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Keywords:

  • DEHP;
  • Oryzias melastigma;
  • accumulation;
  • elimination;
  • endocrine-disruptive effects

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

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 17β-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 endocrine-disrupting effects. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 116–127, 2016.


Abbreviation
ChgH

Choriogenin H

ChgL

Choriogenin L

CYP19

Cytochrome P450 19

DEHP

Di(2-ethylhexyl) phthalate

DMSO

Dimethyl sulfoxide

dpf

Days post fertilization

EDCs

Endocrine disrupting chemicals

E2

17β-estradiol

ER

Estrogen receptor

PPAR

Peroxisome proliferator-activated receptor

PAEs

Phthalate esters

VTG

Vitellogenin

INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

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 μg/L in surface marine water, 3 μg/g in surface marine sediment, and 4.07 ng/g in marine 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 μg/L in surface water, 1.74–182 μg/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 μg/kg dw in marine surface sediments, 0.16 μg/L in marine water and 1.8 μg/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 anti-estrogenic 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 ERα, β1 and β2) and metabolism (PPARα, β, γ) (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 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., ERα, ERβ, ERγ, 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 endocrine-disrupting 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 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 17β-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

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

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 (d4-DEHP) 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 ± 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 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 −80°C for subsequent experiment.

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 µg 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 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 d4-DEHP).

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 2−ΔΔCt 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 log-transformed to approximate normality when necessary. A p value <0.05 was considered to indicate a statistically significant difference. The data are presented as the mean ± SEM.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Mortality and Hatching Analysis

Medaka embryos were statically exposed to waterborne DEHP and the positive control E2 from 1 to 10 dpf. Table 1 shows the mortality and hatching of embryos and larvae in each of the treatment groups. As shown, DEHP and E2 showed no significant effects on the mortality and hatching rates of exposed embryos. DEHP had no effect on the hatching 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 ± 0.3 dpf. Embryonic exposure to DEHP and E2 exhibited no significant effects on the mortality rates of recovered larvae.

Table 1. Effects of DEHP on survival and hatching of medaka embryos exposed to DEHP (0.01, 0.1, and 1 mg/L) from 1 to 10 dpf and recovered larvae hatching from embryos exposed to DEHP (0.01, 0.1, and 1 mg/L).
 Control0.01 mg/L DEHP0.1 mg/L DEHP1 mg/L DEHP0.01 mg/L E2
  1. The experiment was performed three times and the representative data are expressed as the mean ± SEM. **p < 0.01 vs. control

Mortality rates of embryos (%)16.3 ± 1.818.3 ± 3.419.3 ± 4.324.3 ± 4.226.7 ± 1.2
Hatching rates of embryos (%)83.7 ± 1.881.7 ± 3.480.7 ± 4.375.7 ± 4.273.3 ± 1.2
Hatching time of embryos (dpf)11.4 ± 0.211.3 ± 0.311.5 ± 0.410.8 ± 0.413.6 ± 0.3**
Mortality rates of larvae (%)14.8 ± 4.315.2 ± 4.98.9 ± 1.921.9 ± 1.830.1 ± 7.6

Quantification of DEHP

Figure 1 shows that DEHP accumulated in the medaka embryos. Significantly higher DEHP embryonic burdens were detected in the 1 mg/L treated group than in the control group at various embryonic stages, but DEHP significantly accumulated only at 10 dpf in embryos exposed to 0.1 mg/L DEHP. At 3 dpf, the DEHP levels in the embryos reached 0.99 ± 0.12, 2.96 ± 0.53, and 17.99 ± 1.50 µg/g wt for the 0.01, 0.1 and 1 mg/L DEHP exposure levels, respectively. At 10 dpf, the levels were 3.12 ± 0.75, 4.47 ± 0.83, and 28.97 ± 3.42 µg/g wt for the 0.01, 0.1, and 1 mg/L DEHP exposure levels, respectively, which were all higher than the levels at 3 dpf for the corresponding doses. During a subsequent depuration phase, no significant differences of DEHP in larvae were observed among all treatment groups, DEHP was eliminated from larvae to near control levels at each sampling time. At 22 dpf, the levels were 0.56 ± 0.03, 0.88 ± 0.26, and 0.23 ± 0.03 µg/g wet weight for the 0.01, 0.1, and 1 mg/L DEHP exposure, respectively, which were all lower than the levels observed at 10 dpf for the corresponding doses, showing an elimination tendency of DEHP from exposed embryo to recovered larvae.

image

Figure 1. The concentrations of DEHP in the marine medaka embryos from various experimental groups at different embryonic stages of (3, 5, 7, and 10 dpf) and larvae from the depuration period (14, 18, and 22 dpf). The experiment was performed three times and ten embryos or five larvae were included in each replicate. The representative data are expressed as the mean ± SEM. **p < 0.01,*p < 0.05 vs. control, n = 10 for each replicate.

