Aquatic toxicology effects of TDCPP or t

Aquatic Toxicology 134–135 (2013) 104–111 Contents lists available at SciVerse ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Effects of TDCPP or TPP on gene transcriptions and hormones of HPG axis, and their consequences on reproduction in adult zebrafish (Danio rerio) Xiaoshan Liu a , Kyunghee Ji a,b , Areum Jo a , Hyo-Bang Moon c , Kyungho Choi a,∗ a Graduate School of Public Health and Institute of Health and Environment, 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 College of Science and Technology, Hanyang University, Ansan 426-791, Republic of Korea b a r t i c l e i n f o Article history: Received November 2012 Received in revised form 13 March 2013 Accepted 14 March 2013 Keywords: Organophosphate flame retardants Sex steroid VTG Reproduction Hypothalamus–pituitary–gonad axis a b s t r a c t Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) belong to the group of triester organophosphate flame retardants (OPFRs), which have been used in a wide range of consumer products These chemicals have been frequently detected in effluents, surface water, and fish, and hence their potential adverse effects on aquatic ecosystem are of concern The present study was conducted to investigate the reproduction-related effects and possible molecular mechanisms of TDCPP and TPP using a 21 day reproduction test employing adult zebrafish (Danio rerio) After 21 d of exposure to TDCPP or TPP, significant decrease in fecundity along with significant increases of plasma 17␤-estradiol (E2) concentrations, vitellogenin (VTG) levels, and E2/testosterone (T) and E2/11-ketotestosterone (11-KT) ratios were observed The transcriptional profiles of several genes of the hypothalamus–pituitary–gonad (HPG) axis changed as well after the exposure, but the trend was sex-dependent In male fish, gonadotropin-releasing hormone2 (GnRH2), GnRHR3, cytochrome P450 (CYP) 19B, estrogen receptor ˛ (ER˛), ER2 ˇ1, and follicle stimulating hormone ˇ (FSHˇ) were upregulated in the brain, while luteinizing hormone ˇ (LHˇ) and androgen receptor (AR) were downregulated Corresponding to the upregulation of FSHˇ and downregulation of LHˇ in the brain, FSHR was upregulated and LHR was downregulated in the testis Among the genes that regulate the steroidogenesis pathway, transcription of hydroxyl methyl glutaryl CoA reductase (HMGRA), steroidogenic acute regulatory protein (StAR), and 17ˇ-hydroxysteroid dehydrogenase (17ˇHSD) decreased, while transcription of CYP11A, CYP17, and CYP19A increased In female fish, transcription ofGnRH2 and GnRHR3 decreased, but FSHˇ, LHˇ, CYP19B, ER˛, ER2ˇ1, and AR transcription increased in the brain In the ovary, FSHR and LHR were significantly upregulated, and most steroidogenic genes were significantly upregulated The observed disruption of GnRH and GtHs could be further related to subsequent disruption in both sex steroid hormone balance and plasma VTG levels, as well as reproductive performance Overall, our observation indicates that both TDCPP and TPP could disturb the sex hormone balance by altering regulatory mechanisms of the HPG axis, eventually leading to disruption of reproductive performance in fish © 2013 Elsevier B.V All rights reserved Introduction Abbreviations: AR, androgen receptor; CYP11A, cytochrome P450 side-chain cleavage; 3␤HSD, 3␤-hydroxysteroid dehydrogenase; CYP17, cytochrome P450 17; 17␤HSD, 17␤-hydroxysteroid dehydrogenase; CYP19, cytochrome P450 19; Ct, threshold cycle; DMSO, dimethyl sulfoxide; E2, 17␤-estradiol; ER, estrogen receptor; FSH␤, follicle stimulating hormone ␤; FSHR, follicle stimulating hormone receptor; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; HMGR, hydroxymethylglutaryl CoA reductase; LH␤, luteinizing hormone ␤; LHR, luteinizing hormone receptor; FSH␤, follicle stimulating hormone ␤; FSHR, follicle stimulating hormone receptor; StAR, steroidogenic acute regulatory protein; T, testosterone; 11-KT, 11-ketotestosterone ∗ Corresponding author at: School of Public Health, Seoul National University, Gwanakro, Gwanak, Seoul 151-742, Republic of Korea Tel.: +82 880 2738; fax: +82 745 9104 E-mail address: kyungho@snu.ac.kr (K Choi) 0166-445X/$ – see front matter © 2013 Elsevier B.V All rights reserved http://dx.doi.org/10.1016/j.aquatox.2013.03.013 Due to the phase-out of major commercial polybrominated diphenyl ether (PBDE) mixtures, such as pentaBDE and octaBDE products, and more recently decaBDE, from the markets of USA and Europe since 