Yajie Chen

Exposure to DEHP and MEHP from hatching to adulthood causes reproductive dysfunction and endocrine disruption in marine medaka (Oryzias melastigma).

Concern has increased regarding the adverse effects of di-(2-ethylhexyl)-phthalate (DEHP) on reproduction. However, limited information is available on the effects of DEHP in marine organisms. The aim of the present study was to examine whether long-term exposure to DEHP and its active metabolite mono-(2-ethylhexyl)-phthalate (MEHP) disrupts endocrine function in marine medaka (Oryzias melastigma). Marine medaka larvae were exposed to either DEHP (0.1 and 0.5mg/L) or MEHP (0.1 and 0.5mg/L) for 6 months, and the effects on reproduction, sex steroid hormones, liver vitellogenin (VTG), gonad histology and the expression of genes involved in the hypothalamic-pituitary-gonad (HPG) axis were investigated. Exposure to DEHP, but not MEHP, from hatching to adulthood accelerated the start of spawning and decreased the egg production of exposed females. Moreover, exposure to both DEHP and MEHP resulted in a reduction in the fertilization rate of oocytes spawned by untreated females paired with treated males. A significant increase in plasma 17β-estradiol (E2) along with a significant decrease in testosterone (T)/E2 ratios was observed in males, which was accompanied by the upregulation of ldlr, star, cyp17a1, 17βhsd, and cyp19a transcription in the testis. Increased concentrations of T and E2 were observed in females, which was consistent with the upregulation of ldlr. The expression of brain gnrhr2, fshβ, cyp19b and steroid hormone receptor genes also corresponded well with hormonal and reproductive changes. The liver VTG level was significantly increased after DEHP and MEHP exposure in males. DEHP induced histological changes in the testes and ovaries: the testes displayed a reduced number of spermatozoa, and the ovaries displayed an increased number of atretic follicles. In addition, the tissue concentrations of MEHP, MEHHP and MEOHP in DEHP-exposed groups were much higher than those in MEHP-exposed groups, and there were no dose- or sex-specific effects. Thus, DEHP exerts more obvious toxic effects compared with MEHP. There were some commonalities in the toxic effects and molecular mechanisms of DEHP and MEHP, suggesting that some of the toxic effects of DEHP may be induced by both DEHP itself and DEHP metabolites (including MEHP). Taken together, these results indicate that exposure to DEHP and MEHP from hatching to adulthood causes endocrine disruption with sex-specific effects in marine medaka, with males being more sensitive than females.

Perinatal exposure to bisphenol A exacerbates nonalcoholic steatohepatitis-like phenotype in male rat offspring fed on a high-fat diet

Bisphenol A (BPA) is one of the environmental endocrine disrupting chemicals, which is present ubiquitously in daily life. Accumulating evidence indicates that exposure to BPA contributes to metabolic syndrome. In this study, we examined whether perinatal exposure to BPA predisposed offspring to fatty liver disease: the hepatic manifestation of metabolic syndrome. Wistar rats were exposed to 50 μg/kg per day BPA or corn oil throughout gestation and lactation by oral gavage. Offspring were fed a standard chow diet (SD) or a high-fat diet (HFD) after weaning. Effects of BPA were assessed by examination of hepatic morphology, biochemical analysis, and the hepatic expression of genes and/or proteins involved in lipogenesis, fatty acid oxidation, gluconeogenesis, insulin signaling, inflammation, and fibrosis. On a SD, the offspring of rats exposed to BPA exhibited moderate hepatic steatosis and altered expression of insulin signaling elements in the liver, but with normal liver function. On a HFD, the offspring of rats exposed to BPA showed a nonalcoholic steatohepatitis-like phenotype, characterized by extensive accumulation of lipids, large lipid droplets, profound ballooning degeneration, impaired liver function, increased inflammation, and even mild fibrosis in the liver. Perinatal exposure to BPA worsened the hepatic damage caused by the HFD in the rat offspring. The additive effects of BPA correlated with higher levels of hepatic oxidative stress. Collectively, exposure to BPA may be a new risk factor for the development of fatty liver disease and further studies should assess whether this finding is also relevant to the human population.

