Journal of Hazardous Materials 254–255 (2013) 242–251
Contents lists available at SciVerse ScienceDirect
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
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
b
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
Article history:
Received 8 November 2012
Received in revised form 13 March 2013
Accepted 16 March 2013
Available online 22 March 2013
Keywords:
Endocrine disruption
Hypothalamic-pituitary-gonad (HPG) axis
Ibuprofen
NSAIDs
Reproduction
Transgenerational effects
a b s t r a c t
This study was conducted in two experiments, to identify non-steroidal anti-inflammatory drugs
(NSAIDs) with high endocrine disruption potentials, and to understand consequences of exposure to
such NSAIDs in fish. In the first experiment, the effects of five NSAIDs on hormones and gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis were evaluated after 14 d exposure of adult
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. Significant up-regulation of fshˇ, lhˇ, fshr and lhr were observed in females, whereas down-regulation was
observed in males exposed to each NSAID. In the second experiment, ibuprofen was chosen as a model
chemical. Adult zebrafish pairs were exposed to ibuprofen for 21 d, and the effects on reproduction and
development of offspring were examined. The egg production was significantly decreased at ≥1 ␮g/L
ibuprofen, and parental exposure resulted in delayed hatching even when they were transferred to clean
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
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).
0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhazmat.2013.03.036
frequently detected pharmaceuticals in treatment plants and surface 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, ibuprofen, 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, respectively [3]. In Korea, concentrations of acetylsalicylic acid, diclofenac,
ibuprofen, mefenamic acid, and naproxen were reported at as great
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 ibuprofen [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 behavior 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 influence sex hormone balance, but detailed mechanisms in aquatic
vertebrates are not well understood.
In vertebrates, reproduction is regulated by coordinated interactions 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 hormone (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 testosterone (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 fluorotelomer alcohol [12] or 2,4-dichlorophenol [13] could disrupt
gonadotropins like FSH and LH, and sex steroid hormones, subsequently 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 chosen 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 reproduction 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 exposure medium were measured at the beginning of, and after the
48 h exposure, using high performance liquid chromatography tandem mass spectrometry (LC–MS/MS). NSAIDs were separated on a
2.0 mm × 100 mm Unison UK-C18 column (Imtakt USA, Philadelphia, PA, USA). The injection volume was 5 ␮L and the flow rate
was 200 ␮L/min. The mobile phase consisted of a binary mixture (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 electrospray ionization in negative mode (API 4000 triple MS/MS system,
Applied Biosystems, Forster City, CA, USA) in the following conditions: ion source voltage 4.5 kV and ESI temperature 400 ◦ C. The
mass analyzer was operated in the MRM mode: for acetylsalicylic 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 including dissolved oxygen (YSI 5000; YSI Inc., OH, USA), pH (Orion 3-star
pH benchtop meter; Thermo Fisher Scientific, MA, USA), conductivity (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 control (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 reproduction 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 euthanized 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 measurement of hormones and gene transcription, 4 male and 4 female fish
were randomly sampled from two replicate tanks of each treatment.
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 developmental 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
was used for quantification of sex hormones. For the second experiment, 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
was evaporated under a stream of nitrogen, and the residues were
dissolved in 120 ␮L ELISA buffer. E2 (Cat No. 582251) and T (Cat
No. 582701) were quantified by use of ELISA kit (Cayman Chemical Company, Ann Arbor, MI, USA), following the manufacturer’s
instructions.
2.4. Real-time polymerase chain reaction (PCR) assay
Brain and gonad were collected from each fish and preserved
in 250 ␮L RNAlater reagent (QIAGEN, Korea Ltd., Seoul, Korea) at
−20 ◦ C until analysis. Transcriptions of 22 genes were measured
as well as one housekeeping gene (ˇ-actin) which is reported to
be stably expressed following chemical treatment [15] (Table S1).
Full names of the determined genes are shown in Table S2. Total
RNA was isolated from the sample by use of the RNeasy minikit (QIAGEN). Two micrograms of total RNA for each sample were
used for reverse transcription by use of the iScriptTM cDNA Synthesis Kit (BIORAD, Hercules, CA, USA). The ABI 7300 Fast Real-Time
PCR System (Applied Biosystems, Foster City, CA, USA) was used to
perform quantitative real time PCR. PCR reaction mixtures (20 ␮L)
contained 1.8 ␮L (0.9 ␮M) of forward and reverse primers each,
3 ␮L of cDNA sample, 3.4 ␮L of RNase-free water (QIAGEN), and
10 ␮L of 2× SYBR GreenTM PCR master mix (Applied Biosystems).
Samples were denatured at 95 ◦ C for 10 min, followed by 40 cycles
of denaturation for 15 s at 95 ◦ C, annealing together with extension for 1 min at 60 ◦ C. For each sample a dissociation step was
performed at 95 ◦ C for 15 s, 60 ◦ C for 15 s, and 95 ◦ C for 15 s at the
end of the amplification phase to ensure amplification of a single
product. Efficiency of each quantitative real time PCR assay was
assessed by construction of standard curves with serially diluted
cDNA standards. For quantification of PCR results, the threshold
cycle (Ct) was determined for each reaction. Ct values for each gene
of interest were normalized to the endogenous control gene, ˇactin, by use of the Ct method [16]. ˇ-actin was chosen as the
most stable gene among the six candidate housekeeping genes, i.e.,
ˇ-actin, tubulin alpha 1 (tuba1), glyceraldehydes-3-phosphate dehydrogenase (gapdh), elongation factor 1-alpha (elfa), 18s ribosomal RNA
(18S rRNA), and ribosomal protein L8 (rpl8), in both brain and gonads
of male and female fish, using geNorm analysis (results not shown).
Normalized values were used to calculate the degree of induction or
inhibition expressed as a “fold difference” compared to normalized
control values.
2.5. Statistical analysis
Normality and homogeneity of variances of observations were
analyzed by the Kolmogorov–Smirnov test and Levene’s test,
respectively. One-way analysis of variance (ANOVA) with Dunnett’s test was performed by use of SPSS 15.0 for Windows® (SPSS,
Chicago, IL, USA) to determine significant differences between
control and exposure groups. The value p < 0.05 was used as
the criterion for statistical significance. All data are expressed as
mean ± standard deviation.