2003 (Covaci et al., 2011), the production and use of alternative flame retardants (FRs), such as organophosphate flame retardants (OPFRs), have increased (Reemtsma et al., 2008) Among the OPFRs, tris(1,3-dichloro-2-propyl)phosphate (TDCPP) and triphenyl phosphate (TPP) have been widely used in polyurethane foams since 1970s, which are commonly found in sofas, chairs, and car upholstery (Marklund et al., 2003; Reemtsma et al., 2008; Stapleton et al., 2009) These chemicals have also been detected in effluents from German sewage treatment plants (STPs), X Liu et al / Aquatic Toxicology 134–135 (2013) 104–111 indicating inputs from households, industrial sites, and storm water (Regnery and Puttmann, 2010) High concentrations of TDCPP, up to several ␮g/L, have been detected in water at waste disposal sites (Kawagoshi et al., 1999) TDCPP has also been detected at ␮g/L in the effluents from an STP (Marklund et al., 2005) and between 36 and 140 ng/g lipid weight in perch (Perca fluviatilis) caught from ambient water near STPs (Sundkvist et al., 2010) TPP degrades more easily than TDCPP, but the concentrations of TPP that have been detected in marine organisms are in a range similar to those detected in the perch, i.e., between 21 and 180 ng/g (Sundkvist et al., 2010) Metabolites of TDCPP and TPP have also been reported in human urine (Cooper et al., 2011) While information on the endocrine disruption capacity of OPFRs is still generally limited (UNEP, 2002; NRC, 2000; U.S EPA, 2005), available reports suggest that some OPFRs may cause adverse effects on sex hormone balances and possibly endocrine systems Our previous study (Liu et al., 2012) showed that TDCPP and TPP could disturb the sex steroid hormone balance in human adrenal cell (H295R) through several mechanisms including synthesis, metabolism or activation of the sex steroid hormones Sex steroid hormones are important regulators of reproduction processes, and play direct roles during gametogenesis and reproductive maturation (Devlin and Nagahama, 2002) However experimental evidences that show reproduction damages of these chemicals or mechanisms of endocrine disruption in vivo have rarely been reported The present study was conducted to determine the effects of subchronic exposure to TDCPP or TPP on reproduction and reproduction related parameters using zebrafish (Danio rerio) Zebrafish is an attractive model organism for evaluating the reproductive toxicity and endocrine-disrupting effects of xenobiotics because of its small size, ease of culture, short life cycle, and prolific egg production with high fertilization and hatching rates (Segner, 2009) In fish, reproduction is closely regulated by the hypothalamus–pituitary–gonadal (HPG) axis (Sofikitis et al., 2008), Villeneuve et al (2007) described the teleost HPG axis as a novel graphical model in ecotoxicogenomics research on endocrine disrupting chemicals Therefore we also measured transcriptional level changes of several important genes of HPG axis following the exposure The results of this study will help understand the reproduction related disruption of TDCPP and TPP on zebrafish and associated mechanisms of neuroendocrine alteration Materials and methods 2.1 Zebrafish maintenance Wild-type adult male and female zebrafish (4–5 months old) were obtained from a local supplier (Gangnam Aquaria, Suwon Korea) and acclimated for approximately 40 days in a temperaturecontrolled room (27 ◦ C ± ◦ C) in the Environmental Toxicology Laboratory at Seoul National University (Seoul, Korea) The fish were cultured in 30 L glass tanks, filled with the fish culture water The culture water was renewed three times a week Fish were maintained under a photoperiod of 14:10 h light:dark and fed with Artemia nauplii (24 h) tap water The culture water was not detected for residual chlorine and fluoride Just before the exposure, fish were separated by sex for days in a density of 12 adult zebrafish in a 15-L glass tank Then one male and one female fish were selected randomly from the glass tanks and placed in 2-L mating chambers containing exposure media Test concentrations were 0, 0.04, 0.2, or 1.0 mg/L of TDCPP or TPP, which were determined to be non-lethal based on preliminary range finding tests For 21 days of exposure period six pair of fish in six mating chambers were allocated for each treatment or control, and were allowed for mating Exposure medium was renewed in every 48 h Spawned eggs were collected from the bottom of the mating chamber h