Evaluation of the endocrine disrupting potential of the binary exposure to trilostane and prochloraz...
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Evaluation of the endocrine disrupting potential of the binary exposure to trilostane and prochloraz in H295R cells and Medaka fish Chunsheng Liu Chunsheng Liu 1 1 , , Xiaowei Zhang Xiaowei Zhang 2 2 *, *, Saerom Parkl Saerom Parkl 3 3 , , Jonathon Doering Jonathon Doering 2 2 , , Hong Chang Hong Chang 2 2 , Jong Seong Kim , Jong Seong Kim 3 3 , Paul Jones , Paul Jones 2,4 2,4 , Bingsheng Zhou , Bingsheng Zhou 1* 1* , , Markus Markus Hecker Hecker 4 4 John P. Giesy John P. Giesy 2,3,4,5 2,3,4,5 1 1 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan China, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan China, 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada. 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada. 3 Korea 3 Korea University / Division of Environmental Science and Ecological Engineering; University / Division of Environmental Science and Ecological Engineering; 4 4 School of Environment and Sustainability, University of School of Environment and Sustainability, University of Saskatchewan; 5 Saskatchewan; 5 ENTRIX Inc. Saskatoon, SK, Canada; 6 ENTRIX Inc. Saskatoon, SK, Canada; 6 Dep Dep t. Biomedical Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 5 Dept. t. Biomedical Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 5 Dept. Biology Biology & & C C hemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China hemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China Introduction Although living organisms are exposed to mixtures of environmental chemicals, most of previous studies on endocrine disrupting chemical (EDCs) have only evaluated individual chemical -induced effects. The objective of this study was to evaluate the mixture effect of two model EDCs, trilostane and prochloraz, on the reproduction axis, especially the steroidogenesis. Prochloraz is a commonly used imidazole fungicide, which has been reported to affect reproduction and development in fish and wildlife by inhibiting CYP genes, including steroidogenic cytochrome P450 c 17α hydroxylase, 17,20-lyase (CYP17) and aromatase (CYP19). Trilostane is an inhibitor of 3 β-hydroxysteroid dehydrogenase (3β HSD) that is upstream of CYP17 and CYP19 on the steroidogenesis pathway. The testable hypothesis is that co-exposure of prochloraz and trilostane could synergistically affect the production steroids, and the thereby the reproduction of fish. Single and binary exposure of prochloraz and trilostane were conducted in both the H295R cells and small fish model Medaka. Trilostane (0.01-0.3 μM) and prochloraz (0.001-0.03 μM) synergistically inhibited the production of several steroid hormones by H295R cells after 12 h exposure. In medaka, co- exposure with trilostane (60 and 600 μg/L) caused no further inhibition on fish egg production by prochloraz (30 μg/L). Results Discussions 1.Advantage of Nu-Serum- free H295R cell culture system in chemical testing. H295R cells was more stable in the unsupplemented culture system, including (1) comparatively unchanged cell number, (2) steroidogenic gene expression. 2. Prochloraz had higher inhibitory potency in Serum-free H295R culture system . 1) Deoxycorticosterone, 2) 17α OH Progesterone, 3) Androstanedione. 3. Prochloraz synergistically enhanced the inhibitory effect of trilostane on steroidogenesis. 1) Corticosterone, 2) Androstanedione. 4.Synergistic effect of co-exposure of trilostane and prochloraz was not observed at the reproduction tract of Medaka fish. Future works should investigate the effects of binary of exposure on the hypothalamus pitutary adrenal(HPA) axis in animals. Reference Zhang et al., Environ. Sci. Technol. 