Rosiglitazone

Keywords:
Di (2-ethylhexyl) phthalate Monoethylhexyl phthalate

A b s t r a c t

An increasing number of epidemiologic studies show that women have a special exposure profile to phthalates, and the exposures have attracted attention regarding their potential health hazards. Here, we developed a model for studying the ovarian action of di-(2-ethylhexyl) phthalate (DEHP) and its major metabolite monoethylhexyl phthalate (MEHP). In vivo, treatment with DEHP (250, 500, and 1000 mg kg^-1) induced decreased thickness of ovarian granulosa cell layer and mitochondrial damage in quail, caused oxidative stress, interfered with the tran- scription of hypothalamic-pituitary-ovarian axis (HPOA) steroid hormone-related factors (increased transcrip- tion of StAR, 3β-HSD, P450scc, and LH and decreased transcription of 17β-HSD, P450arom, FSH, and ERβ), and blocked the secretion of steroid hormones (decreased FSH, E2, and T levels and increased LH, P, and PRL levels). In vitro, granulosa cells were cultured with MEHP (50, 100, and 200 μM), activator of PPARγ (rosiglitazone, 50 μM), or antagonist of PPARγ (GW9662, 10 μM) for 24 h and gene and protein expression were analyzed by real time RT-PCR and western blot. Rosiglitazone, like MEHP, significantly decreased mRNA and protein levels of P450arom. Antagonist GW9662 partially blocked the suppression of P450arom by MEHP, suggesting that MEHP acts through PPARγ, but not exclusively. Our model shows that MEHP acts on granulosa cells in quail by stimu- lating PPARs, which leads to decreased gene and protein expression of P450arom. Therefore, the environmental endocrine disruptor DEHP and its major metabolite MEHP act through a receptor-mediated signaling pathway to inhibit the production of estradiol, interfere with the modulation of HPOA, suppress the synthesis of sex hor- mones, and cause sex hormone secretion disorders, resulting in severe toxicity in the female reproductive sys- tem. A framework for an adverse outcome pathway of DEHP/MEHP-induced ovarian toxicity was constructed, which can facilitate an improved understanding of the mechanism of female reproductive toxicity.

1. Introduction

Di-2-ethylhexyl phthalate (DEHP) is commonly used as a plasticizer in many daily used products (Rowdhwal and Chen, 2018). Because of annual production of over 2 million tons, DEHP has become one of the most widespread environmental contaminants (Halden, 2010). The common use and high production volume of DEHP have led humans to frequent exposure to DEHP (Lai et al., 2017). Although numerous ep- idemiological and toxicological studies have demonstrated the neural, nephrotoxic, hepatotoxic, cardiotoxic, endocrine, testicular, and ovarian effects of DEHP on humans and animal models (Ward et al., 1998; Rusyn and Corton, 2012; Posnack, 2014; Swan et al., 2015; Xu et al., 2015; Jia et al., 2016; Absalan et al., 2017), little is known about the reproductive and developmental toxicity in bird species. Quail are sensitive to envi- ronmental contaminants, and hence used as experimental animal model species in evaluation of ecotoxicology studies. Recently, it was demonstrated that DEHP produced reproductive and developmental toxicity by inducing various cellular responses including modulation of the expression and regulation of steroid hormone receptors and tran- scription and paracrine factors (Cheon, 2020). However, the effect and underlying molecular mechanism of DEHP in the female reproductive system of quail is still unclear.

The ovary plays a key role in the release of sex hormones and female function and follicular development in female mice and cows (Kalo et al., 2015; Li et al., 2016; Rattan et al., 2018). DEHP diminishes mouse antral follicle functionality by suppressing growth, causing atre- sia, and inhibiting steroidogenesis (Hannon et al., 2015). Several studies have reported that DEHP and its metabolites may affect ovarian steroid synthesis and metabolism (Svechnikova et al., 2011; Inada et al., 2012; Hannon et al., 2015). The close correlation between ovarian steroido- genesis, the hypothalamus and the pituitary are well documented, and the female reproductive lifespan depends on strict hormonal modula- tion of this axis. Although accumulated evidence has suggested an asso- ciation between DEHP and adverse reproductive health outcomes (Caserta et al., 2011; Messerlian et al., 2016b), it is unclear how expo- sure to DEHP influences the follicular development and hypothalamic- pituitary-ovarian axis (HPOA) in the ovary of quail. Therefore, we de- signed this study wherein we treated quail (Coturnix japonica) with DEHP throughout sexual immaturity to maturity to explore the effects on the HPOA, follicles, and the development of female reproductive or- gans with the goal of clarifying the mechanism of DEHP-induced ovar- ian toxicity. It is well known that peroxisome proliferator-activated receptors (PPARs) are targets for phthalate di- and monoesters (Sarath Josh et al., 2014). By regulating the levels of peroxisomal lipid metabolism and growth regulatory genes, stimulation of PPARs plays a significant role in the metabolism of xenobiotics. PPARγ ligands might markedly inhibit P450arom in human granulosa cells (GCs). Mono-(2-ethylhexyl) phthalate (MEHP) is a reproductive toxicant that down-regulates the levels of P450arom, the enzyme that converts testosterone (T) into 17β-estradiol (E2). In ovarian GCs of quail, however, the interaction among MEHP, PPARs, and P450arom is still unclear. Therefore, we treated GCs with MEHP, agonist of PPARγ (rosiglitazone), and antago- nist of PPARγ (GW9662) to verify the mechanism of DEHP-induced ovarian toxicity. Our study constituted a useful model for predicting the potential reproductive and developmental toxicity of DEHP in quail (ovaries and ovarian granulosa cells) and might be significant for the assessment of the potential toxicity of DEHP in wildlife.

