Effects of non-steroidal anti-inflammatory drugs on hormone production
and gene expression in hypothalamic-pituitary-gonad axis of zebrafish
Kyunghee Ji1,2*, Xiaoshan Liu1, Saeram Lee1, Sungeun Kang1, John P. Giesy2, Kyungho Choi1
School of Public Health, Seoul National University, Seoul, 151-742 Korea
2 Toxicology Centre and Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, S7J 5B3 Canada
1
♀
♂
Without IBP
With IBP
E2/T ratio fold-change
relative to DMSO control
4
6
*
*
*
*
*
4
*
2
Hormone
2
T
Female
Male
0
0
more than 3 fold up-regulation
3
3
1.5-3 fold up-regulation
Hypothalamus
CYP19B
Pituitary
ERα
ERβ
E2
AR
GnRH2
GnRH3
GnRHR1
GnRHR2
GnRHR4
FSHΒ
LHΒ
FSH
LH
Blood
FSH
LH
less than 0.33 fold down-regulation
2
2
1
1
*
*
0.66-0.33 fold down-regulation
*
Gonad
No criteria met
Statistical significance (p<0.05)
* *
*
* *
0
mRNA expression standard
ASA
DCF
IBP
MFA
NPX
0
15
15
*
12
12
*
9
6
Gene
9
*
*
6
* *
3
3
* *
* *
*
10 100
10 100
10 100
10 100
10 100
ASA
DCF
IBP
MFA
NPX
FSH
LH
CYP17
FSHR
Progesterone
HDL
LDL
LHR
Female
Male
2-5 fold up-regulation
HMGRB
17-hydroxyprogesterone
CYP17
3βHSD
Prognenolone
HMGRA
more than 5 fold up-regulation
StAR
Cholesterol
Outer mitochondrial
membrane
Aldostenedione
17βHSD
CYP11A
Cholesterol
Testosterone (T)
Inner mitochondrial
membrane
T
CYP19A
less than 0.2 fold down-regulation
0
0
0
0
10 100
10 100
10 100
10 100
10 100
0.5-0.2 fold down-regulation
ASA
DCF
IBP
MFA
NPX
No criteria met
Concentration (µg/L)
6
14
Male
Female
2.0
12
10
8
*
6
4
2
0
2.0
1.5
1.0
*
0.5
0.0
Ctrl SC
0.1
1
10
Ctrl SC
IBP (µg/L)
0.1
1
0.1
Female
Male
1
10
Ctrl SC
IBP (µg/L)
IBP (µg/L)
0.1
1
Estradiol (E2)
E2
Statistical significance (p<0.05)
Concentration (µg/L)
3000
*
2000
*
1000
Ctrl SC
0.1
1
10
IBP (µg/L)
Ctrl SC
0.1
1
10
*
1500
1000
*
*
IBP (µg/L)
Ctrl SC
0.1
1
10
IBP (µg/L)
Ctrl SC
0.1
1
*
5
0.1
1
10
Ctrl SC
10
IBP (µg/L)
0.1
1
4
*
*
*
2
Female
6
4
**
2
0
**
*
LHβ
CYP19b
ERα
0.1
1
10
IBP (µg/L)
Ctrl SC
0.1
1
Control
IBP 0.1 µg/L
IBP 1 µg/L
IBP 10 µg/L
*
*
2
AR
*
*
*
*
FSHR
8
*
**
*
*
LHR
HMGRA HMGRB
StAR
CYP11a
3β HSD
CYP17
17β HSD CYP19a
Female gonad
*
7
**
6
5
4
3
2
*
*
*
*
*
*
*
1
10
IBP (µg/L)
*
1
9
Female brain
*
*
ER2β
1
Ctrl SC
*
0
GnRH3 GnRHR1 GnRHR2 GnRHR4 FSHβ
4
3
6
2
5
* *
*
• Average number of eggs spawned was significantly less at ≥1 μg/L of IBP.
• Continuous exposure to 10 μg/L of IBP significantly reduced the rate of hatching.
• Parental exposure to ≥1 μg/L of IBP resulted in significant delay in hatching, even
when they were transferred to clean water.
• Continuous exposure to IBP through F1 generation increased malformation rates.
• These data indicate that parental exposure to low concentrations of IBP could
influence the F1 generation, resulting in increased adverse effects in the offspring.