2008, 42 (22), 8614. Figure 2. Comparison of cell numbers (determined as MTT activity) to supplemented and Nu-Serum- and ITS+ Premix-free H295R culture system after changing medium. Values represent mean±S.E.M. of four replicate samples. Significant differences between 0 h and other time points is indicated by *P<0.05. Figure 1, Steroidogenesis pathway affected by endocrine disrupting chemicals. Prochloraz is an inhibitor of CYP17 and CYP19 as indicated by the red bold font; Trilostane is an inhibitor of 3β HSD as indicated by the blue bold font. Corticosterone Aldosterone 3 β- HSD CYP21 CYP11B2 DHEA Androstenedione CYP17 CYP19 CYP21 11 - Deoxycortisol CYP11B1 Cortisol Pregnenolone CYP11A Cholesterol Progesterone Testosterone Estrone CYP19 3 β- HSD CYP17 CYP17 CYP11B2 3 β- HSD 17?- OH Pregnenolone 17?- OH Progesterone CYP17 11- Deoxycorticosterone 17 β- HSD 17?-estradiol 17 β- HSD Corticosterone Aldosterone 3 3 β β - - HSD HSD CYP21 CYP11B2 DHEA Androstenedione CYP17 CYP17 CYP19 CYP19 CYP21 11 - Deoxycortisol CYP11B1 Cortisol Pregnenolone CYP11A Cholesterol Progesterone Testosterone Estrone CYP19 CYP19 CYP17 CYP17 CYP17 CYP17 CYP11B2 17α- OH Pregnenolone 17α- OH Progesterone CYP17 CYP17 11- Deoxycorticosterone 17 β- HSD 17β-estradiol 17 β- HSD 3 3 β β - - HSD HSD 3 3 β β - - HSD HSD 0 2 4 6 8 0 20 40 60 80 100 120 D ay E gg production/fem ale Blank S olvent P ro(30ug/L) Tril(60 µg/L) Tril(600 µg/L) P ro(30ug/L)+ Tril(60 µg/L) P ro(30ug/L)+ Tril(600 µg/L) 5e-04 2e-03 5e-03 2e-02 0.0 0.5 1.0 1.5 2.0 C orticosterone Prochloraz(uM) Fold change 5e-04 2e-03 5e-03 2e-02 0.0 0.5 1.0 1.5 2.0 A ndrostanedione Prochloraz(uM) Fold change * * * * * 5e-04 2e-03 5e-03 2e-02 0 1 2 3 4 5 D eoxycorticosterone Prochloraz(uM) Fold change * * * * * 5e-04 2e-03 5e-03 2e-02 0.0 0.5 1.0 1.5 2.0 2.5 3.0 17a-O H P rogesterone Prochloraz(uM) Fold change * * * * * S upplem ented m edium U nsupplem ented m edium Result 1. Comparison between supplemented and serum-free culture systems 2e-04 1e-03 5e-03 2e-02 1e-01 0.1 0.2 0.5 1.0 2.0 C orticosterone Trilostane(uM) Fold change * * * * * -P rochloraz + P rochloraz(0.001uM ) 2e-04 1e-03 5e-03 2e-02 1e-01 0.02 0.10 0.50 2.00 A ndrostanedione Trilostane(uM) Fold change * * * * * -P rochloraz + P rochloraz(0.001uM ) 2e-04 1e-03 5e-03 2e-02 1e-01 0.05 0.20 1.00 D eoxycorticosterone Trilostane(uM) Fold change * * * * * -P rochloraz + P rochloraz(0.001uM ) 2e-04 1e-03 5e-03 2e-02 1e-01 0.1 0.2 0.5 1.0 2.0 17a-O H P rogesterone Trilostane(uM) Fold change * * * * -P rochloraz + P rochloraz(0.001uM ) Figure 6. Effect of binary exposure of trilostane and prochloraz on fecundity of the medaka fish (O. latipes) within a 8 days exposure. The concentration of chemicals were selected to cause minor effects on fish fecundity in a singular exposure manner, based on the previous results (Zhang et al., 2008; Villeneuve et al., 2008 ) Figure 3. Comparison of steroidogenic gene expression to (A) supplemented and (B) Nu-Serum- and ITS+ Premix-free H295R culture system after changing medium. Values represent mean±S.E.M. of two replicate samples. Significant differences between 0 h and other time points is indicated by *P<0.05. A A B B Figure 4. Comparison of prochloraz induced effects on steroid production by H295R cells from supplemented and Nu-Serum- and ITS+ Premix-free H295R culture system after changing medium. Values represent mean±S.E.M. of four replicate samples. Significant differences between control and different concentrations is indicated by *P<0.05. Figure 5. Trilostane inhibited steroidogenesis in Serum-free H295R culture system in a concentration dependent manner, with or without co- exposure of prochloraz (0.001 μM, NOEC). Values represent mean±S.E.M. of four replicate samples. Significant differences between control and different concentrations is indicated by *P<0.05. Result 2. Effect of binary exposure to trilostane and prochlor in H295R cells (serum-free culture) and Medaka fish
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Transcript of Evaluation of the endocrine disrupting potential of the binary exposure to trilostane and prochloraz...