2. Materials and methods

2.1. Animals and treatments

DEHP was dissolved in corn oil for gavage administration. Experi- mental grouping (n = 60) and the treatment doses are shown in Ta- ble S1. Previous research (Du et al., 2017; Luo et al., 2019; Zhang et al., 2019; Zhao et al., 2019) and our preliminary data supported concentra- tions of 250, 500 and 1000 mg kg^-1 for examining DEHP toxicity in the quail ovary. Although selected dosages of DEHP in this study were higher than the environmental levels or levels commonly seen in human diet, these high dosages were chosen to reveal the target organ and organelle and the adverse effects. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Northeast Agricultural University and approved by the Animal Ethics Committee of Northeast Agricultural University. 45 days later, quail were euthanized with carbon dioxide. The serum, hypothalamus, pituitary, ovary, and oviduct were stored at −80 °C. De- tailed methods are declared in Supplementary 1.1.

2.2. Detection of the content of DEHP and its metabolites in the ovary and oviduct

To detect the content of DEHP and its metabolites, we use the im- proved method (Takatori et al., 2004; Chang et al., 2013) (n = 3). De- tails are reported in Supplementary 1.2.

2.3. GC isolation and culture

The in vitro experiment was independent from the in vivo experi- ments. The GC layer was collected only from other individuals that were not part of the in vivo exposure experiment. The GC layer was iso- lated from preovulatory follicles in aseptic conditions, as described pre- viously (Gilbert et al., 1977). The dosages of MEHP, GW9662, and rosiglitazone selected for the in vitro experiment depend on our prelim- inary data and previous research (Seargent et al., 2004). Detailed methods are provided in Supplementary 1.3.

2.4. Histopathological assessment

Samples (n = 3) were processed routinely and were embedded in paraffin blocks, as described previously (Li et al., 2017; X.N. Li et al., 2018; Lin et al., 2018; Q. Zhang et al., 2020). Imaging of sections was per- formed with the Aperio CS2 image capture device (Leica, Germany), and data were analyzed with pathology slide viewing software Aperio ImageScope (Leica, Germany). We classified the follicles according to the methods proposed by Rodler (Rodler et al., 2012). The follicular di- ameter (n = 3) was measured using an Aperio ImageScope.

2.5. Transmission electron microscopy

Detailed methods are provided in Supplementary 1.4. Ultrathin sec- tions of ovary (n = 3) were processed for ultrastructural analysis with an H-7650 transmission electron microscopy (Hitachi, Japan).

2.6. Radioimmunoassay (RIA)

To assess the hormone levels (n = 4) in DEHP-induced quail, serum follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), E2, progesterone (P), and T were measured with commercially available RIA kits (HAT Co., Ltd. China).

2.7. Determination of the protein content

To determine the protein content of ovary samples, we use the pro- tein assay kits. Details are declared in Supplementary 1.5.

2.8. Determination of oxidative stress indices

The oxidative stress indices (n = 4) were assayed by colorimetric and hydroxylamine method. Details of determination are described in Supplementary 1.6.

2.9. RNA extraction and real-time PCR analysis

Total RNA was extracted from hypothalamus, pituitary, and ovary tissue homogenate and transcribed into cDNA as described by previous study (Ge et al., 2019; Zhao et al., 2020a, 2020c). Analysis of the mRNA levels (n = 3) was done using qRT-PCR. Details are described in Supplementary 1.7.

2.10. Western blot analysis

Nuclear, cytoplasmic and membrane protein fractions (n = 3) were extracted following the protocols provided by a kit (Beyotime Institute of Biotechnology, P.R. China). Western blot was then performed accord- ing to previous protocols (Li et al., 2017; C. Zhang et al., 2020; Zhao et al., 2018; Zhao et al., 2020b). Details are described in Supplementary 1.8.

2.11. Statistical analysis

All data were analyzed using GraphPad Prism 5.1 software (USA). The heatmap and principal component analysis (PCA) were plotted using the heatmap R package (version 3.2.1) and SPSS Statistics 17.0 (SPSS Inc., USA), respectively. Asterisks (*) indicate statistically signifi- cant differences to Vcon; * P b 0.05, ** P b 0.01 and *** P b 0.001. Detailed methods are provided in Supplementary 1.9.

3. Results

3.1. Clinical indicators of female reproductive system development

We performed the study as shown by a workflow schematic (Fig. 1a). As shown in Fig. 1b, DEHP increased the average age of the first egg laying (P b 0.01 and P b 0.001). The body weight increased grad- ually with age in all groups (Fig. 1c). Quail showed a dose-dependent decrease in body weight, ovary weight, and ovary coefficient at 45 days (P b 0.05, P b 0.01, and P b 0.001) (Fig. 1d). The concentrations of DEHP and its metabolites in the quail serum, ovary, and oviduct in- creased significantly in the DEHP-treated groups (P b 0.05 and P b 0.001) (Fig. 1e–g).

3.2. Effects of DEHP on morphometric, histological, and ultrastructural fea- tures in the ovary

To further analyze the structures of the ovary, we performed mor- phological (Fig. 2a), histological (Fig. 2b, c), and ultrastructural analyses (Fig. 2d) of ovaries from female quail at 45 days. Morphometric changes in the ovary and oviduct indicated that DEHP exposure induced a delay in reproductive organ development in quail (Fig. 2a). In the Vcon and control groups (Fig. 2b, c), visible primordial follicles, great volumes of the large follicles, and thick GCs were observed relative to those of the treatment groups. There were decreased numbers and volumes of large follicles in the DEHP groups, and they had thinner GCs than what was observed in the other groups. To assess ovarian damage, the percentage of five categories of follicles was counted in various groups (Fig. 2e). The percentages of ovarian primordial follicles (POFs) and perivitelline follicles (PVFs) were greater in the DEHP groups (250, 500, and 1000 mg kg^-1) than they were in the other groups. The per- centages of small white follicles (SWFs), small yellow follicles (SYFs), and follicles 5 (F5) in the Vcon and control groups were higher than those in all DEHP groups. Meanwhile, the ovarian GC layer was less thick in the DEHP group than it was in the other groups (P b 0.001) (Fig. 2f). The GCs in the Vcon and control groups had normal morphol- ogy and organelle distribution (Fig. 2d). The cristae of the mitochondria were reduced and fractured in the DEHP-treated groups. In addition, an increase in the level of vacuolization, irregular nuclei, and mitochondrial malformation was observed in the DEHP groups; further, the nuclei had condensed chromatin and chromatin margination was apparent.

