Introduction

1. Evaluation of the endocrine disrupting potential of the binary exposure to trilostane and prochloraz in H295R cells and Medaka fish Chunsheng Liu1, Xiaowei Zhang2*, Saerom Parkl3, Jonathon Doering2, Hong Chang2, Jong Seong Kim3, Paul Jones2,4, Bingsheng Zhou1*, Markus Hecker4John P. Giesy2,3,4,5 1Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan China, 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada. 3 Korea University / Division of Environmental Science and Ecological Engineering;4 School of Environment and Sustainability, University of Saskatchewan; 5 ENTRIX Inc. Saskatoon, SK, Canada; 6Dept. Biomedical Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 5 Dept. Biology & Chemistry, 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 Result 2. Effect of binary exposure to trilostane and prochloraz in H295R cells (serum-free culture) and Medaka fish Result 1. Comparison between supplemented and serum-free culture systems 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. 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. B A 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. • Discussions • 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. • 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. • Villeneuve et al., Toxicol Sci. 2008,104(1),113-23. CYP11A CYP11A CYP17 CYP17 CYP17 CYP17 Cholesterol Cholesterol Pregnenolone Pregnenolone 17 17 ? α - - OH OH DHEA DHEA Pregnenolone Pregnenolone 3 - HSD β 3β-HSD 3β-HSD 3β-HSD 3 - HSD β 3 - HSD β Progesterone Progesterone CYP17 CYP17 17 17 ? α - - OH OH Androstenedione Androstenedione CYP17 CYP17 CYP21 CYP21 Progesterone Progesterone 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. CYP19 CYP19 17 17 - - HSD HSD β β 11 11 - - Deoxycorticosterone Deoxycorticosterone CYP21 CYP21 Estrone Estrone Testosterone Testosterone 11 11 - - Deoxycortisol Deoxycortisol CYP11B2 CYP11B2 Corticosterone Corticosterone 17 17 - - HSD HSD β β CYP11B1 CYP11B1 CYP19 CYP19 CYP11B2 CYP11B2 17 17 β ? - - estradiol estradiol Cortisol Cortisol Aldosterone Aldosterone 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.