Accumulation of Di(2-ethylhexyl) Phthalate Causes Endocrine-Disruptive Effects in Marine Medaka (Oryzias melastigma) Embryos Ting Ye, Mei Kang, Qiansheng Huang, Chao Fang, Yajie Chen, Liangpo Liu, Sijun Dong Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, People’s Republic of China
Received 8 January 2014; revised 6 July 2014; accepted 13 July 2014 ABSTRACT: Di (2-ethylhexyl) phthalate (DEHP) is extensively distributed in marine environments. However, limited research on the toxicological and molecular effects of DEHP on marine organisms has been conducted. Our study investigated the accumulation, elimination, and endocrine-disruptive effects of DEHP on embryonic marine medaka (Oryzias melastigma). The medaka embryos were continuously exposed to DEHP (0.01, 0.1, and 1 mg/L) or 17b-estradiol (E2, 0.01 mg/L) until hatching, and the newly hatched larvae were then transferred to clean sea water for 12 days of depuration. DEHP and E2 appeared to have no significant effects on the mortality and hatching rates of medaka embryos, but E2 exposure significantly delayed the hatching. Significantly higher DEHP embryonic burdens were detected in the group treated with higher DEHP (0.1 and 1 mg/L) at 10 dpf (days post fertilization). The recovered larvae showed an elimination tendency of DEHP during the recovery period. DEHP had no significant effects on the transcriptional responses of endocrine-disrupting biomarker genes in the 3-dpf embryos. Treatment with 0.1 and 1 mg/L DEHP elicited a significant induction of transcriptional responses of ER, PPAR, and the CYP19 genes in a concentration-dependent manner at 10 dpf, indicating endocrine disruption may be due to bioaccumulation of DEHP. With the elimination of DEHP during the depuration period, all of the effects on these genes showed no significant effects. However, 0.1 mg/L E2 significantly affected the expression of ER, PPAR, and the CYP19 genes in the exposed embryos at both 3 and 10 dpf and recovered larvae. Therefore, these results demonstrate that accumulation of DEHP caused endocrine disruption in medaka embryos and that recovery in clean sea water may weaken the endocrineC 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 116–127, 2016. disrupting effects. V Keywords: DEHP; Oryzias melastigma; accumulation; elimination; endocrine-disruptive effects
INTRODUCTION Correspondence to: Sijun Dong; e-mail: [email protected] Contract grant sponsor: Special Scientific Research Funds for Environmental Protection Commonweal Section. Contract grant number: 201309027. Contract grant sponsor: National Natural Science Foundation of China. Contract grant numbers: 21207127, 21277137. Contract grant sponsor: Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences. Contract grant number: KLUEH-S-201303. Published online 28 July 2014 in Wiley (wileyonlinelibrary.com). DOI: 10.1002/tox.22028
Online
Library
Phthalate esters (PAEs) have been widely used as a plasticizer in PVC formulation, such as in the manufacture of construction products, medical devices, pharmaceuticals, and personal care products (Heudorf et al., 2007). Moreover, these widely produced phthalates are not chemically bound to PVC and can easily leach into the environment (Fromme et al., 2002). Therefore, PAEs are ubiquitous in the environment. PAEs have received attention as a marine pollutant because they are widely distributed in marine environments with concentrations reaching up to 300 lg/L in surface marine water, 3 lg/g in surface marine sediment, and 4.07 ng/g in marine
C 2014 Wiley Periodicals, Inc. V
116
ACCUMULATION OF DEHP CAUSES ENDOCRINE-DISRUPTIVE EFFECTS
organisms (GIAM et al., 1978; Liu et al., 2009). Di (2-ethylhexyl) phthalate (DEHP) is the most widely produced and used phthalate and the most persistent phthalate found in wastewaters (Gavala et al., 2003; Chaler et al., 2004). DEHP accounts for approximately half of the phthalate concentrations, which range from 0.33 to 97.8 lg/L in surface water, 1.74–182 lg/L in sewage effluents, 27.9–154 mg/kg dw (dry weight) in sewage sludge and 0.21–8.44 mg/kg in sediment (Fromme et al., 2002). The DEHP concentrations in marine environments are on the same order of magnitude as those in fresh water environments (Peijnenburg and Struijs, 2006). DEHP was reported to reach 3390 lg/kg dw in marine surface sediments, 0.16 lg/L in marine water and 1.8 lg/kg in marine fish (Klamer et al., 2005; Peijnenburg and Struijs, 2006). The discharge of DEHP into the marine aquatic environment has accumulated in marine fish, and eventually entered the food chain. Thus, a better understanding of the toxic effects of DEHP on marine fish is needed. As one of the most common endocrine-disrupting chemicals (EDC), DEHP has been reported to cause adverse effects on development and