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Gene Expression Analysis

To evaluate the endocrine-disrupting effects of DEHP on marine medaka, the expression levels of genes related to two important EDC-related receptor pathways (ER and PPAR) and the CYP19 genes were selected. These genes include seven ER related genes—ERα, ERβ, ERγ, VTG1, VTG2, ChgH, and ChgL—two CYP19 genes—CYP19a and CYP19b—and three PPAR-related genes—PPARα, PPARβ, and PPARγ.

The transcriptional responses of various genes in the 3-dpf embryos exposed to DEHP and E2 are shown in Figure 2. After 2 days of exposure, DEHP had no significant effects on the expression levels of the tested genes and no significant differences were observed among the different DEHP treatment groups, with the exception of CYP19b. The relative mRNA expression of CYP19b was significantly down-regulated by 1 mg/L DEHP exposure. Likewise, mRNA expression levels of CYP19b were significantly decreased by E2 treatment for 2 days. In contrast, the expression of CYP19a mRNA, the predominant regulator of the rate of estrogen production in gonads, was significantly upregulated after exposure to E2 for 2 days. However, exposure to E2 from 1 to 3 dpf markedly increased the mRNA expression of ERα, ERβ, VTG1, VTG2, ChgH, and ChgL compared with that of the controls. The mRNA expression of genes encoding the PPARβ receptor was significantly down-regulated by E2.

image

Figure 2. The relative expression levels of ER, PPAR and the CYP19 genes at the early developmental stage (3 dpf) of O. melastigma embryos exposed to DEHP (0.01, 0.1 and 1 mg/L) or E2 (0.01 mg/L) from 1 dpf to 3 dpf. Total RNA was extracted from 10 embryos and qRT-PCR was performed to quantify mRNA expression of each gene, and RPL7 was used as an internal standard. Expression levels of each gene at 3 dpf without DEHP and E2 exposure were used as a control. Significant differences between experimental groups are denoted by different letters (p < 0.05), n = 10 for each replicate.

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The mRNA expression profiles of the late developmental stage (10 dpf) after treatment for 9 days are shown in Figure 3. The effect of DEHP on 10-dpf embryos was readily observed after 9 days of exposure to higher concentrations (0.1 and 1 mg/L). One mg/L DEHP induced the mRNA expression of genes encoding both ER receptors (ERα and ERβ) and PPAR receptors (PPARα and PPARγ), but induction was not observed at lower concentration of DEHP (0.01 and 0.1 mg/L). Likewise, the mRNA expression of genes encoding both ER receptors (ERα and for ERγ) and PPARγ were significantly induced by E2 exposure. Treatment with 0.1 and 1 mg/L DEHP for 9 days significantly increased the mRNA expression levels of VTG1, VTG2, ChgH, ChgL, CYP19a, and CYP19b in a concentration-dependent manner. Similarly, positive control E2 exposure also significantly induced the expression of these genes. However, the 0.01 mg/L DEHP treatment group showed no significant effects on any of the tested genes. No significant differences in the changes in transcription of the target genes were found between the low (0.01 and 0.1 mg/L) DEHP treatment groups. However, the transcription of ERα, VTG1, VTG2, ChgH, ChgL, CYP19a, and PPARα were found to be markedly induced by exposure to 1 mg/L DEHP compared to exposure to 0.01 or 0.1 mg/L DEHP, and the transcription of CYP19b was also significantly induced by exposure to 1 mg/L DEHP compared with exposure to 0.01 mg/L DEHP.

image

Figure 3. The relative expression levels of ER, PPAR and the CYP19 genes at the late developmental stage (10 dpf) of O. melastigma embryos exposed to DEHP (0.1 and 1 mg/L) or E2 (0.01 mg/L) from 1 dpf to 10 dpf. Total RNA was extracted from 10 embryos and qRT-PCR was performed to quantify mRNA expression of each gene, and RPL7 was used as an internal standard. Expression levels of each gene at 10 dpf without DEHP and E2 exposure were used as a control. The values represent the mean ± SEM of three replicates relative to each control. Significant differences between experimental groups are denoted by different letters (p < 0.05), n = 10 for each replicate.