7
3
*
GnRH2
8
Male gonad
4
0
6
8
5
3
10
IBP (µg/L)
Male
Control
IBP 0.1 µg/L
IBP 1 µg/L
IBP 10 µg/L
1
0
IBP (µg/L)
Female
500
* *
10
0.5
Ctrl SC
0
0
*
1.0
9
Male brain
5
15
IBP (µg/L)
Male
* *
4000
1.5
10
2000
5000
20
0.0
Ctrl SC
10
Female
Fold change
Male
Female
Fold change
Male
E2/T ratio relative to control
• F0 fish endpoints:
Mortality, Egg production, Somatic index,
Sex hormone concentration, mRNA
expressions of genes along the HPG axis
• F1 fish endpoints:
Hatchability, Time to hatch, malformation
*
6
Brain
ASA
DCF
IBP
MFA
NPX
*
• Godanosomatic index in both male and female fish was significantly decreased • Exposure to IBP affected transcription of genes of the HPG axis.
- Significant up-regulation of CYP19b along with greater plasma concentrations of E2 does not correspond
at 10 μg/L and ≥0.1 μg/L of IBP, respectively.
well with anti-ovulatory properties of IBP.
• Concentrations of plasma E2 were significantly increased in both male and
- A possible explanation is that down-regulation of ERα, ER2β, and AR mRNA in males might be
female fish at ≥1 μg/L of IBP.
compensation to greater production of E2 as a negative feedback.
• Significant decrease of T was observed in both males and females at ≥1 μg/L and - Changes in gene transcriptions were observed at 0.1 μg/L of IBP, within the environmentally relevant range.
10 μg/L of IBP, respectively.
Gonadosomatic index
 Experiment 2: fish exposure
• One most potent NSAID identified among the five NSAIDs was chosen
and its effects on reproduction and development of the next generation
were evaluated.
• Four male and six female fish were placed in a spawning aquarium, and
exposed to 0, 0.1, 1, or 10 μg/L of IBP for 21 d following OECD TG 229.
• On 18th day, thirty eggs collected from each tank were placed in 48-well
plate containing exposure water or clean water for 6 d.
Hormone production standard
Female zebrafish
8
 Experiment 2: Effects of ibuprofen on F0 and F1 fish
Hepatosomatic index
 Experiment 1: fish exposure
• Four male and four female adult fish per group were exposed to 0, 10,
100, and 1,000 μg/L of five NSAIDs for 14 d with static-renewal method.
• Concentration of sex hormone in plasma and transcriptions of several
genes along the HPG axis (brain and gonad tissue) were measured.
- Transcriptions of FSHβ, LHβ, FSHR, and LHR genes in females increased after the exposure to NSAIDs
which could subsequently accelerate gametogenesis and maturation of oocytes.
- Test NSAIDs induced down-regulation of FSHβ, LHβ, FSHR, and LHR genes in males, suggesting
possible delay in spermatogenesis as well as maturation.
- Possible explanation for the lesser concentrations of plasma T in male zebrafish:
1) lesser transcription of 3βHSD, 17βHSD, and CYP17, 2) greater transcription of CYP19a
Testosterone (pg/mL)
 Test compounds
• Five NSAIDs, i.e., acetylsalicylic acid (ASA), diclofenac (DCF),
ibuprofen (IBP), mefenamic acid (MFA), and naproxen (NPX) were
selected based on the frequency of detection in Korea waterways and
their toxicity.
• Concentrations of 17β-estradiol (E2) in plasma were significantly increased in
both male and female fish after exposure to IBP and MFA.
• Concentrations of plasma testosterone (T) were significantly greater in female
fish, while T was decreased among the males.
• E2/T ratio was significantly greater in females exposed to ASA, IBP, MFA, and
NPX, while more pronounced increase was observed in male fish exposed to IBP
and MFA.
• Effects of NSAIDs exposure on gene transcription were sex-dependent.
Condition factor
 We investigated the effects of NSAIDs on the hypothalamic-pituitarygonadal (HPG) axis and related mechanisms in freshwater fish.
 Experiment 1: Effects of five NSAIDs on hormone and gene transcription
17β-estradiol (pg/mL)
 Non-steroidal anti-inflammatory drugs (NSAIDs) have shown estrogenic
activity in vitro and in vivo, however, the mechanism of this activity is
not fully understood.
Testosterone fold-change 17β-estradiol fold-change
relative to DMSO control relative to DMSO control
Male zebrafish
8
0
0
GnRH2
GnRH3 GnRHR1 GnRHR2 GnRHR4 FSHβ
LHβ
CYP19b
ERα
ER2β
AR
FSHR
LHR
HMGRA HMGRB
StAR
CYP11a
3β HSD
CYP17
17β HSD CYP19a
- Left: Control embryo at 80 hour post fertilization
- Middle: Embryo continuously exposed to 10 μg/L
of IBP with cardiac edema
- Right: Embryo continuously exposed to 0.1 μg/L
of IBP with cardiac edema
 IBP 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.
 Potential ecosystem consequences of endocrine disruption by NSAIDs deserve further investigation.
For questions or comments please email me at jkh526@gmail.com