Evaluation of the endocrine disrupting potential of the binary exposure to trilostane and prochloraz in H295R cells and Medaka fish
Chunsheng LiuChunsheng Liu11, , Xiaowei ZhangXiaowei Zhang22*, *, Saerom ParklSaerom Parkl33, , Jonathon DoeringJonathon Doering22, , Hong ChangHong Chang22, Jong Seong Kim, Jong Seong Kim33, Paul Jones, Paul Jones2,42,4, Bingsheng Zhou, Bingsheng Zhou1*1*, , Markus HeckerMarkus Hecker44 John P. GiesyJohn P. Giesy2,3,4,52,3,4,5 11Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan China, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan China, 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada. 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada. 3 Korea University / Division of 3 Korea University / Division of Environmental Science and Ecological Engineering;Environmental Science and Ecological Engineering;4 4 School of Environment and Sustainability, University of Saskatchewan; 5 School of Environment and Sustainability, University of Saskatchewan; 5 ENTRIX Inc. Saskatoon, SK, Canada; 6 ENTRIX Inc. Saskatoon, SK, Canada; 6 DepDept. Biomedical t. Biomedical Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 5 Dept. Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 5 Dept. Biology Biology & C& Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR Chinahemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China
IntroductionAlthough living organisms are exposed to mixtures of environmental chemicals, most of previous studies on endocrine disrupting chemical (EDCs) have only evaluated individual chemical -induced effects. The objective of this study was to evaluate the mixture effect of two model EDCs, trilostane and prochloraz, on the reproduction axis, especially the steroidogenesis. Prochloraz is a commonly used imidazole fungicide, which has been reported to affect reproduction and development in fish and wildlife by inhibiting CYP genes, including steroidogenic cytochrome P450 c 17α hydroxylase, 17,20-lyase (CYP17) and aromatase (CYP19). Trilostane is an inhibitor of 3 β-hydroxysteroid dehydrogenase (3β HSD) that is upstream of CYP17 and CYP19 on the steroidogenesis pathway. The testable hypothesis is that co-exposure of prochloraz and trilostane could synergistically affect the production steroids, and the thereby the reproduction of fish. Single and binary exposure of prochloraz and trilostane were conducted in both the H295R cells and small fish model Medaka. Trilostane (0.01-0.3 μM) and prochloraz (0.001-0.03 μM) synergistically inhibited the production of several steroid hormones by H295R cells after 12 h exposure. In medaka, co-exposure with trilostane (60 and 600 μg/L) caused no further inhibition on fish egg production by prochloraz (30 μg/L).
Results
Discussions1.Advantage of Nu-Serum- free H295R cell culture system in chemical testing.
H295R cells was more stable in the unsupplemented culture system, including (1) comparatively unchanged cell number, (2) steroidogenic gene expression.2. Prochloraz had higher inhibitory potency in Serum-free H295R culture system .
1) Deoxycorticosterone, 2) 17α OH Progesterone, 3) Androstanedione. 3. Prochloraz synergistically enhanced the inhibitory effect of trilostane on steroidogenesis.
1) Corticosterone, 2) Androstanedione.
4.Synergistic effect of co-exposure of trilostane and prochloraz was not observed at the reproduction tract of Medaka fish. Future works should investigate the effects of binary of exposure on the hypothalamus pitutary adrenal(HPA) axis in animals.
ReferenceZhang et al., Environ. Sci. Technol. 2008, 42 (22), 8614. Villeneuve et al., Toxicol Sci. 2008,104 (1),113-23.
Figure 2. Comparison of cell numbers (determined as MTT activity) to supplemented and Nu-Serum- and ITS+ Premix-free H295R culture system after changing medium. Values represent mean±S.E.M. of four replicate samples. Significant differences between 0 h and other time points is indicated by *P<0.05.
Figure 1, Steroidogenesis pathway affected by endocrine disrupting chemicals. Prochloraz is an inhibitor of CYP17 and CYP19 as indicated by the red bold font; Trilostane is an inhibitor of 3β HSD as indicated by the blue bold font.