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The mRNA expression profiles detected in medaka larvae with an 8-d recovery in clean sea water are shown in Figure 4. After 8 d of recovery in clean sea water, DEHP did not affect gene expression of genes in the ER and PPAR pathways or the CYP19-related genes tested as evaluated by RT-PCR. Additionally, no significant differences were found between the different DEHP treatment groups. In contrast, positive control E2 exposure significantly down-regulated the mRNA expression levels of ERα, ERγ, CYP19a, and three PPAR receptors (PPARα, PPARβ and PPARγ), but ChgL was still significantly upgulated by E2 exposure.

image

Figure 4. The relative expression levels of ER, PPAR and the CYP19 genes of O. melastigma larvae recovered in clean sea water for 8 days (18 dpf). Total RNA was extracted from two larvae and qRT-PCR was performed to quantify mRNA expression of each gene, and RPL7 was used as an internal standard. Expression levels of each gene of recovered larvae (18 dpf) without DEHP and E2 exposure were used as a control. The values represent the mean ± SEM of three replicate relative to each control. Significant differences between experimental groups are denoted by different letters (p < 0.05), n = 10 for each replicate.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The tested concentrations of DEHP showed no significant effects on the mortality and hatching of O. melastigma embryos, and thus it exhibited no obvious toxicity. The tougher chorion of the embryo and the narrower perivitelline space between the embryo and the chorion can enhance the resistance to toxicants (Chen et al., 2009), which may lead to less obvious toxicity of DEHP. There are both common and unique effects of DEHP on marine and freshwater fish species. Our results showed that DEHP exhibited no significant effects on the hatching time of marine medaka embryos. On the contrary, DEHP was reported to delay the hatching time of embryos of the medaka (Oryzias latipes), a fresh water Japanese fish, without dose-dependence (Chikae et al., 2004). This may be one of the differences in the effects of DEHP on marine and freshwater fish species. DEHP showed no lethal effects on marine medaka embryos. This result was, to a certain extent, in agreement with previous findings on DEHP in the early stages of freshwater Japanese medaka embryos (Chikae et al., 2004). Additionally, E2 exposure significantly delayed the hatching time of marine medaka. It was reported that the dopaminergic mechanism of the hypothalamic-pituitary-axis regulated secretion of a hatch enzyme that controls the hatching time of fish (Schoots et al., 1983). E2 may delay the hatching of marine medaka by disrupting the mechanism of the hypothalamic-pituitary-axis in marine medaka embryos. Exposure to DEHP during the embryonic stage of Japanese medaka increased the mortality of larvae and led to irreversible biological effects in adulthood (Chikae et al., 2004). However, in contrast to freshwater Japanese medaka, DEHP and 0.01 mg/L E2 did not significantly affect the mortality of the recovered marine medaka larvae in this study.

Although DEHP is highly lipophilic, the low water solubility (0.003 mg/L) and high log Kow values of DEHP (7.50) resulted in the accumulation of DEHP in marine medaka of μg/g levels, which can also be explained by an effective biotransformation (Barron et al., 1989). Metabolism plays an important role in the accumulation and elimination of DEHP (Karara and Hayton, 1988), and DEHP is quickly metabolized in fish (Norman et al., 2007). Additionally, metabolic ability is not yet well developed in the embryonic stages, which may result in the bioaccumulation of DEHP in medaka embryos upon DEHP exposure from 1 to 10 dpf. As studied in many fish species, such as cod (Solbakken et al., 1984) and rainbow trout (Geyer et al., 2000), accumulated lipophilic xenobiotics in embryos are transferred to the larvae upon hatching. Therefore, in our study, DEHP in embryos may be transferred to the larvae on hatching. DEHP showed an elimination tendency in medaka larvae, which may be due to the developed metabolic ability of the larvae. The depuration of the larvae also plays a role in the elimination of DEHP. Similarly, a previous study on Atlantic salmon (Salmo salar) showed that a rapid elimination of DEHP to nearly background levels within 1 week after exposure to DEHP (Norman et al., 2007). In previous studies using zebrafish and rainbow trout (Petersen and Kristensen, 1998; Geyer et al., 2000), chemical elimination rates were found to change during early developmental stages and may increase dramatically after hatching, which may also explain the phenomenon of the time-dependent increase of DEHP burdens during embryonic stages and the decrease of DEHP burdens in larval stages in this study. Therefore, DEHP exhibited similarities in the accumulation and elimination in the studied marine medaka and other freshwater fish species.