Correlations between gene transcripts were investigated by use
of Spearman correlation analysis using SAS (Version 9.2, Cary, NC,
USA). Principal component analysis was conducted to transform a
number of possibly correlated gene variables into a smaller number
of uncorrelated variables called “principal components” using SAS.
With the first and second principal components, we assessed the
relationship between gene transcriptions and sex steroid hormone
concentrations, and visualized the differences between different
levels of exposure.
3. Results
3.1. Experiment 1: effects of five NSAIDs on hormone production
and gene transcription
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
nominal and measured concentrations were still less than 20%
for all tested NSAIDs except for acetylsalicylic acid (Table S3). For
simplicity, the nominal concentrations were used for statistical
analysis and presentation of the results throughout the present
study.
During the 14 d exposure period, mortalities were observed
at the maximum concentration of diclofenac, mefenamic acid,
and naproxen (Fig. S2). The concentration 1000 ␮g/L caused
significant mortality and therefore was not used for measurement of hormone production and gene transcription for all test
NSAIDs.
Concentrations of E2 in blood plasma were significantly greater
in both male and female fish after exposure to ibuprofen or mefenamic acid (10 and 100 ␮g/L) (Fig. 1A). Concentrations of plasma T
were also significantly greater in female fish, while T was decreased
among the males (Fig. 1B). The E2/T ratio was significantly greater
in female fish after exposure to acetylsalicylic acid, ibuprofen,
mefenamic acid, or naproxen. Such effects were more pronounced
among male fish by the exposure to ibuprofen or mefenamic acid
(Fig. 1C).
The sex- and organ-specific profiles of gene transcription are
summarized for each NSAID (Table 1 and Fig. S3). The effects
of NSAIDs exposure on gene transcription were generally sexdependent. Among female fish, significant up-regulation of cyp19b,
gnrh2, gnrh3, gnrhr1, gnrhr2, and gnrhr4 was observed after exposure to ibuprofen or mefenamic acid. Transcription of er˛, er2ˇ,
and ar in brain of female fish were significantly up-regulated by
exposure to ibuprofen or mefenamic acid, but were significantly
down-regulated in male fish by all tested NSAIDs. Similar sexdependent differences were observed for fshˇ and lhˇ in brain and
fshr and lhr in gonad. Up-regulation was observed in these genes
among females, while down-regulation was observed in males.
In ovary, significant up-regulation of fshr, lhr, hmgra, and star
mRNA was observed in females exposed to 10 or 100 ␮g/L ibuprofen
or mefenamic acid. Transcriptions of 17ˇhsd and cyp19a genes were
significantly up-regulated in females exposed to 100 ␮g/L acetylsalicylic acid, 10 or 100 ␮g/L ibuprofen, or 100 ␮g/L mefenamic
acid. In testis, however transcription of fshr, lhr, 3ˇhsd, cyp17, or
17ˇhsd was significantly down-regulated in males exposed to the
test NSAIDs.
3.2. Experiment 2: effects of ibuprofen on F0 and F1 fish
Significant decrease of GSI was observed in males at 10 ␮g/L
and females at >0.1 ␮g/L ibuprofen (Fig. 2). However, the exposure
range tested in the present study did not result in significant effects
on K, BSI, and HSI (Fig. 2).
Concentrations of plasma E2 were significantly increased in both
male and female fish at ≥1 ␮g/L ibuprofen (Fig. 3A). Significant
decrease of T was observed in males at ≥1 ␮g/L and significant
increase of T was observed in females at 10 ␮g/L ibuprofen (Fig. 3B).
The E2/T ratio was significantly increased at ≥1 ␮g/L ibuprofen in
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
Female
Gene
Acetylsalicylic acid
Diclofenac
10 ␮g/L
10 ␮g/L
100 ␮g/L
Ibuprofen
100 ␮g/L
Mefenamic acid
10 ␮g/L
100 ␮g/L
10 ␮g/L
Naproxen
100 ␮g/L
10 ␮g/L
100 ␮g/L
Brain
gnrh2
gnrh3
gnrhr1
gnrhr2
gnrhr4
fshˇ
lhˇ
cyp19b
er˛
er2ˇ
ar
1.60
1.06
0.80
1.18
0.86
2.04
2.06
1.87
0.56
0.74
0.79
±
±
±
±
±