3.3. DEHP exposure induced oxidative stress disorder

To analyze the redox homeostasis of the ovary, several antioxidant enzymes and molecules were assessed. There was an increasing trend in GSH and MDA in the 250, 500, and 1000 mg kg^-1 DEHP groups (P b 0.05, P b 0.01, and P b 0.001) (Fig. 3a and g). However, CAT was not altered in response to DEHP exposure (Fig. 3e). GPX, GST, T-AOC, and T-SOD were significantly reduced in response to DEHP, demonstrat- ing disruption of redox homeostasis (P b 0.05, P b 0.01, and P b 0.001) (Fig. 3b–d and f).

3.4. DEHP exposure alters gonadal hormones

To analyze whether DEHP affects gonadal hormone levels, RIA was performed (Fig. 4a–f). We observed significant reductions in FSH, E2, and T levels following DEHP exposure (P b 0.01 and P b 0.001) (Fig. 4a, d, and f). There was a significant (P b 0.05, P b 0.01, and P b 0.001) increase in LH, and P levels in the 500 and 1000 mg kg^-1 DEHP exposure groups (Fig. 4b, e) and in PRL levels in the 1000 mg kg^-1 group (Fig. 4c). The data demonstrate that DEHP signif- icantly influences hormone secretion.

3.5. DEHP exposure alters gene expression in the HPOA

Based on these results, we then investigated the influence of DEHP on the gene expression levels in the HPOA. Under our experimental con- ditions, 15 differentially expressed genes were observed when compar- ing quail exposed to DEHP with quail exposed to the vehicle control (Fig. 4g). Gene expression analyses showed that 9 genes (PRLH in the hypothalamus; FSH, LH, PRL, and PGR in the pituitary; and StAR, 3β- HSD, P450scc, and LH in the ovary) were upregulated, while 5 genes were downregulated (GnRH in the hypothalamus; and 17β-HSD, P450arom, FSH, and ERβ in the ovary) (Fig. S1–3) (P b 0.05, P b 0.01, and P b 0.001). However, ERα was not altered in 500 and 1000 mg kg^-1 DEHP groups (Fig. S1–3). To analyze the role of DEHP in the gonadal hormones and HPOA, PCA was performed (Fig. 4h). The principal components were 76.875, 10.559, and 5.320%, respectively (Table S3). An anti-clockwise direction in the gonadal hormones and HPOA-related indices demonstrated a dose-dependent trend (Fig. 4h).

3.6. Effects of MEHP exposure on the proliferation of ovarian GCs and ex- pression of the PPAR signaling pathway

To analyze the effects of DEHP on the ovarian GCs in quail, we as- sumed that MEHP exposure is mainly due to metabolism of DEHP after absorption. Under an inverted phase-contrast microscope, the GCs appeared spherical immediately after plating, and they began ad- hering to the wall 8 h later. The adherence rate reached 70% at 24 h and 90% after 2 days of culture. The cellular morphology was irregular, with cells being star-like or spindle-shaped. The cells were connected by intercellular-extended pseudopodia. The nuclei were large and round, and the cytoplasm was full and was highly transparent. The cells showed clustered growth. For GCs plated with uniform cell density, after 24 h of preculture, MEHP was added according to gradient concen- trations. At 48 h, the proliferation and extension of GC pseudopodia in the group treated with MEHP were markedly decreased (Fig. 5a). changed in the MEHP group (Fig. 5b). Gene expression analyses showed that 4 genes (RXRα, PPARγ, FABP, and PPARα) were up- regulated, while 7 genes were downregulated (P450arom, RXRγ, ERα, ERβ, FSHR, FSH, and 17β-HSD) in response to MEHP exposure (Fig. S4) (P b 0.05, P b 0.01, and P b 0.001). MEHP treatment (50 μM, 100 μM, and 200 μM) resulted in the induction of nuclear protein expression of PPARα and PPARγ (P b 0.01 and P b 0.001) (Fig. 5c–e). The cytoplasmic protein levels of PPARα and PPARγ showed the opposite trend (P b 0.001). The expression of the protein P450arom in the membrane following MEHP was markedly decreased (P b 0.001) (Fig. 5c, f). To evaluate the role of MEHP in the PPAR signaling pathway, PCA was performed (Fig. 5g). The principal components were 82.509 and 11.970%, respectively (Table S4). An anti-clockwise direction from the Con group, 50, 100, and 200 μM MEHP-exposed groups in the PPAR-related indices demonstrated a significant dose-dependent trend (Fig. 5g).

3.7. Effect of the PPARγ agonist rosiglitazone and the PPARγ antagonist GW9662 on the proliferation of ovarian GCs and the PPAR
signaling pathway

To test this hypothesis, comparative analysis of gene and protein ex- pression was performed on ovarian GCs treated with MEHP, rosiglitazone, and GW9662. For GCs plated with uniform cell density, after 24 h of preculture, rosiglitazone (50 μM), GW9662 (10 μM), the combination of MEHP (200 μM) and GW9662 (10 μM), or the combina- tion of MEHP (200 μM) and rosiglitazone (50 μM) were used to treat the cells. At 48 h, the proliferation and degree of pseudopodia extension of GCs in the group treated with GW9662 were markedly higher than those in the control group (Fig. 6a). In contrast, rosiglitazone induced cell growth suppression. GW9662 did not prevent MEHP-induced proliferation and extension inhibition. MEHP acted to enhance the role of rosiglitazone. Rosiglitazone or GW9662 treatment resulted in the induction or in- hibition of PPARα and PPARγ mRNA expression, respectively (P b 0.05 and P b 0.001) (Fig. 6b, c). Cotreatment with both MEHP and rosiglitazone resulted in statistically higher PPARα and PPARγ mRNA levels compared to the control group (P b 0.001). No activation or inhi- bition of PPARα and PPARγ mRNA expression was detected following MEHP and GW9662 treatment. In contrast, GW9662 or rosiglitazone treatment resulted in the induction or inhibition of P450arom mRNA ex- pression, respectively (P b 0.01 and P b 0.001) (Fig. 6d). The levels of the P450arom gene were markedly decreased following treatment with the combination of MEHP and rosiglitazone and the combination of MEHP and GW9662 (P b 0.001).