To study the endocrine-disruptive effects of DEHP on medaka embryos, the effects of DEHP on two major EDC-responsive receptors (ER and PPAR) and the CYP19 genes at embryonic developmental stages and recovered larvae were investigated. At the early embryonic developmental stage, DEHP showed no transcriptional effects on genes in the ER and PPAR pathways, but the positive control E2 significantly increased the mRNA abundance of the estrogenic biomarker genes ERα, ERβ, ChgH, ChgL, VTG1, and VTG2. Estrogenic chemicals are well established as a potent inducer of VTG in fish by acting via the ER pathway (Pawlowski et al., 2004). VTG1 and VTG2 are yolk precursors, and ChgH and ChgL are egg envelope precursors, which are all regulated by E2 through ERα in many teleost fish (Scholz et al., 2004; Zhang et al., 2008; Liang and Fang 2012). The similarity of upregulation found between ERα and other estrogen-response genes in the early developmental stage of the medaka embryos may be because ERα is involved in activating estrogen-responsive genes such as VTG and Chg. It has also been reported that VTG1 expression is regulated only by the action of ERα, whereas VTG2 expression is regulated by both ERα and ERβ in Japanese medaka (Yamaguchi et al., 2009). The role of ERβ and ERγ in mediating the effects of estrogenic chemicals on vitellogenesis remains controversial (Soverchia et al., 2005). The induction of VTG and Chg may be activated by ERα after E2 exposure. The CYP19 genes, which encode two different aromatase isoforms, CYP19a and CYP19b, are preferentially expressed in the ovary and the brain. Modulation of CYP19 expression and/or activity, whether upregulation or downregulation, can dramatically alter the rate of estrogen production, causing endocrine-disrupting effects (Cheshenko et al., 2008). In this study, E2 significantly upregulated the expression levels of CYP19a of 3-dpf medaka embryos, and CYP19b mRNA abundance was significantly reduced after E2 and 1 mg/L DEHP exposure. Therefore, E2 exposure can increase the production of estrogen by upregulating the expression of CYP19a, leading to diffusion of the estrogen into the target tissue and/or cell and binding to ERs, resulting in the upregulation of ERs (ERα and ERβ), which facilitates their activation, dimerization and binding to estrogen-responsive elements (EREs) located in most E2-responsive genes such as VTG and Chg.

Our results suggest that gene expression may be more sensitive than gross endpoints (e.g., mortality rates, hatching rates, and hatching time). Furthermore, estrogen-responsive gene induction levels varied in different developmental stages, and so different stages should be considered when evaluating environmental endocrine-disrupting activities of chemicals in the aquatic environment. Therefore, the expression levels of the responsive genes in the late developmental stage were also investigated. With the contents of DEHP increasing in a dose-dependent manner from early to late embryonic developmental stages, the endocrine-disrupting responses of biomarker genes such as VTG1, VTG2, ChgH, ChgL, CYP19a, and CYP19b showed dose-dependent effects of induction on 10-dpf embryos exposed to DEHP. 0.1 and 1 mg/L DEHP was more effective than 0.01 mg/L in causing endocrine-disrupting effects through regulating transcriptional responses in the ER, PPAR pathways, and the CYP19 genes, which may be due to the high accumulation of DEHP in embryos exposed to 0.1 and 1 mg/L DEHP. E2 also induced the expression of these endocrine-disrupting biomarker genes on 10-dpf embryos. The expression levels of ERα, ERβ, PPARα, and PPARγ were upregulated by 1 mg/L of DEHP. Consistently, E2 exposure increased ERα, ERγ, and PPARγ mRNA abundance. Therefore, DEHP may mimic the estrogenic activity of E2 on the 10-dpf marine medaka embryos; namely, DEHP upregulated the expression of the rate-limiting enzymes including CYP19, increased biosynthesis of estrogen such as E2, induced the expression of ERs and then activated E2-responsive genes such as VTG and Chg. The same was not observed in embryos exposed to DEHP at the early embryonic developmental stage of 3 dpf. DEHP was also reported to exhibit E2-like action in zebrafish primary hepatocyte cultures (Maradonna et al., 2013), in which different ER isoform modulation has been suggested to be associated with an increase in VTG. Similarly, estrogens and estrogenic EDC upregulate CYP19a and CYP19b mRNA in many fish species such as zebrafish, Japanese medaka, and fathead minnows (Cheshenko et al., 2008). These may be responsible for the similarities of the endocrine-disrupting activity of DEHP on the studied marine fish and some freshwater fish species. DEHP has also been reported to cause a reduction in vitellogenin synthesis in female Japanese medaka (Kim et al., 2002), which were different from that in marine medaka. It has been reported that DEHP is a peroxisome proliferator and that its active metabolite mono-(2-ethylhexyl) phthalate (MEHP) activates PPARα and PPARγ in cell transactivation assays (Lovekamp-Swan et al., 2003). Thus, the activation of PPARα and PPARγ following treatment with DEHP may be due to not only the parent compound but also MEHP. Further study on the reproductive and developmental toxicity of MEHP medaka is needed to confirm the above hypothesis. However, MEHP was reported to suppress aromatase by activating the PPARγ/RXR complex (Lovekamp-Swan et al., 2003). In contrast, DEHP and E2 exposure simultaneously activated both PPARs (PPARα and PPARγ) and the CYP19 genes in the present study. The effects on aromatase expression may differ depending on the experimental design difference and the species difference. This result could be explained as the endocrine-disrupting property of DEHP. There were both similarities and differences in the endocrine-disrupting effects of DEHP in marine and freshwater fish species.