±
±
±
±
±
±
0.53
0.43
0.27
0.12
0.50
0.45*
0.79
0.60
0.05
0.18
0.40
1.94
1.85
1.12
2.01
1.22
3.02
2.82
1.90
0.71
1.23
1.78
±
±
±
±
±
±
±
±
±
±
±
0.68
0.41
0.26
0.32
0.25
0.21*
0.56*
0.12
0.04
0.31
0.48
1.41
1.05
0.84
1.27
1.11
3.10
1.59
1.16
0.75
1.19
1.12
±
±
±
±
±
±
±
±
±
±
±
0.44
0.46
0.43
0.44
0.49
0.15
0.40
0.62
0.22
0.49
0.46
1.16
1.21
0.87
1.44
1.05
2.78
2.05
1.25
1.28
1.56
1.40
±
±
±
±
±
±
±
±
±
±
±
0.54
0.31
0.28
0.41
0.32
0.84*
0.26
0.20
0.17
0.32
0.64
3.50
5.54
1.80
2.18
2.17
1.92
3.18
5.36
2.11
1.54
2.08
±
±
±
±
±
±
±
±
±
±
±
0.81
0.84*
0.65
0.27*
0.72*
0.23*
1.05*
1.88*
0.45*
0.21
1.09
3.28
6.45
2.36
2.76
1.80
2.76
3.09
6.35
2.85
2.31
2.74
±
±
±
±
±
±
±
±
±
±
±
1.05
2.21*
1.09*
0.32*
0.52
0.40*
0.07*
1.82*
0.30*
0.25*
0.55*
3.85
7.02
1.96
3.26
2.04
2.23
3.75
5.28
2.49
2.53
2.13
±
±
±
±
±
±
±
±
±
±
±
0.0 8
0.59*
0.54
0.72*
0.20*
0.45*
1.27*
2.20*
0.22*
0.85*
0.93
4.24
8.07
1.75
4.23
2.27
3.21
5.50
6.86
3.40
2.65
3.43
±
±
±
±
±
±
±
±
±
±
±
2.41
0.37*
0.49
1.21*
0.76*
0.38*
1.04*
0.68*
0.56*
0.27*
0.46*
1.96
1.23
0.66
0.99
2.05
1.85
2.51
1.74
1.24
1.09
0.74
±
±
±
±
±
±
±
±
±
±
±
1.14
0.81
0.33
0.72
0.54*
0.26
1.09
0.44
0.36
0.68
0.40
2.03
1.04
0.84
1.79
0.89
2.49
2.43
1.86
1.23
1.58
0.97
±
±
±
±
±
±
±
±
±
±
±
0.91
0.32
0.16
0.23
0.38
0.79*
0.60
0.31
0.19
0.10
0.07
Gonad
fshr
lhr
hmgra
hmgrb
star
cyp11a
3ˇhsd
cyp17
17ˇhsd
cyp19a
0.97
2.65
2.20
1.58
1.52
0.52
0.59
0.62
0.55
2.66
±
±
±
±
±
±
±
±
±
±
0.29
1.51
1.08
0.80
0.35
0.14
0.32
0.47
0.11
0.74
2.15
2.82
2.67
0.96
1.97
0.87
0.93
1.33
3.03
3.35
±
±
±
±
±
±
±
±
±
±
0.29
0.32
0.93
0.40
0.17
0.38
0.45
0.56
1.06*
0.51*
1.23
1.31
0.71
1.26
0.49
1.00
1.67
1.11
0.91
1.25
±
±
±
±
±
±
±
±
±
±
0.28
0.41
0.50
0.25
0.04
0.64
0.71
0.38
0.05
1.10
2.30
2.49
1.49
0.86
2.16
0.97
1.76
1.69
1.95
1.70
±
±
±
±
±
±
±
±
±
±
0.32
0.13
0.59
0.17
0.32
0.39
0.39
0.38
0.06
0.19
4.82
20.29
7.37
1.23
4.56
1.08
1.56
1.55
2.80
5.98
±
±
±
±
±
±
±
±
±
±
1.05*
2.39*
1.02*
0.38
0.55*
0.51
0.50
0.68
1.33*
0.61*
5.16
25.36
7.90
1.30
4.47
0.98
1.25
1.13
3.38
8.51
±
±
±
±
±
±
±
±
±
±
0.75*
3.52*
0.85*
0.37
0.77*
0.07
0.18
0.13
0.48*
1.96*
6.03
9.27
2.95
1.49
2.85
0.77
1.01
0.89
2.46
2.56
±
±
±
±
±
±
±
±
±
±
1.45*
1.23*
0.29*
0.38
1.03*
0.16
0.58
0.43
0.93*
0.46*
5.82
14.00
5.89
1.09
5.77
1.04
1.30
0.98
3.81
5.66
±
±
±
±
±
±
±
±
±
±
1.26*
2.03*
2.05*
0.43
1.45*
0.17
0.40
0.39
0.99*
1.85*
0.86
2.63
1.77
1.01
1.72
0.48
0.92
1.08
0.83
2.69
±
±
±
±
±
±
±
±
±
±
0.35
1.02
0.70
0.10
0.70
0.21
0.17
0.81
0.51
0.74
1.43
2.46
2.27
0.71
3.35
0.99
1.00
1.41
2.25
2.93
±
±
±
±
±
±
±
±
±
±
0.33
0.22
0.55
0.14
0.39
0.39
0.23
0.61
0.55
0.95
Brain
gnrh2
gnrh3
gnrhr1
gnrhr2
gnrhr4
fshˇ
lhˇ
cyp19b
er˛
er2ˇ
ar
1.75
1.46
1.28
1.49
1.29
0.48
0.40
0.76
0.81
0.58
0.57
±
±
±
±
±
±
±
±
±
±
±
1.04
1.01
0.54
1.35
0.77
0.07*
0.11*
0.64
0.37
0.27
0.27*
1.59 ± 0.47
1.46 ± 0.45
1.03 ± 0.09
1.73 ± 0.58
1.63 ± 0.48
0.33 ± 0.13*
0.34 ± 0.06*
1.27 ± 0.55
0.37 ± 0.15*
0.41 ± 0.08*
0.32 ± 0.02*
1.68
0.81
1.15
1.59
0.99
0.90
0.69
1.57
0.43
0.59
0.37
±
±
±
±
±
±
±
±
±
±
±
1.01
0.26
0.89
0.81
0.76
0.26
0.16
1.15
0.05
0.36
0.05
1.75
2.21
1.38
2.82
1.63
0.84
0.56
1.16
0.33
0.43
0.31
±
±
±
±
±
±
±
±
±
±
±
0.87
0.38
0.49
0.16*
0.46
0.15
0.25*
0.28
0.17*
0.05*
0.09*
1.19
2.15
1.18
0.94
0.71
0.49
0.29
1.30
0.46
1.04
0.39
±
±
±
±
±
±
±
±
±
±
±
0.98
0.33
0.64
0.68
0.37
0.12*
0.08*
0.20
0.12*
0.37
0.11*
2.08
4.48
1.24
2.29
1.37
0.39
0.34
1.56
0.39
0.82
0.32
±
±
±
±
±
±
±
±
±
±
±
0.73
0.33
0.31
0.54
0.62
0.06*
0.11*
0.41
0.10*
0.13
0.04*
2.26
2.36
1.70
1.69
1.30
0.44
0.39
2.04
0.40
0.84
1.09
±
±
±
±
±
±
±
±
±
±
±
1.16
0.98*
0.36
0.58
0.60
0.18*
0.20*
0.28
0.07*
0.18
0.23
2.30
3.75
1.82
2.25
1.30
0.27
0.25
1.68
0.43
0.71
0.41
±
±
±
±
±
±
±
±
±
±
±
0.23
1.21*
0.64
0.45
0.64
0.08*
0.13*
0.78
0.10*
0.10
0.06*
1.92
1.41
0.96
1.98
1.67
0.81
0.94
1.60
0.83
0.64
0.54
±
±
±
±
±
±
±
±
±
±
±
1.34
0.35
0.49
1.15
0.42
0.12
0.31
0.63
0.21
0.20
0.24*
1.94
1.17
1.10
2.03
1.25
0.65
0.77
1.33
0.78
0.54
0.42
±
±
±
±
±
±
±
±
±
±
±
0.71
0.37
0.33
0.61
0.22
0.06*
0.13
0.35
0.25
0.06*
