). The results are shown as mean ± standard
deviation. Mean and standard deviation of (C) time to hatch and (D) malformation rates were calculated from sixty eggs randomly selected. Asterisk (*) indicates significant
difference from control and # indicates significant difference between F1 generation fish with continuous exposure and in clean water (p < 0.05).

concentrations, especially in ibuprofen. Potential consequences of Appendix A. Supplementary data

endocrine disruption by NSAIDs deserve further investigation.
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.jhazmat.2013.03.036.
Acknowledgement

This study was supported by the National Research Foundation

References
of Korea (NRF) funded by the Ministry of Education, Science and
Technology (357-2011-1-D00133). Prof. Giesy was supported by [1] N. Nakada, T. Tanishima, H. Shinohara, K. Kiri, H. Takada, Pharmaceuti-
the Canada Research Chair program, at large Chair Professorship at cal chemicals and endocrine disrupters in municipal wastewater in Tokyo
the Department of Biology and Chemistry and State Key Laboratory and their removal during activated sludge treatment, Water Res. 40 (2006)
3297–3303.
in Marine Pollution, City University of Hong Kong, The Einstein Pro- [2] D. Ashton, M. Hilton, K.V. Thoman, Investigating the environmental transport of
fessor Program of the Chinese Academy of Sciences and the Visiting human pharmaceuticals to streams in the United Kingdom, Sci. Total Environ.
Professor Program of King Saud University. 333 (2004) 167–184.
 K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251
[3] T.A. Ternes, Occurrence of drugs in German sewage treatment plants and rivers,
Water Res. 32 (1998) 3245–3260.
[4] National Institute of Environmental Research, Risk Assessment of Pharmaceut-
icals in the Environment (IV), Ministry of Environment, Korea, 2011.
[5] A.C. Martini, L.M. Vincenti, M.E. Santillán, G. Stutz, R. Kaplan, R.D. Ruiz, M.F.
de Cuneo, Chronic administration of nonsteroidal-antiinflammatory drugs
(NSAIDS): effects upon mouse reproductive functions, Rev. Fac. Cien. Med. Univ.
Nac. Cordoba 65 (2008) 47–59.
[6] J. Lee, K. Ji, Y.L. Kho, P. Kim, K. Choi, Chronic exposure to diclofenac on two fresh-
water cladocerans and Japanese medaka, Ecotoxicol. Environ. Saf. 74 (2011)
1216–1225.
[7] J.L. Flippin, D. Huggett, C.M. Foran, Changes in the timing of reproduction
following chronic exposure to ibuprofen in Japanese medaka, Oryzias latipes,
Aquat. Toxicol. 81 (2007) 73–78.
[8] S. Han, K. Choi, J. Kim, K. Ji, S. Kim, B. Ahn, J. Yun, K. Choi, J.S. Khim, X. Zhang, J.P.
Giesy, Endocrine disruption and consequences of chronic exposure to ibupro-
fen in Japanese medaka (Oryzias latipes) and freshwater cladocerans Daphnia
magna and Moina macrocopa, Aquat. Toxicol. 98 (2010) 256–264.
[9] D.L. Villeneuve, L.S. Blake, J.D. Brodin, J.E. Cavallin, E.J. Durhan, K.M. Jensen, M.D.
Kahl, E.A. Makynen, D. Martinović, N.D. Mueller, G.T. Ankley, Effects of 3␤-
hydroxysteroid dehydrogenase inhibitor, trilostane, on the fathead minnow
reproductive axis, Toxicol. Sci. 104 (2008) 113–123.
[10] Y. Nagahama, M. Yamashita, Regulation of oocyte maturation in fish, Dev.
Growth Differ. 50 (2008) S195–S219.
[11] N. Sofikitis, N. Giotitsas, P. Tsounapi, D. Baltogiannis, D. Giannakis, N. Parda-
lidis, Hormonal regulation of spermatogenesis and spermiogenesis, J. Steroid
Biochem. 109 (2008) 323–330.