Nuclear protein expression of PPARα following treatment with rosiglitazone, GW9662 or a combination of MEHP and GW9662 was sig- nificantly suppressed (P b 0.001) (Fig. 6e, f). The nuclear protein levels of PPARα were markedly elevated in the group treated with a combina- tion of MEHP and rosiglitazone (P b 0.001). The cytoplasmic protein levels of PPARα showed the opposite trend (P b 0.001). PPARγ nuclear protein expression was consistent with its gene levels (P b 0.05 and P b 0.001) (Fig. 6e, g). All treated groups had markedly decreased levels of cytoplasmic PPARγ protein (P b 0.001). The membrane protein levels of P450arom following treatment with rosiglitazone, GW9662 or a com- bination of MEHP and GW9662 were markedly increased (P b 0.001)

4. Discussion

DEHP is one of the most regularly detected environmental contami- nants (Nardelli et al., 2017). Humans and animals are exposed in many ways, after which DEHP enters the blood circulation (Liu et al., 2017). Phthalates and their metabolites are routinely detected in liver, kidney, testicular, and ovarian tissue. This study investigated whether DEHP and MEHP affect the development and function of the female reproductive system in quail. Adverse effects on the ovary of following DEHP and MEHP exposure were linked to the activation of the PPAR response and interference in E2 production that resulted in sex hormone secre- tion disorders. reduced body weight and egg production (Wood and Bitman, 1984). The low body weight and low organ coefficient of ovaries exposed to DEHP agree with previ- ously published results (Liu et al., 2014). Mice exposed to DEHP had low birth weight (Zong et al., 2015). In this study, DEHP and its major me- tabolites were detected in quail ovaries and oviducts, indicating that exposure to DEHP could cause injury to the female reproductive system of quail (Kalo et al., 2015; Messerlian et al., 2016a) 2015).

Consistent with previous studies, this research further indicated that DEHP may affect various components of the HPOA and may have a direct ef- fect on ovarian function. There is an association between steroid hormone secretion and steroidogenic factor expression. Increasing evidence has suggested that GnRH expression levels were diminished with DEHP treatments (Qin et al., 2018). Consistent with increased serum PRL levels, the expression levels of the prolactin-releasing hormone (PRLH) and PRL mRNA were observed to be induced by DEHP exposure in the hypothalamus and pi- tuitary. In line with serum FSH and LH levels, DEHP affected FSH and LH transcription in the pituitary and ovary. However, our study demon- strated that DEHP caused elevation of FSH levels in the pituitary. Due to negative feedback of the HPO axis, the FSH level in the pituitary was increased. DEHP did not cause higher levels of PGR mRNA (Kim et al., 2018). Interestingly, our data were in contrast to previous re- search: PGR mRNA expression was markedly upregulated following DEHP treatment compared with that of the control. Our results reveal that either direct effects of DEHP exposure or an HPOA response may cause developmental abnormalities in the ovary by stimulating the ex- pression of steroidogenic genes, including StAR, 3β-HSD, and P450scc, and by inhibiting 17β-HSD, P450arom, and ERβ. E2 is essential for ovar- ian folliculogenesis (Dewailly et al., 2016), and its biosynthesis requires the expression of several proteins, such as 3β- and 17β-HSD, P450scc, StAR, and P450arom. In our study, the decreased levels of T and E2 may have been induced by a reduction in the expression of P450arom and 17β-HSD compared with their levels in control groups. Our results were similar to those of previous research (Ernst et al., 2014; Helal, 2014), which revealed that DEHP decreased mRNA levels of P450arom and 17β-HSD. It is likely that the increased levels of StAR, P450scc, and 3β-HSD after DEHP treatment may have upregulated the serum level of P. It is well documented that MEHP markedly increased key enzymes in P production and accelerated P secretion (N. Li et al., 2018).

In the present study, the levels of ERβ were decreased. As the expression of ERα and ERβ is normally enhanced (Y. Chen et al., 2009), decreased levels may be due to delayed ovarian development or negative feedback from ER activation. These results demonstrate that exposure to DEHP interferes with hormonal homeostasis in quail, especially with the ex- pression of steroidogenic factors in the HPOA, which is critical to the growth and development of follicles. MEHP, the primary metabolite of DEHP, is a female reproductive toxicant. Some results demonstrated that MEHP inhibited ovarian GCs proliferation (Li et al., 2015; N. Li et al., 2018). MEHP increased reproductive hormone receptor (ERα, ERβ, and FSHR) levels in GCs at the protein level (N. Li et al., 2018). Our result is not consistent with this previous study. This may be because of posttranscriptional control. MEHP significantly suppressed FSH levels in GCs at the gene level. MEHP exposure in vitro and the present research regarding DEHP treatment in vivo produce the same results. Moreover, our data showed that MEHP increased the mRNA and protein expression of PPARα and PPARγ and decreased the mRNA and protein levels of P450arom. Our results were in accordance with previous research (Lovekamp-Swan et al., 2003). PPARγ and RXR ligands synergisti- cally inhibit P450arom in human granulosa cancer cells (Mu et al., 2001). Present study revealed that MEHP may inhibit P450arom by activating PPARα, PPARγ, and RXRα and by inhibiting RXRγ. MEHP decreased the mRNA expression of 17β-HSD and P450arom, which control the rate-limiting steps in E2 synthesis (Gregoraszczuk et al., 2011). Significant suppression in serum E2 was observed, which might have led to a decline in 17β-HSD and P450arom transcription. DEHP and MEHP are ligands of fatty acid binding protein (FABP) (Lovekamp-Swan and Davis, 2003). Activation of PPAR upregulates the levels of FABP in GCs (Lovekamp-Swan et al., 2003), forming binding proteins for PPAR activators. Our results were in accordance with previous reports. Based on our findings, we hypothesized that MEHP inhibits P450arom by stimulating PPARα and PPARγ in quail GCs.