0.04*
Gonad
fshr
lhr
hmgra
hmgrb
star
cyp11a
3ˇhsd
cyp17
17ˇhsd
cyp19a
0.27
0.40
0.74
0.65
0.89
0.56
0.19
0.25
0.18
0.70
±
±
±
±
±
±
±
±
±
±
0.24*
0.15*
0.39
0.23
0.74
0.39
0.11*
0.23*
0.14*
0.28
0.19
0.27
0.55
0.80
0.57
0.67
0.18
0.18
0.13
2.46
±
±
±
±
±
±
±
±
±
±
0.48
1.11
1.25
0.83
2.13
0.86
0.42
0.89
0.88
1.35
±
±
±
±
±
±
±
±
±
±
0.11
0.59
0.28
0.32
0.47
0.32
0.13
0.12
0.39
0.53
0.39
0.35
0.59
0.82
1.20
0.88
0.32
0.60
0.63
1.61
±
±
±
±
±
±
±
±
±
±
0.13
0.10*
0.22
0.15
0.40
0.26
0.19*
0.17
0.32
0.42
0.50
0.97
0.62
0.53
0.48
0.59
0.59
0.69
1.13
2.37
±
±
±
±
±
±
±
±
±
±
0.27
0.33
0.47
0.39
0.34
0.40
0.51
0.21
0.20
0.59*
0.30
0.24
0.67
0.98
0.78
1.28
0.18
0.52
0.53
2.83
±
±
±
±
±
±
±
±
±
±
0.03*
0.06*
0.18
0.39
0.31
0.21
0.06*
0.08
0.05*
1.17*
0.31
1.29
0.54
0.57
0.47
0.68
0.54
0.41
0.42
1.71
±
±
±
±
±
±
±
±
±
±
0.25*
0.18
0.21
0.21
0.41
0.25
0.24
0.21*
0.12*
0.31
0.18
0.30
0.49
0.75
0.69
1.24
0.18
0.49
0.28
2.22
±
±
±
±
±
±
±
±
±
±
0.02*
0.07*
0.11
0.26
0.29
0.58
0.03*
0.24*
0.07*
0.33*
0.97
0.12
1.33
1.78
1.66
1.01
0.51
0.62
0.17
1.51
±
±
±
±
±
±
±
±
±
±
0.87
0.07*
0.50
1.59
1.06
0.79
0.36*
0.53
0.20*
0.17
0.44
0.10
0.86
0.81
0.68
0.77
0.55
0.54
0.32
1.83
±
±
±
±
±
±
±
±
±
±
0.21
0.02*
0.33
0.12
0.24
0.25
0.23
0.32
0.11*
0.41
0.09*
0.08*
0.16
0.23
0.23
0.26
0.04*
0.08*
0.04*
0.32*
*
*
*
*
K. Ji et al. / Journal of Hazardous Materials 254–255 (2013) 242–251
Male
Tissue
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
17β-estradiol fold-change
relative to DMSO control
A
Male zebrafish
Female zebrafish
8
8
*
6
*
6
*
*
4
*
*
4
*
2
2
0
0
0
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
0
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
Concentration ( μg/L)
Testosterone fold-change
relative to DMSO control
B
Concentration ( μg/L)
3
3
2
2
1
*
*
*
* *
0
0
E2/T ratio fold-change
relative to DMSO control
*
*
1
*
0
C
*
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
0
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
Concentration ( μg/L)
Concentration ( μg/L)
15
15
*
12
12
*
9
9
*
*
6
6
*
3
*
0
0
* *
3
*
*
* *
*
0
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
Concentration ( μg/L)
0
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
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 upregulation 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 correlated 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 successfully at 4 dpf. Parental exposure to ≥1 ␮g/L ibuprofen resulted
in significant delay in hatching, even when they were transferred 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 fertilization (Fig. 6D). Phenotypic malformation, e.g., cardiac edema,
was observed in embryos subsequently exposed to 10 ␮g/L ibuprofen, 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
Male zebrafish
Hepatosomatic index
C
Gonadosomatic index
D
12
12
10
10
8
8
6
6
4
4
2
2
0
0
2.0
2.0
1.5
A 5000
17β-estradiol (pg/mL)
14
1.0
0.5
0.5
0.0
0.0
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
2.0
20
1.5
*
5
0.0
0
Ctrl SC
0.1 1
IBP ( µg/L)
10
4000
3000
3000
*
2000
*
* *
2000
1000
1000
0
0.1
1
10
Ctrl SC
IBP ( μg/L)
Ctrl SC
0.1 1
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. Condition 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 ibuprofen 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 synthesis and release of gonadotropin hormone and also acts as a
IBP ( μg/L)
1000
1000
*
*
500
500
0
0.1
1
10
Ctrl SC
IBP ( μg/L)
8
0.1
1
10
IBP ( μg/L)
4
* *
6
3
4
2
2
1
0
0
Ctrl SC
0.1
1
10
IBP ( μg/L)
10
10
*
C
*
1
1500
1500
Ctrl SC
* *
0.1
2000
0
10
0.5
4000
B 2000
15
1.0
Female zebrafish
5000
Ctrl SC
1.5
1.0
Male zebrafish
0
Testosterone (pg/mL)
Brainsomatic index
B
Female zebrafish
14
E2/T ratio relative to control
Condition factor
A
247
Ctrl SC
0.1
1
10
IBP ( μg/L)
Fig. 3. Effects of ibuprofen on (A) 17␤-estradiol (E2) hormone concentration,
(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 difference from control (p < 0.05).
neuro-modulater to regulate reproductive behaviors [17,18].