reproductive function through activation of the estrogen receptor (ER) and peroxisome proliferator-activated receptor (PPAR) in mammals (Lyche et al., 2009; Magdouli et al., 2013). DEHP showed antiestrogenic activity in female medaka, which was presented by decreased vitellogenin (VTG) levels and retardation of oocyte development (Kim et al., 2002). DEHP deeply impaired fecundity with serious impacts on zebrafish oogenesis and embryo production in female zebrafish by affecting signals involved in oocyte growth, maturation and ovulation (Carnevali et al., 2010). Furthermore, DEHP disrupts spermatogenesis by decreasing the ability to fertilize oocytes spawned by untreated females via PPAR signaling pathways in the testis and oestrogen signaling pathways in the liver of adult male zebrafish (Uren-Webster et al., 2010). DEHP was also found to show estrogenic potency in both male and female hepatocyte cultures using target molecules involved in fish reproduction (VTG and ERa, b1 and b2) and metabolism (PPARa, b, g) (Maradonna et al., 2013). Additionally, DEHP caused endocrine-disrupting effects by modulating the transcription of genes involved in steroidogenesis, subsequently altering sex hormone levels in some freshwater fish Abbreviation ChgH ChgL CYP19 DEHP DMSO dpf EDCs E2 ER PPAR PAEs VTG
Choriogenin H Choriogenin L Cytochrome P450 19 Di(2-ethylhexyl) phthalate Dimethyl sulfoxide Days post fertilization Endocrine disrupting chemicals 17b-estradiol Estrogen receptor Peroxisome proliferator-activated receptor Phthalate esters Vitellogenin
117
species, such as the Chinese rare minnow (Gobiocypris rarus) (Wang et al., 2013), carp (Cyprinus carpio) (Thibaut and Porte 2004), fathead minnows (Pimephales promelas) (Crago and Klaper, 2012). However, previous studies that detected the endocrine-disruptive effects of DEHP generally focused on a few of estrogen-responsive genes and only evaluated adult fish. Transcript analysis of individual genes is transient and may not reflect the true nature of xenoestrogen exposure (Arukwe et al., 2001). Therefore, the utilization of multiple estrogen-responsive genes holds great importance for the reliable and accurate evaluation of the endocrine-disruptive effects of DEHP in the aquatic environment. Several estrogenic biomarker genes, e.g., ERa, ERb, ERg, VTG1, VTG2, choriogenin H (ChgH), choriogenin L (ChgL), have been shown to be sensitive biomarkers associated with estrogen and estrogen-like pollutants in fish (Chen et al., 2008; Jin et al., 2011). Additionally, two CYP19 (cytochrome P450 aromatase) genes, CYP19a and CYP19b, which regulate the rate of estrogen production, are considered to be a potential target of estrogen-like pollutants. Alteration of the expression of these CYP19 genes can dramatically alter the rate of estrogen production, consequently leading to disturbances of reproductive processes (Cheshenko et al., 2008). Fish are most sensitive to pollutants during early developmental stages, and exposure to EDC during these stages may cause adverse consequences and permanent damage (Hamlin and Guillette, 2011). Investigation of DEHP during embryonic stages may be critical for understanding the mechanisms of the endocrine-disrupting effects of DEHP. Moreover, embryo toxicity analysis has been widely applied as an alternative for fish toxicity tests due to its cost-effectiveness, adequate throughput, straightforward assay and good reproducibility (Brannen et al., 2010; Embry et al., 2010). Nevertheless, most research studies regarding the toxicity of DEHP have focused on fresh water and adult fish, and the effects in marine fish during early developmental stages, especially during the embryonic stage, have received limited attention. Thus, a marine fish embryo is urgently needed to investigate the endocrinedisrupting effects in the marine environment. The marine medaka, Oryzias melastigma, has been widely used as a marine fish model for ecotoxicological studies because of its wide salinity and temperature adaptation, short generation time and high reproductive rate. Additionally, the marine medaka embryo has a small size, is easy to culture and has high transparency, all of which make it a valuable and sensitive species for environmental risk assessment in marine environments (Bo et al., 2011; Chen et al., 2009, 2011). The toxicity of xenobiotics on fish is determined partly by their bioaccumulation and elimination process, and the toxicant uptake/elimination kinetic process is determined partly by fish metabolic rate (Landrum et al., 2013). Because early developmental stages of fish demonstrate sensitivity to many compounds, understanding these processes in embryonic and larval fish is particularly important. Investigation of
Environmental Toxicology DOI 10.1002/tox
118
YE ET AL.