Corticosterone
Aldosterone
3 β- HSD
CYP21
CYP11B2
DHEA
Androstenedione
CYP17
CYP19
CYP21
11 - Deoxycortisol
CYP11B1
Cortisol
PregnenoloneCYP11A
Cholesterol
Progesterone
Testosterone Estrone
CYP19
3 β- HSD
CYP17
CYP17
CYP11B2
3 β- HSD
17?- OH
Pregnenolone
17?- OH
Progesterone
CYP17
11- Deoxycorticosterone 17 β- HSD
17?- estradiol
17 β- HSDCorticosterone
Aldosterone
33ββ--HSDHSD
CYP21
CYP11B2
DHEA
Androstenedione
CYP17CYP17
CYP19CYP19
CYP21
11 - Deoxycortisol
CYP11B1
Cortisol
PregnenoloneCYP11A
Cholesterol
Progesterone
Testosterone Estrone
CYP19CYP19
CYP17CYP17
CYP17CYP17
CYP11B2
17α- OH
Pregnenolone
17α- OH
Progesterone
CYP17CYP17
11- Deoxycorticosterone 17 β- HSD
17β- estradiol
17 β- HSD
33ββ--HSDHSD
33ββ--HSDHSD
0 2 4 6 8
02
04
06
08
01
00
12
0
Day
Eg
g p
rod
uct
ion
/fem
ale
BlankSolventPro(30ug/L)Tril(60 µg/L) Tril(600 µg/L) Pro(30ug/L) + Tril(60 µg/L)Pro(30ug/L) + Tril(600 µg/L)
5e-04 2e-03 5e-03 2e-02
0.0
0.5
1.0
1.5
2.0
Corticosterone
Prochloraz(uM)
Fold
change
5e-04 2e-03 5e-03 2e-02
0.0
0.5
1.0
1.5
2.0
Androstanedione
Prochloraz(uM)
Fold
change
**
*
**
5e-04 2e-03 5e-03 2e-02
01
23
45
Deoxycorticosterone
Prochloraz(uM)
Fold
change
**
*
**
5e-04 2e-03 5e-03 2e-02
0.0
0.5
1.0
1.5
2.0
2.5
3.0
17a-OH Progesterone
Prochloraz(uM)
Fold
change
** *
* *
Supplemented mediumUnsupplemented medium
Result 1. Comparison between supplemented and serum-free culture systems
2e-04 1e-03 5e-03 2e-02 1e-01
0.1
0.2
0.5
1.0
2.0
Corticosterone
Trilostane(uM)
Fol
d ch
ange *
*
*
*
*
-Prochloraz+ Prochloraz(0.001uM)
2e-04 1e-03 5e-03 2e-02 1e-01
0.02
0.10
0.50
2.00
Androstanedione
Trilostane(uM)
Fol
d ch
ange *
**
**
-Prochloraz+ Prochloraz(0.001uM)
2e-04 1e-03 5e-03 2e-02 1e-01
0.05
0.20
1.00
Deoxycorticosterone
Trilostane(uM)
Fol
d ch
ange
*
**
*
*-Prochloraz+ Prochloraz(0.001uM)
2e-04 1e-03 5e-03 2e-02 1e-01
0.1
0.2
0.5
1.0
2.0
17a-OH Progesterone
Trilostane(uM)
Fol
d ch
ange
** *
*-Prochloraz+ Prochloraz(0.001uM)
Figure 6. Effect of binary exposure of trilostane and prochloraz on fecundity of the medaka fish (O. latipes) within a 8 days exposure. The concentration of chemicals were selected to cause minor effects on fish fecundity in a singular exposure manner, based on the previous results (Zhang et al., 2008; Villeneuve et al., 2008 )
Figure 3. Comparison of steroidogenic gene expression to (A) supplemented and (B) Nu-Serum- and ITS+ Premix-free H295R culture system after changing medium. Values represent mean±S.E.M. of two replicate samples. Significant differences between 0 h and other time points is indicated by *P<0.05.
AA BB
Figure 4. Comparison of prochloraz induced effects on steroid production by H295R cells from supplemented and Nu-Serum- and ITS+ Premix-free H295R culture system after changing medium. Values represent mean±S.E.M. of four replicate samples. Significant differences between control and different concentrations is indicated by *P<0.05.
Figure 5. Trilostane inhibited steroidogenesis in Serum-free H295R culture system in a concentration dependent manner, with or without co-exposure of prochloraz (0.001 μM, NOEC). Values represent mean±S.E.M. of four replicate samples. Significant differences between control and different concentrations is indicated by *P<0.05.
Result 2. Effect of binary exposure to trilostane and prochloraz in H295R cells (serum-free culture) and Medaka fish
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