Among the five NSAIDs, ibuprofen or mefenamic acid exposure
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, transcriptions 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 maturation 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 regulating 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]. Downregulation 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 steroidogenic 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 exposure 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 transcription 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 activity can influence the concentration and balance of sex hormones
in zebrafish [25,26]. In fish brain, cyp19b gene is known to be controlled 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 vertebrates including fish, there are two COX isozymes, COX-1 and COX2. 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 tissue 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 estrogenicity 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 expression of sex steroid hormone receptor in male and female zebrafish.
Increase of er˛ and er2ˇ transcription in female zebrafish following 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 hormone 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 accompanied 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 malformation 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 exposure, 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 disrupting chemicals: following prenatal exposure to sublethal concentrations of nonylphenol [39] and endosulfan [40], adverse morphological 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 mechanisms 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
endocrine disruption by NSAIDs deserve further investigation.
Acknowledgement
This study was supported by the National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science and
Technology (357-2011-1-D00133). Prof. Giesy was supported by
the Canada Research Chair program, at large Chair Professorship at
the Department of Biology and Chemistry and State Key Laboratory
in Marine Pollution, City University of Hong Kong, The Einstein Professor Program of the Chinese Academy of Sciences and the Visiting
Professor Program of King Saud University.
Appendix A. Supplementary data
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.
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SUPPLEMENTARY DATA
Effects of non-steroidal anti-inflammatory drugs on hormones and genes of
the hypothalamic-pituitary-gonad axis, and reproduction of zebrafish
Kyunghee Ji1,2, Xiaoshan Liu1, Saeram Lee1, Sungeun Kang1, Younglim Kho3, John P Giesy2,
Kyungho Choi1*
1
School of Public Health, Seoul National University, Seoul, 151-742, Korea
2
Department of Biomedical Veterinary Sciences and Toxicology Centre, University of
Saskatchewan, Saskatoon, SK, S7N 5B3, Canada
3
School of Human & Environmental Sciences, Eulji University, Gyeonggi, 461-713, Korea
* Corresponding author
I
Contents of Supporting Information
Supporting Materials ................................................................................. III
Fish exposures ......................................................................................... III
Figure S1 ....................................................................................... III
Real-time PCR assay ............................................................................... IV
Table S1 ......................................................................................... IV
Table S2 ......................................................................................... VI
Supporting Results .................................................................................... VII
Measured concentrations of test pharmaceuticals ................................. VII
Table S3 ........................................................................................ VII
Survival of fish ..................................................................................... VIII
Figure S2 .................................................................................... VIII
Effects of five NSAIDs on gene and hormone levels in F0 fish ............... IX
Figure S3 ....................................................................................... IX
Table S4 ......................................................................................... XI
Table S5 ........................................................................................ XV
II
Supporting Materials
Fish exposures
Figure S1. Exposure design of the 1st and 2nd set of experiments.
III
Real-time PCR assay
Table S1. Sequences of primers for the genes measured.
Gene name Accession No.
Description
Sequence (5’-3’)
β-actin
Forward
TGCTGTTTTCCCCTCCATTG
Reverse
TCCCATGCCAACCATCACT
Forward
CTGAGACCGCAGGGAAGAAA
Reverse
TCACGAATGAGGGCATCCA
Forward
TTGCCAGCACTGGTCATACG
Reverse
TCCATTTCACCAACGCTTCTT
Forward
ACCCGAATCCTCGTGGAAA
Reverse
TCCACCCTTGCCCTTACCA
Forward
CAACCTGGCCGTGCTTTACT
Reverse
GGACGTGGGAGCGTTTTCT
Forward
CACCAACAACAAGCGCAAGT
Reverse
GGCAACGGTGAGGTTCATG
Forward
GCTGTCGACTCACCAACATCTC
Reverse
GTGACGCAGCTCCCACATT
Forward
GGCTGCTCAGAGCTTGGTTT
Reverse
TCCACCGATACCGTCTCATTTA
Forward
GTCGTTACTTCCAGCCATTCG
Reverse
GCAATGTGCTTCCCAACACA
Forward
CAGACTGCGCAAGTGTTATGAAG
Reverse
CGCCCTCCGCGATCTT
Forward
TTCACCCCTGACCTCAAGCT
gnrh2
gnrh3
gnrhr1
gnrhr2
gnrhr4
fshβ
lhβ
cyp19b
erα
er2β
NM_131031
AY657018
NM_182887
NM_001144980
NM_001144979
NM_001098193
NM_205624
NM_205622
AF183908
NM_152959
NM_174862
IV
ar
fshr
lhr
hmgra
hmgrb
star
cyp11a
3βhsd
cyp17
17βhsd
cyp19a
ptgs2
NM_001083123
NM_001001812
AY424302
BC155135
NM_001014292
NM_131663
NM_152953
AY279108
AY281362
AY306005
AF226620
AY028585
Reverse
TCCATGATGCCTTCAACACAA
Forward
TCTGGGTTGGAGGTCCTACAA
Reverse
GGTCTGGAGCGAAGTACAGCAT
Forward
CGTAATCCCGCTTTTGTTCCT
Reverse
CCATGCGCTTGGCGATA
Forward
GGCCATCGCCGGAAA
Reverse
GGTTAATTTGCAGCGGCTAGTG
Forward
GAATCCACGGCCTCTTCGT
Reverse
GGGTTACGGTAGCCACAATGA
Forward
TGGCCGGACCGCTTCTA
Reverse
GTTGTTGCCATAGGAACATGGA
Reverse
GGTCTGAGGAAGAATGCAATGAT
Reverse
CCAGGTCCGGAGAGCTTGT
Forward
GGCAGAGCACCGCAAAA
Reverse
CCATCGTCCAGGGATCTTATTG
Forward
AGGCACGCAGGAGCACTACT
Reverse
CCAATCGTCTTTCAGCTGGTAA
Forward
TCTTTGACCCAGGACGCTTT
Reverse
CCGACGGGCAGCACAA
Forward
TGCATCTCGCATCAAATCCA
Reverse
GTCCAAGTTCCGCATAGTAGCA
Forward
GCTGACGGATGCTCAAGGA
Reverse
CCACGATGCACCGCAGTA
Forward
TGGATCTTTCCTGGGTGAAGG
Reverse
GAAGCTCAGGGGTAGTGCAG
V
Table S2. Gene list of HPG axes of zebrafish.