Similar to MEHP, rosiglitazone increased the mRNA expression of PPARα and PPARγ and decreased the mRNA expression of P450arom. Our results were in accordance with previous research (Q. Chen et al., 2009; Pogrmic-Majkic et al., 2019). GW9662 par- tially blocked the inhibition of P450arom induced by MEHP, imply- ing that MEHP acts in a nonexclusive way through PPARγ. Our results were in accordance with previous reports that indicated the effect of PPARγ on reducing P450arom transcripts and produc- tion in MEHP-treated ovarian GCs (Ernst et al., 2014). The results indicated that MEHP may cause abnormal ovarian GC function by activating PPAR-related pathways and disrupting P450arom transcription. An overview of the pathways is summarized in Fig. 7, which summa- rizes the toxic effects of DEHP on the ovary and ovarian GCs in quail. DEHP induces P secretion by elevating LH from the pituitary and by in- creasing the expression of StAR, P450scc, and 3β-HSD. DEHP leads to a decrease in T and E2 secretion, which is regulated by the downregula- tion of 3β-HSD and P450arom. Persistent suppression of serum FSH within the quail has been linked to GnRH in the hypothalamus. MEHP acts on GCs by stimulating PPARs, which results in the suppression of P450arom transcription. In addition, MEHP suppresses the expression of FSHR and ERs mRNA, which may contribute to the inhibition of E2 in ovarian GCs. Our study suggested a putative framework for an ovarian toxicity adverse outcome pathway (AOP) in quail that integrate-gene and protein data, serum sex hormones, histopathology, although further verify is necessary to build an AOP model with sufficient evi- dence (Fig. 8).

In conclusion, this study revealed that DEHP, through its major metab- olite MEHP, acted through a receptor-mediated signaling pathway to in- terfere with the modulation of HPOA and cause sex hormone secretion disorders, resulting in serious toxicity to the female reproductive system of quail. The present findings provide new insights into the molecular mechanisms of DEHP-induced female reproductive toxicity in birds. How- ever, further research on the molecular mechanism of DEHP-induced fe- male reproductive toxicity in birds is still needed. Based on these results, a framework for an AOP of DEHP/MEHP-induced ovarian toxicity was constructed, which can facilitate an improved understanding of the molecular mechanism of female reproductive toxicity in birds.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.

Acknowledgments

This study has received assistance from National Natural Science Foundation of China (No. 31572586), Excellent Youth Foundation of
Heilongjiang Province of China (No. JC2017005) and China Agriculture Research System (No. CARS-35).

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2020.140293.