the relationships between the toxicant accumulation/elimination kinetic process and toxic effects is necessary to understand the toxicity of chemicals. The uptake and elimination of DEHP has been well studied in mammalian species, but there has been limited research on aquatic organisms and especially marine organisms. To address these issues, we investigated the relationships between the toxicant accumulation/elimination kinetic process and endocrine-disrupting effects of DEHP on medaka embryos and recovered larvae. Furthermore, because some fish species have the ability to recover after chemical exposure (de Menezes et al., 2011), we also assessed the ability of marine medaka to recover from endocrine-disrupting effects after embryonic exposure to DEHP. We first investigated the uptake and elimination of DEHP in marine medaka (O. melastigma) embryos upon DEHP (0.01, 0.1, and 1 mg/ L) exposure and larvae recovered in clean seawater. Then, the effects of DEHP and positive control 17b-estradiol (E2, 0.01 mg/L) on endocrine-disrupting biomarker pathways and genes including two important EDC-related receptor pathways (ER and PPAR) and the CYP19 genes were investigated in the marine medaka embryos of early [3 dpf (days post fertilization)] and late (10 dpf) developmental stages and recovered larvae (depuration in clean sea water for 8 days).
MATERIALS AND METHODS
clear artificial sea water and acclimated under the same condition for one day (1 dpf) before experiments.
Exposure Experiment The 1-dpf embryos were statically exposed to DEHP (0.01, 0.1, and 1 mg/L), solvent control (0.1% DMSO) and positive control of E2 (0.01 mg/L) in salt water for 9 days, which were nominal concentrations in all cases. The range of concentrations was selected based on previous reports (Staples et al., 1997), in which DEHP concentrations ranged from 0.01 to 2 mg/L in exposure water in studies with several freshwater species. Three replicates for each treatment were used, and all replicates received 0.1% DMSO. Each replicate consisted of 100 viable embryos. Embryos of each replicate were distributed in 90 mm glass dishes containing 40 mL artificial seawater, and the media were renewed every day. The embryo was considered dead when the color of the embryo turned white. The hatching time and rate and mortality rate were recorded for each treatment every day. After exposure for 9 days, the newly hatched larvae were transferred to clean seawater for depuration of 12 days. For DEHP exposure, the embryos were sampled at 3, 5, 7, and 10 dpf and the larvae were sampled at 14, 18, and 22 dpf during the depuration period. For E2 exposure, the embryos were sampled at 3 and 10 dpf and the larvae were sampled at 18 dpf (recovery in clean sea water for 8 days). The medaka embryos and larvae were frozen and stored at 280 C for subsequent experiment.
Chemicals DEHP obtained from Supelco (Bellefont, PA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Corp. St. Louis, MO). Bis (2-ethylhexyl) Phthalate-3,4,5,6-d4 (d4DEHP) and standard DEHP were obtained from Cambridge Isotope Laboratories (Andover, MA). The chemicals prepared for GC/MS analysis were HPLC grade. All other chemicals used in this study were analytical grade.