Abbreviation
Gene name
gnrh
Gonadotropin-releasing hormone
gnrhr
Gonadotropin-releasing hormone receptor
fshβ
Follicle stimulating hormone β
lhβ
Luteinizing hormone β
cyp19b
Cytochrome P450 19B
er
Estrogen receptor
ar
Androgen receptor
fshr
Follicle stimulating hormone receptor
lhr
Luteinizing hormone receptor
hmgr
Hydroxymethylglutaryl CoA reductase
star
Steroidogenic acute regulatory protein
cyp11a
Cytochrome P450 side-chain cleavage
3βhsd
3β-hydroxysteroid dehydrogenase
cyp17
Cytochrome P450 17
17βhsd
17β-hydroxysteroid dehydrogenase
cyp19a
Cytochrome P450 19A
VI
Supporting Results
Measured concentrations of test pharmaceuticals
Table S3. Nominal and measured concentrations of acetylsalicylic acid, diclofenac, ibuprofen,
mefenamic acid, and naproxen at the beginning of, and after the 48 h exposure.
Experiment
Pharmaceuticals
LODa
Nominal
Measured concentration (μg/L)
(μg/L) concentration (μg/L) beginning of exposure
1st set
Control
0
<LOD
<LOD
DMSO control
0
<LOD
<LOD
10
11.60 ± 0.26
<LOD
100
94.93 ± 12.69
121.63 ± 37.91
10
10.43 ± 0.15
10.10 ± 0.60
100
102.50 ± 9.73
100.30 ± 1.57
10
7.93 ± 1.39
9.15 ± 0.10
100
107.33 ± 6.66
115.00 ± 1.00
10
11.80 ± 0.20
10.27 ± 0.15
100
112.00 ± 12.49
110.33 ± 8.62
10
12.97 ± 0.64
11.13 ± 0.47
100
101.10 ± 6.40
100.93 ± 2.10
Control
0
<LOD
<LOD
MeOH control
0
<LOD
<LOD
10
8.98 ± 0.12
8.87 ± 0.55
Acetylsalicylic acid
Diclofenac
Ibuprofen
Mefenamic acid
Naproxen
2nd set
Ibuprofen
a
after 48 h exposure
8.34
2.16
3.30
2.74
5.13
1.02
LOD: Limit of detection. Values are mean ± standard deviation of three replicate samples.
VII
Survival of fish
Female zebrafish
100
80
80
Survival (%)
Survival (%)
Male zebrafish
100
60
*
40
60
40
*
20
20
*
0
C SC
0.01 0.1 1
ASA
0.01 0.1 1
DCF
0.01 0.1 1
0.01 0.1 1
IBP
MFA
*
0
C SC
0.01 0.1 1
0.01 0.1 1
ASA
NPX
0.01 0.1 1
DCF
0.01 0.1 1
0.01 0.1 1
IBP
MFA
0.01 0.1 1
NPX
Concentration (mg/L)
Concentration (mg/L)
Figure S2. Survival of zebrafish after exposure to acetylsalicylic acid, diclofenac, ibuprofen,
mefenamic acid, or naproxen for 14 d. The results are shown as mean ± standard deviation
(n=4 for treatment and n=8 for control).
Asterisk indicates significant difference from
control (p < 0.05).
VIII
Effects of five NSAIDs on gene and hormone levels in F0 fish
(A)
IX
(B)
Figure S3. Effects of acetylsalicylic acid, diclofenac, ibuprofen, mefenamic acid, and
naproxen on gene transcription of hypothalamic-pituitary-gonad (HPG) axes in female and
male zebrafish. Gene transcriptions in female (upper) and male (lower) zebrafish treated by
10 μg/L (A) and 100 μg/L (B) are shown in a box with colors and stripes. The legend
describes the order of the treated pharmaceuticals and the colors designing different fold
changess. Gene acronyms are defined in Table S2 of Supporting information.
X
Table S4. Spearman correlation coefficients (r) between mRNA expressions of the genes along the HPG axis in zebrafish after ibuprofen exposure.