References

Absalan, F., Saremy, S., Mansori, E., Taheri Moghadam, M., Eftekhari Moghadam, A.R., Ghanavati, R., 2017. Effects of mono-(2-ethylhexyl) phthalate and Di-(2-ethylhexyl) phthalate administrations on oocyte meiotic maturation, apoptosis and gene quanti- fication in mouse model. Cell J. 18, 503–513.
Caserta, D., Mantovani, A., Marci, R., Fazi, A., Ciardo, F., La Rocca, C., Maranghi, F., Moscarini, M., 2011. Environment and women’s reproductive health. Hum. Reprod. Update 17, 418–433.
Chang, Y.J., Lin, K.L., Chang, Y.Z., 2013. Determination of Di-(2-ethylhexyl)phthalate (DEHP) metabolites in human hair using liquid chromatography-tandem mass spec- trometry. Clin. Chim. Acta 420, 155–159.
Chen, Q., Sun, X., Chen, J., Cheng, L., Wang, J., Wang, Y., Sun, Z., 2009a. Direct rosiglitazone action on steroidogenesis and proinflammatory factor production in human granulosa-lutein cells. Reprod. Biol. Endocrinol. 7, 147.
Chen, Y., Breen, K., Pepling, M.E., 2009b. Estrogen can signal through multiple pathways to regulate oocyte cyst breakdown and primordial follicle assembly in the neonatal mouse ovary. J. Endocrinol. 202, 407–417.
Cheon, Y.P., 2020. Di-(2-ethylhexyl) phthalate (DEHP) and uterine histological character- istics. Dev. Reprod. 24, 1–17.
Dewailly, D., Robin, G., Peigne, M., Decanter, C., Pigny, P., Catteau-Jonard, S., 2016. Interac- tions between androgens, FSH, anti-Mullerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Hum. Reprod. Update 22, 709–724.
Du, Z.H., Xia, J., Sun, X.C., Li, X.N., Zhang, C., Zhao, H.S., Zhu, S.Y., Li, J.L., 2017. A novel nu-
clear xenobiotic receptors (AhR/PXR/CAR)-mediated mechanism of DEHP-induced cerebellar toxicity in quails (Coturnix japonica) via disrupting CYP enzyme system homeostasis. Environ. Pollut. 226, 435–443.
Ernst, J., Jann, J.C., Biemann, R., Koch, H.M., Fischer, B., 2014. Effects of the environmental contaminants DEHP and TCDD on estradiol synthesis and aryl hydrocarbon receptor and peroxisome proliferator-activated receptor signalling in the human granulosa cell line KGN. Mol. Hum. Reprod. 20, 919–928.
Ge, J., Zhang, C., Sun, Y.C., Zhang, Q., Lv, M.W., Guo, K., Li, J.L., 2019. Cadmium exposure triggers mitochondrial dysfunction and oxidative stress in chicken (Gallus gallus) kidney via mitochondrial UPR inhibition and Nrf2-mediated antioxidant defense ac- tivation. Sci. Total Environ. 689, 1160–1171.
Gilbert, A.B., Evans, A.J., Perry, M.M., Davidson, M.H., 1977. A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus). J. Reprod. Fertil. 50, 179–181.
Gregoraszczuk, E.L., Jerzak, J., Rak-Mardyla, A., Falandysz, J., 2011. Halowax 1051 affects steroidogenesis, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and cyto- chrome P450arom (CYP19) activity, and protein expression in porcine ovarian folli- cles. Reprod. Toxicol. 32, 379–384.
Halden, R.U., 2010. Plastics and health risks. Annu. Rev. Public Health 31, 179–194. Hannon, P.R., Brannick, K.E., Wang, W., Gupta, R.K., Flaws, J.A., 2015. Di(2-ethylhexyl)
phthalate inhibits antral follicle growth, induces atresia, and inhibits steroid hormone
production in cultured mouse antral follicles. Toxicol. Appl. Pharmacol. 284, 42–53. Helal, M.A., 2014. Celery oil modulates DEHP-induced reproductive toxicity in male rats.
Reprod. Biol. 14, 182–189.
Herreros, M.A., Encinas, T., Torres-Rovira, L., Garcia-Fernandez, R.A., Flores, J.M., Ros, J.M., Gonzalez-Bulnes, A., 2013. Exposure to the endocrine disruptor di(2-ethylhexyl) phthalate affects female reproductive features by altering pulsatile LH secretion. En- viron. Toxicol. Pharmacol. 36, 1141–1149.
Hirosawa, N., Yano, K., Suzuki, Y., Sakamoto, Y., 2006. Endocrine disrupting effect of di-(2- ethylhexyl)phthalate on female rats and proteome analyses of their pituitaries. Prote- omics 6, 958–971.
Inada, H., Chihara, K., Yamashita, A., Miyawaki, I., Fukuda, C., Tateishi, Y., Kunimatsu, T., Kimura, J., Funabashi, H., Miyano, T., 2012. Evaluation of ovarian toxicity of mono- (2-ethylhexyl) phthalate (MEHP) using cultured rat ovarian follicles. J. Toxicol. Sci. 37, 483–490.
Jia, P.P., Ma, Y.B., Lu, C.J., Mirza, Z., Zhang, W., Jia, Y.F., Li, W.G., Pei, D.S., 2016. The effects of disturbance on hypothalamus-pituitary-thyroid (HPT) axis in zebrafish larvae after exposure to DEHP. PLoS One 11, e0155762.
Kalo, D., Hadas, R., Furman, O., Ben-Ari, J., Maor, Y., Patterson, D.G., Tomey, C., Roth, Z., 2015. Carryover effects of acute DEHP exposure on ovarian function and oocyte de- velopmental competence in lactating cows. PLoS One 10, e0130896.
Kim, J., Cha, S., Lee, M.Y., Hwang, Y.J., Yang, E., Ryou, C., Jung, H.I., Cheon, Y.P., 2018. Chronic low-dose nonylphenol or Di-(2-ethylhexyl) phthalate has a different estrogen-like response in mouse uterus. Dev. Reprod. 22, 379–391.
Lai, F.N., Liu, J.C., Li, L., Ma, J.Y., Liu, X.L., Liu, Y.P., Zhang, X.F., Chen, H., De Felici, M., Dyce, P.W., Shen, W., 2017. Di (2-ethylhexyl) phthalate impairs steroidogenesis in ovarian follicular cells of prepuberal mice. Arch. Toxicol. 91, 1279–1292.
Li, S., Dai, J., Zhang, L., Zhang, J., Zhang, Z., Chen, B., 2011. An association of elevated serum prolactin with phthalate exposure in adult men. Biomed. Environ. Sci. 24, 31–39.
Li, N., Liu, K., Yuan, H., Zhu, J., Yu, G., Xie, J., Fu, S., Guo, K., Ye, L., 2015. The effect of mono- (2-ethylhexyl) phthalate on apoptosis of rat ovarian granulosa cells in vitro. Environ. Toxicol. Pharmacol. 39, 643–650.