after the light was turned on every morning The frequency of spawning and the number of eggs per spawning event were recorded throughout the entire test period Up to 50 eggs were randomly selected from each mating chamber on the day of spawning, and were observed for fertilization and hatching success After the 21 day of exposure, all fish in the mating chambers were anesthetized on ice, and body weights and lengths were measured Following Vitale et al (2006), gonadosomatic index (GSI = 100 × [gonad weight (g)/body weight (g)]), hepatosomatic index (HSI = 100 × [gonad weight (g)/body weight (g)]), and condition factor (CF = 100 × [body weight (g)/total length3 (cm)]) values were calculated Samples of 4–10 ␮L of blood per fish were collected from the caudal vein and stored in heparinized microcapillary tubes Blood was sampled from five fish per treatment group or control Collected blood samples were centrifuged at 5000 × g for 20 at ◦ C and the supernatants were separated and stored at −80 ◦ C until analysis The brain, gonads, and liver were dissected out from each fish, weighed, and preserved in 250 ␮L of RNALater reagent (Qiagen, Korea Ltd., Seoul, Korea) at −80 ◦ C until further analysis 2.3 Chemical analysis The actual concentrations of TDCPP or TPP were measured from the exposed media using a gas chromatograph interfaced with a mass spectrometer (GC/MS) Water samples from each treatment group were taken directly from the test beakers before (0 h) and after 48 h of exposure (48 h), and were stored at −80 ◦ C until chemical analysis The following procedures were used for extraction (Okumura and Nishikawa, 1995) Water samples (20 mL) were spiked with surrogate standard (tri-n-butyl-d27 phosphate; CDN Isotope, Pointe-Claire, QB, Canada) which was prepared in methanol (ultra-trace residue analysis grade, J.T Baker, Phillipsburg, NJ, USA) and extracted in mL of dichloromethane (DCM) for 30 with mechanical shaking Then the mixture was allowed to stand for 20 to separate into water and DCM phases The DCM phase was then transferred into 15-mL PP tube (BD Falcon, Franklin Lakes, NJ, USA), and the samples were extracted twice with mL of DCM Approximately g of anhydrous sodium sulfate (pesticide residue analysis grade, Kanto Chemicals, Tokyo, Japan) was added to the DCM extracts to remove any residual water The extracts were concentrated to approximately mL under a stream of nitrogen gas, and were added to mL of n-hexane (ultra-trace residue analysis grade, J.T Baker, Phillipsburg, NJ, USA) for solvent exchange For identification and quantification GC/MS (Agilent 7890A/5975C MSD; Agilent Technologies, Wilmington, DE) was used The MS was operated under positive electron impact mode and ions were monitored using the selected ion monitoring (SIM) mode The capillary column used to separate TDCPP and TPP was 106 X Liu et al / Aquatic Toxicology 134–135 (2013) 104–111 a DB5-MS (30 m length, 0.25 mm inner diameter, 0.25 ␮m film thickness; J&W Scientific, Palo Alto, CA, USA) The recovery rate of spiked tri-n-butyl-d27 phosphate was 106% Solvent (n-hexane) that was injected before and after the injection of standards showed negligible contamination or carryover Procedural blanks were processed with each set of water samples to check for laboratory contamination Blanks did not contain quantifiable amounts of TDCPP or TPP Limits of detection (LOD) for TDCPP (0.06 ng/mL) and TPP (0.12 ng/mL) were calculated as times the signal to noise ratio Details about quantification can be found in Table S1 of supporting information Results 3.1 Measured concentrations of TDCPP or TPP in media The measured concentrations of TDCPP were consistent over the renewal interval, i.e., 48 h (Table S5 of supporting information) However, measured concentrations of TPP after 48 h of exposure were much lower than those of TDCPP, suggesting that TPP was degraded more rapidly than TDCPP For simplicity, nominal concentrations are used when presenting results in this paper 3.2 Effects on reproduction, and growth 2.4 Sex hormones and vitellogenin measurements Plasma sex steroid hormones were measured in both male and female fish by competitive enzyme-linked immunosorbent assay (ELISA) using commercially available kits (testosterone [Cat # 582701], 17␤-estradiol [Cat # 582251]; Cayman Chemical, Ann Arbor, MI, USA) 11-Ketotestosterone (11-KT) was measured by ELISA using a kit (11-ketotestosterone [Cat # 582751; Cayman Chemical]) Intra- and interassay coefficients of variation (CV) were