An understanding of the recovery of toxic effects is essential for the environmental risk assessment of chemicals (Wu et al., 2005). Thus, the reversibility of the endocrine-disrupting effects after cessation of DEHP and E2 exposure was investigated in this study. After cessation of DEHP exposure in marine medaka, all gene expression biomarkers of endocrine-disruption (e.g., ERs, VTG, Chg, CYP19, and PPARs mRNA expression) showed no significant effects compared to control levels within 8 days of recovery, which might be a consequence of the elimination of DEHP from medaka. After cessation of E2 exposure in marine medaka, the estrogenic biomarker gene ChgL mRNA expression was still induced but the mRNA level of some other endocrine-disrupting biomarker genes (e.g., ERα, ERγ, CYP19a, and PPARs) were significantly reduced, suggesting that exposure to E2 during the embryonic stage resulted in endocrine-disruption effects on medaka larvae. A similar phenomenon was observed in studies on zebrafish and sheepshead minnows (Cyprinodon ariegatus) (Du et al., 2009; Hemmer et al., 2002; Schäfers et al., 2007), in which a continuous increase in VTG expression after cessation of exposure to EE2, PFOS, 17-estradiol and p-nonylphenol was observed. The reversibility of the effects of EDC depended on time, duration, the test species, the chemical compound studied, and the concentration of the exposure (Schäfers et al., 2007). The abundance of VTG mRNA in the E2-exposed group was also significantly lower than that in the control group after a single injection of E2 for 30 days (Park et al., 2010).

A comparison of 3-dpf, 10-dpf and 8-day depuration data indicated that with the accumulation of DEHP from 3 to 10 dpf, the endocrine-disruptive effects of DEHP on medaka embryos were increased as reflected by eliciting transcriptional responses in ER, PPAR, and the CYP19; with the elimination of DEHP from medaka embryos to hatched larvae, embryonic exposure to DHEP did not show transcriptional responses in the studied endocrine-disruptive related genes in the recovered larvae. This indicates that the accumulation of DEHP in embryos from 1 to 10 dpf was effective in stimulating certain endocrine-disruptive responses, which may be due to the high accumulation levels of DEHP in 10-dpf embryos. Additionally, elimination of DEHP in the depuration period may weaken the endocrine-disruptive effects on recovered medaka larvae, suggesting that marine medaka have the ability to recover from endocrine-disrupting effects after embryonic exposure to DEHP. Certainly, the effects of DEHP may also be stage-specific effects in marine medaka embryos. However, the mRNA levels of estrogenic-responsive genes, including ERα, ChgH, ChgL, VTG1, and VTG2 were all upregulated at both 3 dpf and 10 dpf in the positive control E2 treatment group, which further confirms that the endocrine-disrupting effects of DEHP exposure on 10-dpf embryos is due to the accumulation of DEHP and not the stage-specific effects of marine medaka embryos. Additionally, E2 exposure also causes weak endocrine-disrupting effects on recovered larvae, which confirms that the elimination of endocrine-disrupting effects on recovered medaka larvae is mainly due to the ability of marine medaka larvae to recover from the endocrine-disrupting effects of DEHP.

In summary, DEHP and E2 showed no significant effects on the mortality and hatching rates of exposed embryos, and E2 exposure significantly delayed the hatching time of marine medaka. Accumulation of DEHP causes induction of endocrine-disrupting biomarkers of the ER and PPAR pathways as well as CYP19-related genes. Elimination of DEHP from marine medaka may weaken the endocrine-disrupting effects (e.g., altered mRNA expression of ERs, VTG, Chg, CYP19, and PPARs) induced by DEHP exposure. However, E2 exposure induced endocrine-disrupting effects at both the embryonic stage and the recovered larval stage. Further analysis is necessary to reveal the molecular mechanism of DEHP and the toxicity of its metabolites on marine medaka development and reproduction.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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