Li, L., Liu, J.C., Lai, F.N., Liu, H.Q., Zhang, X.F., Dyce, P.W., Shen, W., Chen, H., 2016. Di (2-
ethylhexyl) phthalate exposure impairs growth of antral follicle in mice. PLoS One 11, e0148350.
Li, X.N., Lin, J., Xia, J., Qin, L., Zhu, S.Y., Li, J.L., 2017. Lycopene mitigates atrazine-induced cardiac inflammation via blocking the NF-κB pathway and NO production. J. Funct. Foods 29, 208–216.
Li, N., Liu, T., Guo, K., Zhu, J., Yu, G., Wang, S., Ye, L., 2018a. Effect of mono-(2-ethylhexyl) phthalate (MEHP) on proliferation of and steroid hormone synthesis in rat ovarian granulosa cells in vitro. J. Cell. Physiol. 233, 3629–3637.
Li, X.N., Zuo, Y.Z., Qin, L., Liu, W., Li, Y.H., Li, J.L., 2018b. Atrazine-xenobiotic nuclear recep- tor interactions induce cardiac inflammation and endoplasmic reticulum stress in quail (Coturnix coturnix coturnix). Chemosphere 206, 549–559.
Lin, J., Zhao, H.S., Qin, L., Li, X.N., Zhang, C., Xia, J., Li, J.L., 2018. Atrazine triggers mitochon- drial dysfunction and oxidative stress in quail (Coturnix C. coturnix) cerebrum via ac- tivating xenobiotic-sensing nuclear receptors and modulating cytochrome P450 systems. J. Agric. Food Chem. 66, 6402–6413.
Liu, T., Li, N., Zhu, J., Yu, G., Guo, K., Zhou, L., Zheng, D., Qu, X., Huang, J., Chen, X., Wang, S., Ye, L., 2014. Effects of di-(2-ethylhexyl) phthalate on the hypothalamus-pituitary- ovarian axis in adult female rats. Reprod. Toxicol. 46, 141–147.
Liu, J.C., Lai, F.N., Li, L., Sun, X.F., Cheng, S.F., Ge, W., Wang, Y.F., Li, L., Zhang, X.F., De Felici, M., Dyce, P.W., Shen, W., 2017. Di (2-ethylhexyl) phthalate exposure impairs meiotic progression and DNA damage repair in fetal mouse oocytes in vitro. Cell Death Dis. 8, e2966.
Lovekamp-Swan, T., Davis, B.J., 2003. Mechanisms of phthalate ester toxicity in the female reproductive system. Environ. Health Perspect. 111, 139–145.
Lovekamp-Swan, T., Jetten, A.M., Davis, B.J., 2003. Dual activation of PPARalpha and PPARgamma by mono-(2-ethylhexyl) phthalate in rat ovarian granulosa cells. Mol. Cell. Endocrinol. 201, 133–141.
Luo, Y., Li, X.N., Zhao, Y., Du, Z.H., Li, J.L., 2019. DEHP triggers cerebral mitochondrial dys- function and oxidative stress in quail (Coturnix japonica) via modulating mitochon- drial dynamics and biogenesis and activating Nrf2-mediated defense response. Chemosphere 224, 626–633.
Meltzer, D., Martinez-Arguelles, D.B., Campioli, E., Lee, S., Papadopoulos, V., 2015. In utero exposure to the endocrine disruptor di(2-ethylhexyl) phthalate targets ovarian theca cells and steroidogenesis in the adult female rat. Reprod. Toxicol. 51, 47–56.
Messerlian, C., Souter, I., Gaskins, A.J., Williams, P.L., Ford, J.B., Chiu, Y.H., Calafat, A.M., Hauser, R., Earth Study, T., 2016a. Urinary phthalate metabolites and ovarian reserve among women seeking infertility care. Hum. Reprod. 31, 75–83.
Messerlian, C., Wylie, B.J., Minguez-Alarcon, L., Williams, P.L., Ford, J.B., Souter, I.C., Calafat, A.M., Hauser, R., Earth Study, T., 2016b. Urinary concentrations of phthalate metabo- lites and pregnancy loss among women conceiving with medically assisted reproduc- tion. Epidemiology 27, 879–888.
Moyer, B., Hixon, M.L., 2012. Reproductive effects in F1 adult females exposed in utero to moderate to high doses of mono-2-ethylhexylphthalate (MEHP). Reprod. Toxicol. 34, 43–50.
Mu, Y.M., Yanase, T., Nishi, Y., Takayanagi, R., Goto, K., Nawata, H., 2001. Combined treat- ment with specific ligands for PPARgamma:RXR nuclear receptor system markedly inhibits the expression of cytochrome P450arom in human granulosa cancer cells. Mol. Cell. Endocrinol. 181, 239–248.
Nardelli, T.C., Albert, O., Lalancette, C., Culty, M., Hales, B.F., Robaire, B., 2017. In utero and lactational exposure study in rats to identify replacements for Di(2-ethylhexyl) phthalate. Sci. Rep. 7, 3862.
Pan, G., Hanaoka, T., Yoshimura, M., Zhang, S., Wang, P., Tsukino, H., Inoue, K., Nakazawa, H., Tsugane, S., Takahashi, K., 2006. Decreased serum free testosterone in workers ex- posed to high levels of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in China. Environ. Health Perspect. 114, 1643–1648. Pogrmic-Majkic, K., Kosanin, G., Samardzija Nenadov, D., Fa, S., Stanic, B., Trninic Pjevic, A., Andric, N., 2019. Rosiglitazone increases expression of steroidogenic acute regulatory protein and progesterone production through PPARgamma-EGFR-ERK1/2 in human
cumulus granulosa cells. Reprod. Fertil. Dev. 31, 1647–1656.
Posnack, N.G., 2014. The adverse cardiac effects of Di(2-ethylhexyl)phthalate and Bisphenol A. Cardiovasc. Toxicol. 14, 339–357.
Qin, X., Ma, Q., Yuan, J., Hu, X., Tan, Q., Zhang, Z., Wang, L., Xu, X., 2018. The effects of di-2- ethylhexyl phthalate on testicular ultrastructure and hormone-regulated gene ex- pression in male rats. Toxicol. Res. (Camb.) 7, 408–414.
Rattan, S., Brehm, E., Gao, L., Niermann, S., Flaws, J.A., 2018. Prenatal exposure to di(2- ethylhexyl) phthalate disrupts ovarian function in a transgenerational manner in fe- male mice. Biol. Reprod. 98, 130–145.
Rodler, D., Sasanami, T., Sinowatz, F., 2012. Assembly of the inner perivitelline layer, a ho- molog of the mammalian zona pellucida: an immunohistochemical and ultrastruc- tural study. Cells Tissues Organs 195, 330–339.
Rowdhwal, S.S.S., Chen, J., 2018. Toxic effects of di-2-ethylhexyl phthalate: an overview.
Biomed. Res. Int. 2018, 1750368.