Fish Maintenance O.melastigma were raised and kept in artificial seawater at a salinity of 30& and standard laboratory conditions of 28 6 1 C on a 14 : 10 light/dark photoperiod in a recirculation system (7960 Stromesa Court, San Diego, USA). The fish were fed twice daily with freshly hatched Artemia nauplii at 9:00 am and 3:00 pm. Embryos spawned by healthy 6-month-old females paired with healthy males of the same age were collected from the female abdomen. All collected embryos were checked for health conditions and developmental features under a microscope (XTL-340, Changfang Optical Instrument, Shanghai, China PR) mounted with a CCD camera (CF-2098, Changfang Optical Instrument) to ensure the collected embryos were healthy, fertilized, and freshly spawned. Then, all selected embryos were rinsed in
Environmental Toxicology DOI 10.1002/tox
Quantification of DEHP The DEHP concentrations were measured in medaka embryos and larvae. The endpoints were selected as follows: embryonic stages (3, 5, 7, and 10 dpf) and larval stages (14, 18, and 22 dpf). The DEHP concentrations were determined by Gas chromatography (GC) (7890A GC system, Agilent Technologies, USA) in conjunction with tandem mass spectrometry (MS) (5975 inert XL MSD with Triple-Axis Detector, Agilent Technologies, USA). The method for sample preparation was based on a previous study with minor modifications (Lin et al., 2003). A total of 10 embryos or five fishes were pooled from one replicate as a single sample and three replicates were performed for each experimental group. Tissue samples were rinsed twice with 10 mL of deionized water and homogenized using a glass homogenizer. All samples were then spiked with 0.04 mg d4-DEHP as an internal standard. The homogenate was extracted with 1 : 1 (v/v) dichloromethane/hexane (DCM/Hex) in an ultrasonic water-bath for 10 min, shaken on a shaker table for 30 s, and centrifuged at 4 C for 10 min at 3000 RPM to separate the organic layers. The organic layer was removed and the extraction was repeated twice with fresh solvent. The extracts of embryos or larvae were evaporated under a gentle stream of nitrogen, the residue was reconstituted in 0.2 mL
ACCUMULATION OF DEHP CAUSES ENDOCRINE-DISRUPTIVE EFFECTS
of hexane, and the supernatant was removed into an auto sampler vial (7693 Autosampler, Agilent Technologies, USA). The extract was then analyzed by GC-MS in the selected ion mode (m/z 149 for DEHP and m/z 153 for d4DEHP).
119
ing time of embryos. However, E2 exposure significantly delayed the hatching time of medaka embryos, which was delayed by 2.2 days relative to the control group, with an average hatching time of 13.6 6 0.3 dpf. Embryonic exposure to DEHP and E2 exhibited no significant effects on the mortality rates of recovered larvae.
Quantitative RT-PCR Analyses Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of 12 targeted genes. Ten embryos or two larvae per replicate were randomly collected at 3 dpf, 10 dpf, and 18 dpf (8 days depuration in clean sea water). Then, omega kits (Omega Bio-Tek, Inc. Norcross, USA) were used to extract total RNA following the manufacturer’s protocol. The purified total RNA yield and quality was determined with a spectrophotometer. Reverse transcription was performed using the PrimeScript RT master mix perfect real time kit (DRR036A, TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instructions. qRT-PCR was carried out using the SYBR Premix Ex TaqTM kit (TaKaRa Bio, Shiga, Japan) on a Roche Light Cycler 480 II. The primers were used in the qRT-PCR analysis according to our previous study (Fang et al., 2012; Ye et al., 2014). The thermal cycle began with an initial denaturation step at 95 C for 30 s, followed by 40 cycles at 95 C for 5 s and 60 C for 34 s. Finally, a dissociation curve analysis was performed following the thermal cycle. Three technical replicates of PCR reaction for each tested gene were performed. The ribosomal protein l7 (RPL7) expression levels were relative stable under chemicals exposure, and thus RPL7 was used as a reference for the expression calculation of the target genes. The relative expression levels were calculated using the 22DDCt method (Livak and Schmittgen, 2001).