[12] C. Liu, J. Deng, L. Yu, M. Ramesh, B. Zhou, Endocrine disruption and reproduc-
tive impairment in zebrafish by exposure to 8:2 fluorotelomer alcohol, Aquat.
Toxicol. 96 (2010) 70–76.
[13] Y. Ma, J. Han, Y. Guo, P.K.S. Lam, R.S.S. Wu, J.P. Giesy, X. Zhang, B. Zhou, Disrup-
tion of endocrine function in in vitro H295R cell-based and in in vivo assay in
zebrafish by 2,4-dichlorophenol, Aquat. Toxicol. 106–107 (2012) 173–180.
[14] Organization for Economic Co-operation and Development, Fish Short Term
Reproduction Assay, OECD Guideline 229, Paris, France, 2009.
[15] A.T. McCurley, G.V. Callard, Characterization of housekeeping genes in
zebrafish: male-female differences and effects of tissue type, developmental
stage and chemical treatment, BMC Mol. Biol. 9 (2008) 102.
[16] J.K. Livak, T.D. Schmittgen, Analysis of relative gene expression data by use
of real-time quantitative PCR and the 2C(T) method, Methods 25 (2001)
402–408.
[17] P.T. Bosma, F.E.M. Rebers, W. van Dijk, P. Willems, H.J.T. Goos, R.W. Schulz,
Inhibitory and stimulatory interactions between endogenous gonadotripin-
releasing hormones in the Africa catfish (Clarias gariepinus), Biol. Reprod. 62
(2000) 731–738.
[18] K. Okuzawa, K. Gen, M. Bruysters, J. Bogerd, Y. Gothif, H. Kagawa, Seasonal
variation of the three native Gonadotropin-releasing hormone messenger
ribonucleic acids concentrations in the brain of female red seabream, Gen.
Comp. Endocrinol. 130 (2003) 324–332.
[19] R.S. Kumar, J.M. Trant, Piscine glycoprotein hormone (gonadotropin and thy-
rotropin) receptors: a review of recent developments, Comp. Biochem. Physiol.
129B (2001) 347–355.
[20] H.F. Kwok, W.K. So, Y. Wang, W. Ge, Zebrafish gonadotropins and their recep-
tors: I. Cloning and characterization of zebrafish follicle-stimulating hormone
and luteinizing hormone receptors-evidence for their distinct functions in fol-
licle development, Biol. Reprod. 72 (2005) 1370–1381.
[21] E. Clelland, C. Peng, Endocrine/paracrine control of zebrafish ovarian develop-
ment, Mol. Cell. Endocrinol. 312 (2009) 42–52.
251
[22] R.W. Schulz, L.R. França, J. Lareyre, F. LeGac, H. Chiarini-Garcia, R.H. Nobrega, T.
Miura, Spermatogenesis in fish, Gen. Comp. Endocrinol. 165 (2010) 390–411.
[23] T. Ohta, H. Miyake, C. Miura, H. Kamei, K. Aida, T. Miura, Follicle-stimulating hor-
mone induces spermatogenesis mediated by androgen production in Japanese
eel, Anguilla japonica, Biol. Reprod. 77 (2007) 970–977.
[24] D. Fernandes, S. Schnell, C. Porte, Can pharmaceuticals interfere with the syn-
thesis of active androgens in male fish? An in vitro study, Mar. Pollut. Bull. 62
(2011) 2250–2253.
[25] M. Uchida, T. Yamashita, T. Kitano, T. Iguchi, An aromatase inhibitor or high-
water temperature induce oocyte apoptosis and depletion of P450 aromatase
activity in the gonads of genetic female zebrafish during sex-reversal, Comp.
Biochem. Physiol. 137A (2004) 11–20.
[26] M. Fenske, H. Segner, Aromatase modulation alters gonadal differentiation in
developing zebrafish (Danio rerio), Aquat. Toxicol. 67 (2004) 105–126.