Rusyn, I., Corton, J.C., 2012. Mechanistic considerations for human relevance of cancer hazard of di(2-ethylhexyl) phthalate. Mutat. Res. 750, 141–158.
Sarath Josh, M.K., Pradeep, S., Vijayalekshmi Amma, K.S., Balachandran, S., Abdul Jaleel, U.C., Doble, M., Spener, F., Benjamin, S., 2014. Phthalates efficiently bind to human peroxisome proliferator activated receptor and retinoid X receptor alpha, beta, gamma subtypes: an in silico approach. J. Appl. Toxicol. 34, 754–765.
Seargent, J.M., Yates, E.A., Gill, J.H., 2004. GW9662, a potent antagonist of PPARgamma, in- hibits growth of breast tumour cells and promotes the anticancer effects of the PPARgamma agonist rosiglitazone, independently of PPARgamma activation. Br.
J. Pharmacol. 143, 933–937.
Sekaran, S., Jagadeesan, A., 2015. In utero exposure to phthalate downregulates critical genes in Leydig cells of F1 male progeny. J. Cell. Biochem. 116, 1466–1477.
Svechnikova, I., Svechnikov, K., Soder, O., 2007. The influence of di-(2-ethylhexyl) phthal- ate on steroidogenesis by the ovarian granulosa cells of immature female rats.
J. Endocrinol. 194, 603–609.
Svechnikova, K., Svechnikova, I., Soder, O., 2011. Gender-specific adverse effects of mono- ethylhexyl phthalate on Steroidogenesis in immature granulosa cells and rat Leydig cell progenitors in vitro. Front. Endocrinol. (Lausanne) 2, 9.
Swan, S.H., Sathyanarayana, S., Barrett, E.S., Janssen, S., Liu, F., Nguyen, R.H., Redmon, J.B., Team, T.S., 2015. First trimester phthalate exposure and anogenital distance in new- borns. Hum. Reprod. 30, 963–972.
Takatori, S., Kitagawa, Y., Kitagawa, M., Nakazawa, H., Hori, S., 2004. Determination of di (2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in human serum using liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 804, 397–401.
Wang, W., Craig, Z.R., Basavarajappa, M.S., Gupta, R.K., Flaws, J.A., 2012a. Di (2-ethylhexyl) phthalate inhibits growth of mouse ovarian antral follicles through an oxidative stress pathway. Toxicol. Appl. Pharmacol. 258, 288–295.
Wang, W., Craig, Z.R., Basavarajappa, M.S., Hafner, K.S., Flaws, J.A., 2012b. Mono-(2- ethylhexyl) phthalate induces oxidative stress and inhibits growth of mouse ovarian antral follicles. Biol. Reprod. 87, 152.
Ward, J.M., Peters, J.M., Perella, C.M., Gonzalez, F.J., 1998. Receptor and nonreceptor- mediated organ-specific toxicity of di(2-ethylhexyl)phthalate (DEHP) in peroxisome proliferator-activated receptor alpha-null mice. Toxicol. Pathol. 26, 240–246.
Wood, D.L., Bitman, J., 1984. The effect of feeding di-(2-ethylhexyl) phthalate and related compounds on lipids in the laying hen. Poult. Sci. 63, 469–477.
Xu, X., Yang, Y., Wang, R., Wang, Y., Ruan, Q., Lu, Y., 2015. Perinatal exposure to di-(2- ethylhexyl) phthalate affects anxiety- and depression-like behaviors in mice. Chemosphere 124, 22–31.
Zhang, X.F., Zhang, T., Han, Z., Liu, J.C., Liu, Y.P., Ma, J.Y., Li, L., Shen, W., 2015.
Transgenerational inheritance of ovarian development deficiency induced by mater- nal diethylhexyl phthalate exposure. Reprod. Fertil. Dev. 27, 1213–1221.
Zhang, Q., Zhao, Y., Talukder, M., Han, Y., Zhang, C., Li, X.N., Li, J.L., 2019. Di(2-ethylhexyl) phthalate induced hepatotoxicity in quail (Coturnix japonica) via modulating the mi- tochondrial unfolded protein response and NRF2 mediated antioxidant defense. Sci. Total Environ. 651, 885–894.
Zhang, C., Wang, L.L., Cao, C.Y., Li, N., Talukder, M., Li, J.L., 2020a. Selenium mitigates cadmium-induced crosstalk between autophagy and endoplasmic reticulum stress via regulating calcium homeostasis in avian leghorn male hepatoma (LMH) cells. En- viron. Pollut. 265, 114613.
Zhang, Q., Zhang, C., Ge, J., Lv, M.W., Talukder, M., Guo, K., Li, Y.H., Li, J.L., 2020b. Amelio- rative effects of resveratrol against cadmium-induced nephrotoxicity via modulating nuclear xenobiotic receptor response and PINK1/Parkin-mediated Mitophagy. Food Funct. 11, 1856–1868.
Zhao, Y., Du, Z.H., Talukder, M., Lin, J., Li, X.N., Zhang, C., Li, J.L., 2018. Crosstalk between unfolded protein response and Nrf2-mediated antioxidant defense in Di-(2- ethylhexyl) phthalate-induced renal injury in quail (Coturnix japonica). Environ. Pollut. 242, 1871–1879.
Zhao, Y., Fan, J.H., Luo, Y., Talukder, M., Li, X.N., Zuo, Y.Z., Li, J.L., 2019. Di-(2-ethylhexyl) phthalate (DEHP)-induced hepatotoxicity in quail (Coturnix japonica) via suppres- sion of the heat shock response. Chemosphere 228, 685–693.
Zhao, Y., Li, M.Z., Shen, Y., Lin, J., Wang, H.R., Talukder, M., Li, J.L., 2020a. Lycopene prevents DEHP-induced Leydig cell damage with the Nrf2 antioxidant signaling pathway in mice. J. Agric. Food Chem. 68, 2031–2040.
Zhao, Y., Lin, J., Talukder, M., Zhu, S.Y., Li, M.Z., Wang, H.R., Li, J.L., 2020b. Aryl hydrocarbon receptor as a target for lycopene preventing DEHP-induced spermatogenic disorders.
J. Agric. Food Chem. 68, 4355–4366.
Zhao, Y., Li, M.Z., Talukder, M., Luo, Y., Shen, Y., Li, J., Wang, H.R., Li, J.L., 2020c. Effect of mitochondrial quality control on lycopene antagonizing DEHP-induced mitophagy in spermatogenic cells. Food Funct. https://doi.org/10.1039/ D0FO00554A.
Zong, T., Lai, L., Hu, J., Guo, M., Li, M., Zhang, L., Zhong, C., Yang, B., Wu, L., Zhang, D., Tang, M., Kuang, H., 2015. Maternal exposure to di-(2-ethylhexyl) phthalate disrupts pla- cental growth and development in Rosiglitazone pregnant mice. J. Hazard. Mater. 297, 25–33.