Data Analysis All statistical analyses were performed using SPSS 16.0 software. One-way ANOVA followed by Tukey’s test (post hoc) were used to determine significant differences between all treatment groups. The data were checked for normality and homogeneity of variance, and the data were logtransformed to approximate normality when necessary. A p value
Received 8 January 2014; revised 6 July 2014; accepted 13 July 2014 ABSTRACT: Di (2-ethylhexyl) phthalate (DEHP) is extensively distributed in marine environments. However, limited research on the toxicological and molecular effects of DEHP on marine organisms has been conducted. Our study investigated the accumulation, elimination, and endocrine-disruptive effects of DEHP on embryonic marine medaka (Oryzias melastigma). The medaka embryos were continuously exposed to DEHP (0.01, 0.1, and 1 mg/L) or 17b-estradiol (E2, 0.01 mg/L) until hatching, and the newly hatched larvae were then transferred to clean sea water for 12 days of depuration. DEHP and E2 appeared to have no significant effects on the mortality and hatching rates of medaka embryos, but E2 exposure significantly delayed the hatching. Significantly higher DEHP embryonic burdens were detected in the group treated with higher DEHP (0.1 and 1 mg/L) at 10 dpf (days post fertilization). The recovered larvae showed an elimination tendency of DEHP during the recovery period. DEHP had no significant effects on the transcriptional responses of endocrine-disrupting biomarker genes in the 3-dpf embryos. Treatment with 0.1 and 1 mg/L DEHP elicited a significant induction of transcriptional responses of ER, PPAR, and the CYP19 genes in a concentration-dependent manner at 10 dpf, indicating endocrine disruption may be due to bioaccumulation of DEHP. With the elimination of DEHP during the depuration period, all of the effects on these genes showed no significant effects. However, 0.1 mg/L E2 significantly affected the expression of ER, PPAR, and the CYP19 genes in the exposed embryos at both 3 and 10 dpf and recovered larvae. Therefore, these results demonstrate that accumulation of DEHP caused endocrine disruption in medaka embryos and that recovery in clean sea water may weaken the endocrineC 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 116–127, 2016. disrupting effects. V Keywords: DEHP; Oryzias melastigma; accumulation; elimination; endocrine-disruptive effects
INTRODUCTION Correspondence to: Sijun Dong; e-mail: [email protected] Contract grant sponsor: Special Scientific Research Funds for Environmental Protection Commonweal Section. Contract grant number: 201309027. Contract grant sponsor: National Natural Science Foundation of China. Contract grant numbers: 21207127, 21277137. Contract grant sponsor: Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences. Contract grant number: KLUEH-S-201303. Published online 28 July 2014 in Wiley (wileyonlinelibrary.com). DOI: 10.1002/tox.22028
Online
Library
Phthalate esters (PAEs) have been widely used as a plasticizer in PVC formulation, such as in the manufacture of construction products, medical devices, pharmaceuticals, and personal care products (Heudorf et al., 2007). Moreover, these widely produced phthalates are not chemically bound to PVC and can easily leach into the environment (Fromme et al., 2002). Therefore, PAEs are ubiquitous in the environment. PAEs have received attention as a marine pollutant because they are widely distributed in marine environments with concentrations reaching up to 300 lg/L in surface marine water, 3 lg/g in surface marine sediment, and 4.07 ng/g in marine
C 2014 Wiley Periodicals, Inc. V
116
ACCUMULATION OF DEHP CAUSES ENDOCRINE-DISRUPTIVE EFFECTS
organisms (GIAM et al., 1978; Liu et al., 2009). Di (2-ethylhexyl) phthalate (DEHP) is the most widely produced and used phthalate and the most persistent phthalate found in wastewaters (Gavala et al., 2003; Chaler et al., 2004). DEHP accounts for approximately half of the phthalate concentrations, which range from 0.33 to 97.8 lg/L in surface water, 1.74–182 lg/L in sewage effluents, 27.9–154 mg/kg dw (dry weight) in sewage sludge and 0.21–8.44 mg/kg in sediment (Fromme et al., 2002). The DEHP concentrations in marine environments are on the same order of magnitude as those in fresh water environments (Peijnenburg and Struijs, 2006). DEHP was reported to reach 3390 lg/kg dw in marine surface sediments, 0.16 lg/L in marine water and 1.8 lg/kg in marine fish (Klamer et al., 2005; Peijnenburg and Struijs, 2006). The discharge of DEHP into the marine aquatic environment has accumulated in marine fish, and eventually entered the food chain. Thus, a better understanding of the toxic effects of DEHP on marine fish is needed. As one of the most common endocrine-disrupting chemicals (EDC), DEHP has been reported to cause