Male
Brain
GnRH2
GnRH3
GnRHR1 GnRHR2 GnRHR4
1.000
0.582
0.142
0.482
0.154
(0.007)
(0.552)
(0.032)
(0.518)
1.000
0.084
0.611
0.230
(0.726)
(0.004)
(0.329)
1.000
-0.081
0.056
(0.736)
(0.816)
1.000
0.341
Gonad
FSHβ
LHβ
CYP19b
ERα
ER2β
AR
FSHR
LHR
HMGRA
HMGRB
StAR
CYP11a
3βHSD
CYP17
17βHSD
CYP19a
-0.298
-0.504
0.385
-0.295
0.227
-0.102
-0.248
-0.116
0.137
0.150
0.165
0.360
0.344
-0.318
-0.425
0.378
(0.207) (0.335) (0.669)
(0.291)
(0.627)
(0.565)
(0.529)
(0.487)
(0.119)
(0.138)
(0.172)
(0.062)
(0.100)
0.049
-0.384
-0.194
-0.658
-0.221
-0.430
0.156
0.680
0.714
-0.555
-0.797
0.633
(<0.000) (0.001) (<0.000) (0.002) (0.838) (0.094)
(0.413)
(0.002)
(0.349)
(0.059)
(0.512)
(0.001)
(0.000)
(0.011)
(<0.000)
(0.003)
0.097
-0.157
-0.197
-0.066
-0.124
-0.091
-0.115
0.163
-0.293
-0.090
(0.875) (0.442) (0.506)
(0.684)
(0.509)
(0.405)
(0.783)
(0.602)
(0.703)
(0.628)
(0.492)
(0.210)
(0.705)
-0.459 -0.012
-0.444
-0.260
-0.690
-0.023
-0.199
0.098
0.697
0.601
-0.719
-0.354
0.755
(0.042) (0.960) (0.050)
(0.268)
(0.001)
(0.925)
(0.399)
(0.682)
(0.001)
(0.005)
(0.000)
(0.126)
(0.000)
0.300
-0.151
-0.282
-0.093
-0.007
0.308
0.092
0.005
-0.123
-0.011
(0.848) (0.198) (0.858)
(0.199)
(0.525)
(0.228)
(0.695)
(0.977)
(0.186)
(0.700)
(0.985)
(0.605)
(0.965)
0.175
0.289
0.575
0.171
0.328
-0.312
-0.662
-0.852
0.403
0.834
-0.668
(0.217)
(0.008)
(0.470)
(0.158)
(0.180)
(0.002)
(<0.000)
(0.079)
(<0.000)
(0.001)
0.189
0.652
0.172
0.176
0.087
-0.719
-0.767
0.722
0.682
-0.824
(0.000) (0.882) (0.006)
(0.425)
(0.002)
(0.468)
(0.457)
(0.717)
(0.000)
(<0.000)
(0.000)
(0.001)
(<0.000)
-0.485 -0.023
-0.466
-0.310
-0.646
-0.193
-0.397
0.266
0.626
0.720
-0.494
-0.800
0.623
(0.030) (0.925) (0.038)
(0.184)
(0.002)
(0.416)
(0.083)
(0.257)
(0.003)
(0.000)
(0.027)
(<0.000)
(0.003)
-0.101
0.612
0.427
0.255
0.224
-0.671
-0.601
0.554
0.666
-0.511
(0.673)
(0.004)
(0.060)
(0.278)
(0.342)
(0.001)
(0.005)
(0.011)
(0.001)
(0.021)
0.384
0.241
0.136
0.070
0.175
-0.160
-0.212
-0.194
-0.053
0.204
-0.147
(0.095)
(0.307)
(0.567)
(0.770)
(0.460)
(0.501)
(0.369)
(0.412)
(0.824)
(0.389)
(0.537)
1.000
0.339
0.492
0.238
0.369
0.423
-0.478
-0.556
0.546
0.524
-0.401
(0.143)
(0.028)
(0.313)
(0.109)
(0.063)
(0.033)
(0.011)
(0.013)
(0.018)
(0.080)
Brain
GnRH2
GnRH3
GnRHR1
GnRHR2
(0.141)
GnRHR4
1.000
(0.202) (0.024)
-0.803
-0.015
-0.700
-0.006
(0.950) (0.980)
-0.445
-0.712
(0.049) (0.000)
0.009
0.024
(0.970) (0.920)
FSHβ
1.000
0.718
(0.094)
0.783
0.172
(0.468)
0.592
(0.006)
0.258
(0.272)
-0.826
-0.625
-0.038 -0.182
-0.046
0.535
0.300
-0.158
0.043
0.405
(0.000) (<0.000) (0.015) (0.462) (0.076)
LHβ
1.000
-0.709
(0.001)
CYP19b
ERα
1.000
0.742
1.000
0.035
0.229
0.592
0.589
(0.331) (0.006)
ER2β
AR
1.000
Gonad
XI
FSHR
1.000
LHR
HMGRA
HMGRB
StAR
CYP11a
3βHSD
CYP17
17βHSD
0.030
-0.177
0.281
-0.299
0.044
-0.203
0.275
0.077
-0.155
(0.900)
(0.454)
(0.231)
(0.201)
(0.855)
(0.390)
(0.240)
(0.748)
(0.514)
1.000
0.245
0.468
0.037
-0.554
-0.545
0.587
0.520
-0.608
(0.297)
(0.037)
(0.879)
(0.011)
(0.013)
(0.007)
(0.019)
(0.005)
1.000
0.498
0.291
-0.105
-0.045
0.056
0.326
-0.089
(0.025)
(0.213)
(0.659)
(0.850)
(0.816)
(0.160)
(0.710)
1.000
0.166
-0.033
-0.271
0.386
0.302
-0.255
(0.485)
(0.890)
(0.249)
(0.093)
(0.196)
(0.278)
1.000
0.123
0.102
0.156
-0.089
0.065
(0.607)
(0.667)
(0.513)
(0.710)
(0.786)
1.000
0.779
-0.552
-0.658
0.753
(<0.000)
(0.012)
(0.002)
(0.000)
1.000
-0.619
-0.744
0.712
(0.004)
(0.000)
(0.000)
1.000
0.282
-0.631
(0.228)
(0.003)
1.000
-0.549
(0.012)
CYP19a
1.000
XII
Female
Brain
GnRH2
GnRH3
GnRHR1 GnRHR2 GnRHR4
1.000
0.747
0.709
0.749
0.579
(0.000)
(0.001)
(0.000)
(0.007)
1.000
0.612
0.795
0.662
(0.004)
(<0.000)
(0.002)
1.000
0.539
0.337
(0.014)
(0.146)
1.000
0.770
Gonad
FSHβ
LHβ
CYP19b
ERα
ER2β
AR
FSHR
LHR
HMGRA
HMGRB
StAR
CYP11a
3βHSD
CYP17
17βHSD
CYP19a
-0.074
0.657
0.762
0.420
0.340
-0.153
0.724
0.809
0.607
0.138
0.765
0.389
-0.167
0.514
0.642