[27] G.V. Collard, A.V. Tchoudakova, M. Kishida, E. Wood, Differential tissue dis-
tribution, developmental programming, estrogen regulation and promoter
characteristics of cyp19 genes in teleost fish, J. Steroid Biochem. Mol. Biol. 79
(2001) 305–314.
[28] L.C. Folmar, N.D. Denslow, V. Rao, M. Chow, D.A. Crain, J. Enblom, J. Marcino, L.J.
Guillete Jr., Vitellogenin induction and reduced serum testosterone concentra-
tions in feral male carp (Cyprinus carpio) captured near a major metropolitan
sewage treatment plant, Environ. Health Perspect. 104 (1996) 1096–1101.
[29] E.F. Orlando, A.S. Kolok, G.A. Binzcik, J.L. Gates, M.K. Horton, C.S. Lambright, L.E.
Gray Jr., A.M. Soto, L.J. Guillete Jr., Endocrine-disrupting effects of cattle feedlot
effluent on an aquatic sentinel species, the fathead minnow, Environ. Health
Perspect. 112 (2004) 353–358.
[30] E.H.H. Shang, R.M.K. Yu, R.S.S. Wu, Hypoxia effects sex differentiation and devel-
opment, leading to a male-dominated population in zebrafish (Danio rerio),
Environ. Sci. Technol. 40 (2006) 3118–3122.
[31] J.R. Vane, Y.S. Bakhle, R.M. Botting, Cyclooxygenase 1 and 2, Annu. Rev. Pharma-
col. Toxicol. 38 (1998) 97–120.
[32] R.W. Brueggemeier, J.C. Hackett, E.S. Diaz-Cruz, Aromatase inhibitors in the
treatment of breast cancer, Endocr. Rev. 26 (2005) 331–345.
[33] B. Cavallaro, B. Burnside, Prostaglandins E1, E2, and D2 induce dark-adaptive
retinomotor movements in teleost retinal cones and RPE, Invest. Ophthalmol.
Vis. Sci. 29 (1988) 882–891.
[34] A.L. Lister, G. Van Der Kraak, An investigation into the role of prostaglandins in
zebrafish oocyte maturation and ovulation, Gen. Comp. Endocrinol. 159 (2008)
46–57.
[35] M. Robichaud, Effects of ibuprofen on rainbow trout (Oncorhynchus mykiss)
following acute and chronic waterborne exposures, Thesis of master degree,
University of Ontario Institute of Technology, 2011.
[36] X. Zhang, M. Hecker, P.D. Jones, J. Newsted, D. Au, R. Kong, R.S.S. Wu, J.P. Giesy,
Responses of the medaka HPG axis PCR array and reproduction to prochloraz
and ketoconazole, Environ. Sci. Technol. 42 (2008) 6762–6769.
[37] Z.B. Zhang, J.Y. Hu, H.J. Zhen, X.Q. Wu, C. Huang, Reproductive inhibition
and transgenerational toxicity of triphenyltin on medaka (Oryzias latipes) at
environmentally relevant tip concentrations, Environ. Sci. Technol. 42 (2008)
8133–8139.
[38] K. Van den Belt, R. Verheyen, H. Witters, Reproductive effects of ethynylestra-
diol and 4t-octylphenol on the zebrafish (Danio rerio), Arch. Environ. Contam.
Toxicol. 41 (2001) 458–467.
[39] F.X. Yang, Y. Xu, Y. Hui, Reproductive effects of prenatal exposure to nonylphe-
nol on zebrafish (Danio rerio), Comp. Biochem. Physiol. C: Toxicol. Pharmacol.
142 (2006) 77–84.
[40] Y.M. Velasco-Santamaría, R.D. Handy, K.A. Sloman, Endosulfan affects health
variables in adult zebrafish (Danio rerio) and induces alterations in larvae devel-
opment, Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 153 (2011) 372–380.