adverse effects on development and reproductive function through activation of the estrogen receptor (ER) and peroxisome proliferator-activated receptor (PPAR) in mammals (Lyche et al., 2009; Magdouli et al., 2013). DEHP showed antiestrogenic activity in female medaka, which was presented by decreased vitellogenin (VTG) levels and retardation of oocyte development (Kim et al., 2002). DEHP deeply impaired fecundity with serious impacts on zebrafish oogenesis and embryo production in female zebrafish by affecting signals involved in oocyte growth, maturation and ovulation (Carnevali et al., 2010). Furthermore, DEHP disrupts spermatogenesis by decreasing the ability to fertilize oocytes spawned by untreated females via PPAR signaling pathways in the testis and oestrogen signaling pathways in the liver of adult male zebrafish (Uren-Webster et al., 2010). DEHP was also found to show estrogenic potency in both male and female hepatocyte cultures using target molecules involved in fish reproduction (VTG and ERa, b1 and b2) and metabolism (PPARa, b, g) (Maradonna et al., 2013). Additionally, DEHP caused endocrine-disrupting effects by modulating the transcription of genes involved in steroidogenesis, subsequently altering sex hormone levels in some freshwater fish Abbreviation ChgH ChgL CYP19 DEHP DMSO dpf EDCs E2 ER PPAR PAEs VTG
Choriogenin H Choriogenin L Cytochrome P450 19 Di(2-ethylhexyl) phthalate Dimethyl sulfoxide Days post fertilization Endocrine disrupting chemicals 17b-estradiol Estrogen receptor Peroxisome proliferator-activated receptor Phthalate esters Vitellogenin
117
species, such as the Chinese rare minnow (Gobiocypris rarus) (Wang et al., 2013), carp (Cyprinus carpio) (Thibaut and Porte 2004), fathead minnows (Pimephales promelas) (Crago and Klaper, 2012). However, previous studies that detected the endocrine-disruptive effects of DEHP generally focused on a few of estrogen-responsive genes and only evaluated adult fish. Transcript analysis of individual genes is transient and may not reflect the true nature of xenoestrogen exposure (Arukwe et al., 2001). Therefore, the utilization of multiple estrogen-responsive genes holds great importance for the reliable and accurate evaluation of the endocrine-disruptive effects of DEHP in the aquatic environment. Several estrogenic biomarker genes, e.g., ERa, ERb, ERg, VTG1, VTG2, choriogenin H (ChgH), choriogenin L (ChgL), have been shown to be sensitive biomarkers associated with estrogen and estrogen-like pollutants in fish (Chen et al., 2008; Jin et al., 2011). Additionally, two CYP19 (cytochrome P450 aromatase) genes, CYP19a and CYP19b, which regulate the rate of estrogen production, are considered to be a potential target of estrogen-like pollutants. Alteration of the expression of these CYP19 genes can dramatically alter the rate of estrogen production, consequently leading to disturbances of reproductive processes (Cheshenko et al., 2008). Fish are most sensitive to pollutants during early developmental stages, and exposure to EDC during these stages may cause adverse consequences and permanent damage (Hamlin and Guillette, 2011). Investigation of DEHP during embryonic stages may be critical for understanding the mechanisms of the endocrine-disrupting effects of DEHP. Moreover, embryo toxicity analysis has been widely applied as an alternative for fish toxicity tests due to its cost-effectiveness, adequate throughput, straightforward assay and good reproducibility (Brannen et al., 2010; Embry et al., 2010). Nevertheless, most research studies regarding the toxicity of DEHP have focused on fresh water and adult fish, and the effects in marine fish during early developmental stages, especially during the embryonic stage, have received limited attention. Thus, a marine fish embryo is urgently needed to investigate the endocrinedisrupting effects in the marine environment. The marine medaka, Oryzias melastigma, has been widely used as a marine fish model for ecotoxicological studies because of its wide salinity and temperature adaptation, short generation time and high reproductive rate. Additionally, the marine medaka embryo has a small size, is easy to culture and has high transparency, all of which make it a valuable and sensitive species for environmental risk assessment in marine environments (Bo et al., 2011; Chen et al., 2009, 2011). The toxicity of xenobiotics on fish is determined partly by their bioaccumulation and elimination process, and the toxicant uptake/elimination kinetic process is determined partly by fish metabolic rate (Landrum et al., 2013). Because early developmental stages of fish demonstrate sensitivity to many compounds, understanding these processes in embryonic and larval fish is particularly important. Investigation of
Environmental Toxicology DOI 10.1002/tox
118
YE ET AL.