Di(2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection

Di(2‐ethylhexyl) phthalate (DEHP) is used as plasticizer and is ubiquitously found in the environment. Exposure to DEHP has been linked to an increased incidence of type 2 diabetes. Pancreatic β‐cell dysfunction is a hallmark of type 2 diabetes; however, it is unknown whether DEHP exposure contributes to this risk. Here, we aimed to investigate the cytotoxic effects of DEHP on INS‐1 cells and to further explore the related underlying mechanisms. INS‐1 cells were exposed to 0, 5, 25, 125 or 625 μM DEHP for 24 hrs. Cell viability, glucose‐stimulated insulin secretion, reactive oxygen species (ROS) generation, cellular antioxidant response, Ca2+ homoeostasis and the levels of genes and proteins involved in endoplasmic reticulum (ER) stress were measured. The results showed that DEHP decreased insulin secretion and content and induced apoptosis in INS‐1 cells in a dose‐dependent manner. Furthermore, ROS generation was increased and Nrf2‐dependent antioxidant defence protection was dysregulated in INS‐1 cells after DEHP exposure. Most importantly, DEHP effectively depleted ER Ca2+ and triggered the ER stress response as demonstrated by the elevated transcription and translation of the ER chaperone GRP78 and GRP94, the increased phosphorylation of protein kinase R‐like endoplasmic reticulum kinase (PERK) and its downstream substrate eukaryotic translation initiation factor 2α (eIF2α), as well as the increased levels of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Taken together, DEHP exerted toxic effects on INS‐1 cells by inducing apoptosis, which is dependent on the activation of the PERK–ATF4–CHOP ER stress signalling pathway and the suppression of Nrf2‐dependent antioxidant protection.

Accumulation of di(2‐ethylhexyl) phthalate causes endocrine‐disruptive effects in marine medaka (Oryzias melastigma) embryos

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.

Embryonic exposure to PFOS induces immunosuppression in the fish larvae of marine medaka

Perfluorooctane sulfonate (PFOS) is a global pollutant that has been studied because of its health risks. PFOS has been shown to have immune toxicity. However, few studies have focused on the immune responses of fish larvae exposed to PFOS at early embryonic stages. In this study, the larvae of marine medaka (Oryzias melastigma) were evaluated for postnatal immune toxicity after embryonic exposure to PFOS (0, 1, 4 and 16mg/L) from 2 days post fertilization (dpf). The physiological indices, survival rates, PFOS elimination kinetics, liver histology and gene transcription in the fish larvae were examined after depuration. The elimination rate constant (ke) of PFOS in the fish larvae ranged from 0.04±0.00 to 0.07±0.01d(-1). Embryonic exposure to PFOS severely compromised the postnatal survival of fish larvae after depuration. The survival rate and body width decreased in a concentration dependent manner. PFOS impaired the liver structure in the fish larvae by enlarging the cell nuclei and damaging the cell structure. To explore the toxic mechanisms that affect the immune responses, fish larvae at 27 days post hatch (dph) were exposed to lipopolysaccharides (LPS) to elicit an inflammatory response. The inflammatory response and immune-related genes were generally up-regulated in the fish larvae following embryonic exposure to 0mg/L PFOS. In contrast, the genes were all markedly down-regulated in the fish larvae following embryonic exposure to 1 and 4mg/L PFOS. These results suggest that early life exposure to PFOS could alter immunoregulation functions, leading to functional dysfunction or weakness of the immune system in fish larvae. The immunosuppression effects caused by PFOS could reduce the efficiency of immune defense mechanisms and increase the susceptibility to infectious agents, which may contribute to various detrimental health effects in the fish larvae.

The inflammation and estrogen metabolism impacts of polychlorinated biphenyls on endometrial cancer cells

Polychlorinated biphenyls (PCBs) are persistent and bio-accumulative chemicals that provoke a wide range of toxic effects. Their adverse impacts on the reproductive system are of great concern, however, the effects of PCBs on endometrium are still unclear. In the study, the endometrial adenocarcinoma Ishikawa cells were exposed to both dioxin-like CB126 and non-dioxin-like CB153 at the nominal concentrations of 0.3, 3, and 30μM. The inflammatory and endocrine effects were detected after treatment by PCBs. Results showed that CB126 stimulated the proliferation of Ishikawa cells at lower concentrations of 0.3 and 3μM. By contrast, CB153 did not affect the viability of the cells. Both congeners exerted the stimulatory effects on the enzymatic activity of SOD1. CB126 decreased the abundance of Interleukin-8 both at the mRNA and protein levels. Blocking of estrogen receptor or aryl hydrocarbon receptor by the antagonist abolished the effects of CB126 on the expressions of inflammatory factors. The levels of testosterone and 17beta-estradiol were not changed after exposure to lower doses of PCBs. In accordance, PCBs did not affect the mRNA expressions of estrogen metabolism-related genes. In all, our study revealed that PCBs affected the expression of inflammatory factors through ER and AHR receptors, however, no toxic effects were observed on estrogen metabolism.