0.726
(0.000)
(<0.000)
(0.005)
(0.563)
(<0.000)
(0.090)
(0.482)
(0.020)
(0.002)
(0.000)
0.471
0.657
0.791
0.260
0.724
0.330
-0.199
0.226
0.582
0.669
(0.036)
(0.002)
(<0.000)
(0.269)
(0.000)
(0.155)
(0.402)
(0.339)
(0.007)
(0.001)
0.647
0.663
0.501
0.056
0.646
0.131
-0.515
0.020
0.696
0.670
(0.993) (0.508) (0.816)
(0.002)
(0.002)
(0.025)
(0.816)
(0.002)
(0.582)
(0.020)
(0.932)
(0.001)
(0.001)
0.388
0.508
0.746
0.821
0.414
0.616
0.388
-0.173
0.333
0.594
0.780
(0.022)
(0.000)
(<0.000)
(0.070)
(0.004)
(0.091)
(0.466)
(0.151)
(0.006)
(<0.000)
0.345
0.628
0.584
0.351
0.500
0.403
-0.103
0.355
0.602
0.541
(0.136)
(0.003)
(0.007)
(0.129)
(0.025)
(0.078)
(0.665)
(0.125)
(0.005)
(0.014)
0.054
0.044
0.130
0.026
-0.186
0.123
0.112
0.038
0.365
-0.059
(0.822)
(0.855)
(0.584)
(0.915)
(0.433)
(0.604)
(0.638)
(0.872)
(0.114)
(0.806)
0.567
0.499
0.689
0.301
0.439
0.568
0.043
0.320
0.633
0.550
(0.009)
(0.025)
(0.001)
(0.197)
(0.053)
(0.009)
(0.858)
(0.169)
(0.003)
(0.012)
0.752
0.820
0.585
0.338
0.672
0.649
0.071
0.476
0.536
0.704
(0.000)
(<0.000)
(0.007)
(0.144)
(0.001)
(0.002)
(0.767)
(0.034)
(0.015)
(0.001)
0.097
0.432
0.497
0.233
0.348
0.174
-0.011
0.621
0.226
0.302
(0.684)
(0.057)
(0.026)
(0.324)
(0.132)
(0.464)
(0.965)
(0.004)
(0.338)
(0.196)
0.349
0.050
0.247
0.115
0.002
0.267
0.296
-0.044
0.139
0.431
0.247
(0.132)
(0.835)
(0.295)
(0.629)
(0.995)
(0.255)
(0.206)
(0.855)
(0.559)
(0.058)
(0.295)
1.000
-0.395
-0.141
-0.049
-0.381
-0.172
-0.241
-0.008
-0.026
0.184
-0.320
(0.085)
(0.552)
(0.838)
(0.097)
(0.247)
(0.305)
(0.975)
(0.915)
(0.438)
(0.169)
Brain
GnRH2
GnRH3
GnRHR1
GnRHR2
(<0.000)
GnRHR4
1.000
(0.757) (0.002) (<0.000) (0.065) (0.143) (0.519)
0.070
0.707
(0.767) (0.001)
0.108
0.360
(0.652) (0.119)
0.013
0.581
(0.957) (0.007)
0.282
0.516
(0.229) (0.020)
FSHβ
LHβ
1.000
0.690
(0.001)
0.475
(0.035)
0.645
(0.002)
0.501
0.359
-0.194
(0.032) (0.120) (0.413)
0.002
0.631
0.157
-0.056
-0.056
(0.003) (0.091) (0.816)
0.802
0.694
0.221
(0.024) (<0.000) (0.001) (0.349)
0.263
0.145
(0.263)
(0.541)
1.000
0.643
(0.002)
CYP19b
0.480
1.000
0.133
0.003
0.245
(0.576) (0.990) (0.297)
0.330
0.216
-0.199
(0.155) (0.361) (0.401)
0.326
0.131
-0.250
(0.160) (0.583) (0.289)
ERα
1.000
0.464
0.148
(0.039) (0.533)
ER2β
AR
1.000
Gonad
XIII
FSHR
1.000
LHR
HMGRA
HMGRB
StAR
CYP11a
3βHSD
CYP17
17βHSD
0.841
0.399
0.438
0.686
0.543
-0.163
0.290
0.553
0.758
(<0.000)
(0.081)
(0.054)
(0.001)
(0.013)
(0.494)
(0.215)
(0.011)
(0.000)
1.000
0.581
0.467
0.796
0.489
-0.265
0.463
0.632
0.841
(0.007)
(0.038)
(<0.000)
(0.029)
(0.260)
(0.040)
(0.003)
(<0.000)
1.000
0.312
0.476
0.330
-0.168
0.306
0.491
0.729
(0.181)
(0.034)
(0.155)
(0.480)
(0.189)
(0.028)
(0.000)
1.000
0.429
0.423
-0.308
0.050
0.247
0.539
(0.059)
(0.063)
(0.186)
(0.833)
(0.294)
(0.014)
1.000
0.368
-0.450
0.357
0.532
0.706
(0.111)
(0.047)
(0.123)
(0.016)
(0.001)
1.000
0.044
0.367
0.509
0.596
(0.853)
(0.112)
(0.022)
(0.006)
1.000
0.069
-0.338
-0.341
(0.772)
(0.145)
(0.141)
1.000
0.149
0.338
(0.531)
(0.144)
1.000
0.597
(0.006)
CYP19a
1.000
The values in parentheses are p values.
XIV
Table S5. Result of principal component analysis.
Males
Females
PC1
PC2
PC1
PC2
Eigenvalues
9.10
2.07
10.55
2.36
% Variance
43.4
9.9
50.3
11.2
Accumulative (%)
43.4
53.3
50.3
61.5
gnrh2
-0.16
0.15
0.27
0.01
gnrh3
-0.30
-0.12
0.29
-0.01
gnrhr1
-0.02
-0.41
0.19
-0.14
gnrhr2
-0.26
0.16
0.27
0.04
gnrhr4
-0.06
-0.23
0.25
0.31
cyp19b
-0.30
-0.06
0.27
-0.09
fshβ
0.26
-0.05
0.09
0.16
lhβ
0.28
-0.17
0.26
-0.00
erα
0.24
0.06
0.17
0.39
er2β
0.11
0.05
0.10
0.41
ar
0.23
0.13
-0.01
0.52
fshr
0.09
-0.20
0.23
-0.26
lhr
0.25
0.13
0.29
-0.08
hmgra
0.11
0.51
0.27
-0.01
hmgrb
0.17
0.29
0.15
-0.20
star
0.01
0.34
0.25
-0.09
cyp11a
-0.29
0.09
0.16
-0.06
3βhsd
-0.27
0.15
-0.07
0.24
Factor loadings
XV
cyp17
0.23
-0.17
0.16
0.23
17βhsd
0.25
0.19
0.26
0.02
cyp19a
-0.27
0.23
0.26
-0.13
Values >0.25 are shown in bold.
XVI