the relationships between the toxicant accumulation/elimination kinetic process and toxic effects is necessary to understand the toxicity of chemicals. The uptake and elimination of DEHP has been well studied in mammalian species, but there has been limited research on aquatic organisms and especially marine organisms. To address these issues, we investigated the relationships between the toxicant accumulation/elimination kinetic process and endocrine-disrupting effects of DEHP on medaka embryos and recovered larvae. Furthermore, because some fish species have the ability to recover after chemical exposure (de Menezes et al., 2011), we also assessed the ability of marine medaka to recover from endocrine-disrupting effects after embryonic exposure to DEHP. We first investigated the uptake and elimination of DEHP in marine medaka (O. melastigma) embryos upon DEHP (0.01, 0.1, and 1 mg/ L) exposure and larvae recovered in clean seawater. Then, the effects of DEHP and positive control 17b-estradiol (E2, 0.01 mg/L) on endocrine-disrupting biomarker pathways and genes including two important EDC-related receptor pathways (ER and PPAR) and the CYP19 genes were investigated in the marine medaka embryos of early [3 dpf (days post fertilization)] and late (10 dpf) developmental stages and recovered larvae (depuration in clean sea water for 8 days).
MATERIALS AND METHODS
clear artificial sea water and acclimated under the same condition for one day (1 dpf) before experiments.
Exposure Experiment The 1-dpf embryos were statically exposed to DEHP (0.01, 0.1, and 1 mg/L), solvent control (0.1% DMSO) and positive control of E2 (0.01 mg/L) in salt water for 9 days, which were nominal concentrations in all cases. The range of concentrations was selected based on previous reports (Staples et al., 1997), in which DEHP concentrations ranged from 0.01 to 2 mg/L in exposure water in studies with several freshwater species. Three replicates for each treatment were used, and all replicates received 0.1% DMSO. Each replicate consisted of 100 viable embryos. Embryos of each replicate were distributed in 90 mm glass dishes containing 40 mL artificial seawater, and the media were renewed every day. The embryo was considered dead when the color of the embryo turned white. The hatching time and rate and mortality rate were recorded for each treatment every day. After exposure for 9 days, the newly hatched larvae were transferred to clean seawater for depuration of 12 days. For DEHP exposure, the embryos were sampled at 3, 5, 7, and 10 dpf and the larvae were sampled at 14, 18, and 22 dpf during the depuration period. For E2 exposure, the embryos were sampled at 3 and 10 dpf and the larvae were sampled at 18 dpf (recovery in clean sea water for 8 days). The medaka embryos and larvae were frozen and stored at 280 C for subsequent experiment.
Chemicals DEHP obtained from Supelco (Bellefont, PA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Corp. St. Louis, MO). Bis (2-ethylhexyl) Phthalate-3,4,5,6-d4 (d4DEHP) and standard DEHP were obtained from Cambridge Isotope Laboratories (Andover, MA). The chemicals prepared for GC/MS analysis were HPLC grade. All other chemicals used in this study were analytical grade.