Dioxin-like rather than non-dioxin-like PCBs promote the development of endometriosis through stimulation of endocrine–inflammation interactions

Polychlorinated biphenyls (PCBs) contain 209 congeners with various structure–activities. Exposure to PCBs was related to disorders of female reproduction. Endometriosis (EM) is an estrogen- and inflammation-dependent disease with high prevalence and severe health outcomes. Epidemiological studies have shown the effects of PCBs exposure on EM in regard to various structures of PCBs. However, little evidence is available from the toxicology considering the structure of PCBs. In the study, environmentally relevant concentrations of PCBs were used to treat primary cultured endometrial cells and an EM mouse model. Dioxin-like CB126, but not non-dioxin-like CB153, significantly enhanced 17β-estradiol (E2) biosynthesis in a dose-dependent manner. Among the genes related to estrogen metabolism, the level of 17β-hydroxysteroid dehydrogenase 7 (HSD17B7) showed significant increase following CB126 exposure. We further found that CB126 exposure decreased the methylation of the HSD17B7 promoter. Elevated expression of HSD17B7 was observed in the eutopic endometrium of EM patients. CB126 rather than CB153 triggered the inflammatory response by directly stimulating the secretion of inflammatory factors and indirectly reducing the level of lipoxin A4 (LXA4). Furthermore, the inflammation enhanced the expression of HSD17B7. Antagonism of the aryl hydrocarbon receptor (AhR) diminished the effects induced by CB126. In vivo, the PCB-treated EM mouse model confirmed that CB126 rather than CB153 increased the levels of both E2 and inflammatory factors in peritoneal fluid and promoted the development of endometriotic lesions. In all, CB126, but not CB153, triggered EM development by stimulating estrogen biosynthesis, inflammation and their interactions and that these effects were mediated by the AhR receptor.

The combined effects of DEHP and PCBs on phospholipase in the livers of mice

Di(2‐ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) are two widely distributed pollutants that are of great concern due to their adverse health effects. However, few studies have investigated the combined effects of DEHP and PCBs. In this study, adult mice were continuously exposed to mixtures of DEHP (15 mg/kg bodyweight/day) and Aroclor 1254 (7.5 mg/kg bodyweight/day) for 12 days to investigate the combined effects of these compounds. The results showed that the ratio of the liver weight to the body weight was higher in the treated group than that in the control group. The effects of combined exposure on three important receptors, the proliferator‐activated receptor (PPAR), estrogen receptor (ER), and aryl hydrocarbon receptor (AHR), were investigated. The mRNA level of PPARγ was significantly up‐regulated after exposure. The expression level of ERα was decreased in the male treated group. In contrast, the expression levels of AHR and related genes (cyp1a1 and cyp1b1) were not markedly affected. The expression level of phospholipase A (PLA) was significantly down‐regulated at both the mRNA and protein levels in male mice after combined treatment. In all, our study demonstrated the combined effects of DEHP and PCBs on the expression levels of key receptors in mice. The combined exposure led to a decrease in phospholipase in male mice.

The Inflammation Response to DEHP through PPARγ in Endometrial Cells

Epidemiological studies have shown the possible link between phthalates and endometrium-related gynecological diseases, however the molecular mechanism(s) behind this is/are still unclear. In the study, both primary cultured endometrial cells and an endometrial adenocarcinoma cell line (Ishikawa) were recruited to investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) at human-relevant concentrations. The results showed that DEHP did not affect the viability of either type of cell, which showed different responses to inflammation. Primary cultured cells showed stronger inflammatory reactions than the Ishikawa cell line. The expression of inflammatory factors was induced both at the mRNA and protein levels, however the inflammation did not induce the progress of epithelial-mesenchymal transition (EMT) as the protein levels of EMT markers were not affected after exposure to either cell type. Further study showed that the mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) wereup-regulated after exposure. In all, our study showed that human-relevant concentrations of DEHP could elicit the inflammatory response in primary cultured endometrial cells rather than in Ishikawa cell line. PPARγ may act as the mediating receptor in the inflammation reaction.

Different effects of bisphenol a and its halogenated derivatives on the reproduction and development of Oryzias melastigma under environmentally relevant doses

Bisphenol A (BPA) and its halogenated compounds (H-BPAs) are widely detected in the environmental media and organisms. However, their toxicological effects, especially chronic exposure at low doses, have not been fully compared. In this study, the effects of BPA and H-BPAs on the reproduction and development of Oryzias melastigma were systematically assessed and compared at various developmental stages. BPA and its derivatives tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) elicited the acceleration of embryonic heartbeat. BPA did not show any significant impact on the hatching time and rate of embryos. In contrast, both TBBPA and TCBPA led to the delayed hatching and decreased hatching rate. Accordingly, the expressions of hatching enzyme significantly decreased upon exposure and TCBPA was found to be more toxic than TBBPA. The body weight and gonadsomatic index (GSI) of the treated fish were relatively lower than the control fish upon long-term (four months from larvae to adult) exposure to BPA rather than H-BPAs. Slowed oocyte development occurred in the ovary, and the estrogen level decreased after exposure to BPA rather than H-BPAs. In male fish, no significant alteration was observed in the testis for all groups. The concentration of testosterone significantly decreased upon exposure to BPA rather than H-BPAs. The effects of these three chemicals on the estrogen-related gene expressions were different under various developmental stages. Our study indicated the importance of considering both the exposure stages and structure-activity relationship when assessing the eco-toxicological impact of pollutants.