Fish Maintenance O.melastigma were raised and kept in artificial seawater at a salinity of 30& and standard laboratory conditions of 28 6 1 C on a 14 : 10 light/dark photoperiod in a recirculation system (7960 Stromesa Court, San Diego, USA). The fish were fed twice daily with freshly hatched Artemia nauplii at 9:00 am and 3:00 pm. Embryos spawned by healthy 6-month-old females paired with healthy males of the same age were collected from the female abdomen. All collected embryos were checked for health conditions and developmental features under a microscope (XTL-340, Changfang Optical Instrument, Shanghai, China PR) mounted with a CCD camera (CF-2098, Changfang Optical Instrument) to ensure the collected embryos were healthy, fertilized, and freshly spawned. Then, all selected embryos were rinsed in
Environmental Toxicology DOI 10.1002/tox
Quantification of DEHP The DEHP concentrations were measured in medaka embryos and larvae. The endpoints were selected as follows: embryonic stages (3, 5, 7, and 10 dpf) and larval stages (14, 18, and 22 dpf). The DEHP concentrations were determined by Gas chromatography (GC) (7890A GC system, Agilent Technologies, USA) in conjunction with tandem mass spectrometry (MS) (5975 inert XL MSD with Triple-Axis Detector, Agilent Technologies, USA). The method for sample preparation was based on a previous study with minor modifications (Lin et al., 2003). A total of 10 embryos or five fishes were pooled from one replicate as a single sample and three replicates were performed for each experimental group. Tissue samples were rinsed twice with 10 mL of deionized water and homogenized using a glass homogenizer. All samples were then spiked with 0.04 mg d4-DEHP as an internal standard. The homogenate was extracted with 1 : 1 (v/v) dichloromethane/hexane (DCM/Hex) in an ultrasonic water-bath for 10 min, shaken on a shaker table for 30 s, and centrifuged at 4 C for 10 min at 3000 RPM to separate the organic layers. The organic layer was removed and the extraction was repeated twice with fresh solvent. The extracts of embryos or larvae were evaporated under a gentle stream of nitrogen, the residue was reconstituted in 0.2 mL
ACCUMULATION OF DEHP CAUSES ENDOCRINE-DISRUPTIVE EFFECTS
of hexane, and the supernatant was removed into an auto sampler vial (7693 Autosampler, Agilent Technologies, USA). The extract was then analyzed by GC-MS in the selected ion mode (m/z 149 for DEHP and m/z 153 for d4DEHP).
119
ing time of embryos. However, E2 exposure significantly delayed the hatching time of medaka embryos, which was delayed by 2.2 days relative to the control group, with an average hatching time of 13.6 6 0.3 dpf. Embryonic exposure to DEHP and E2 exhibited no significant effects on the mortality rates of recovered larvae.
Quantitative RT-PCR Analyses Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of 12 targeted genes. Ten embryos or two larvae per replicate were randomly collected at 3 dpf, 10 dpf, and 18 dpf (8 days depuration in clean sea water). Then, omega kits (Omega Bio-Tek, Inc. Norcross, USA) were used to extract total RNA following the manufacturer’s protocol. The purified total RNA yield and quality was determined with a spectrophotometer. Reverse transcription was performed using the PrimeScript RT master mix perfect real time kit (DRR036A, TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instructions. qRT-PCR was carried out using the SYBR Premix Ex TaqTM kit (TaKaRa Bio, Shiga, Japan) on a Roche Light Cycler 480 II. The primers were used in the qRT-PCR analysis according to our previous study (Fang et al., 2012; Ye et al., 2014). The thermal cycle began with an initial denaturation step at 95 C for 30 s, followed by 40 cycles at 95 C for 5 s and 60 C for 34 s. Finally, a dissociation curve analysis was performed following the thermal cycle. Three technical replicates of PCR reaction for each tested gene were performed. The ribosomal protein l7 (RPL7) expression levels were relative stable under chemicals exposure, and thus RPL7 was used as a reference for the expression calculation of the target genes. The relative expression levels were calculated using the 22DDCt method (Livak and Schmittgen, 2001).
Data Analysis All statistical analyses were performed using SPSS 16.0 software. One-way ANOVA followed by Tukey’s test (post hoc) were used to determine significant differences between all treatment groups. The data were checked for normality and homogeneity of variance, and the data were logtransformed to approximate normality when necessary. A p value
