Vu Hoang Dang

Di-(2 ethylhexyl) phthalate and flutamide alter gene expression in the testis of immature male rats

We previously demonstrated that the androgenic and anti-androgenic effects of endocrine disruptors (EDs) alter reproductive function and exert distinct effects on developing male reproductive organs. To further investigate these effects, we used an immature rat model to examine the effects of di-(2 ethylhexyl) phthalate (DEHP) and flutamide (Flu) on the male reproductive system. Immature male SD rats were treated daily with DEHP and Flu on postnatal days (PNDs) 21 to 35, in a dose-dependent manner. As results, the weights of the testes, prostate, and seminal vesicle and anogenital distances (AGD) decreased significantly in response to high doses of DEHP or Flu. Testosterone (T) levels significantly decreased in all DEHP- treated groups, whereas luteinizing hormone (LH) plasma levels were not altered by any of the two treatments at PND 36. However, treatment with DEHP or Flu induced histopathological changes in the testes, wherein degeneration and disorders of Leydig cells, germ cells and dilatation of tubular lumen were observed in a dose-dependent manner. Conversely, hyperplasia and denseness of Leydig, Sertoli and germ cells were observed in rats given with high doses of Flu. The results by cDNA microarray analysis indicated that 1,272 genes were up-regulated by more than two-fold, and 1,969 genes were down-regulated in response to DEHP, Flu or both EDs. These genes were selected based on their markedly increased or decreased expression levels. These genes have been also classified on the basis of gene ontology (e.g., steroid hormone biosynthetic process, regulation of transcription, signal transduction, metabolic process, biosynthetic process...). Significant decreases in gene expression were observed in steroidogenic genes (i.e., Star, Cyp11a1 and Hsd3b). In addition, the expression of a common set of target genes, including CaBP1, Vav2, Plcd1, Lhx1 and Isoc1, was altered following exposure to EDs, suggesting that they may be marker genes to screen for the anti-androgenic or androgenic effects of EDs. Overall, our results demonstrated that exposure to DEHP, Flu or both EDs resulted in a alteration of gene expression in the testes of immature male rats. Furthermore, the toxicological effects of these EDs on the male reproductive system resulted from their anti-androgenic effects. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of EDs, in regards to anti-androgenic effects in humans and wildlife.

In vitro exposure to xenoestrogens induces growth hormone transcription and release via estrogen receptor-dependent pathways in rat pituitary GH3 cells

In this study, we employed an in vitro model to examine the effects of endocrine disruptors (EDs) in the regulation of growth hormone (GH) gene, an important hormone in growth, development and body composition. The rat pituitary cells, GH3, were treated with alkyl-phenols (APs), i.e., 4-tert-octyl-phenol (OP), p-nonyl-phenol (NP) or bisphenol A (BPA) for 24h in a dose-dependent manner (10(-5), 10(-6) and 10(-7)M) and in a time-dependent fashion (1, 3, 6, 12 and 24h) at a high concentration (10(-5)M). An anti-estrogen, ICI 182,780, was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. Treatment with OP, NP and BPA induced a significant increase in GH gene expression at high and medium doses at 24h. ED-exposure induced a marked increase in GH gene transcription as early as 6h and peaked at 12h. Co-treatment with ICI 182,780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was significantly increased at 24h in response to OP, NP or BPA, whereas co-treatment with ICI 182,780 significantly reversed ED-induced GH secretion, indicating that ER may take part in both GH gene transcription and its release in these cells. In addition, the activation of extracellular signal-regulated kinases (ERKs), protein kinases B (Akt) or G protein in response to OP, NP or BPA at 24h was observed in this study. Exposure to these APs resulted in a rapid and significant activation of ERK phosphorylation, reflecting that EDs-induced response may involve both genomic and non-genomic pathways in these cells. Taken together, these results may provide new insight into the mode of ED-induced action in GH gene regulation as well as the biological pathway underlying these molecular events.

Estrogen regulates the localization and expression of calbindin-D9k in the pituitary gland of immature male rats via the ERα-pathway

Estrogen (E2; estradiol) plays a key role in the regulation of many pituitary hormones. It exerts its effects by binding to the intracellular estrogen receptor (ER), which then functions as a transcription factor. Although E2 has been shown to regulate calbindin-D(9k) (CaBP-9k) in the female reproductive system of rodents, the effects of E2 on the regulation of CaBP-9k in male rats remain to be elucidated. To investigate E2-induced regulation of the pituitary CaBP-9k gene, immature male rats were injected with E2 daily for 3 consecutive days with a dose of 40 microg/kg body weight (BW). The expression levels of CaBP-9k mRNA and protein were analyzed by RT-PCR and Western blot analysis, respectively, in the absence and presence of ICI 182,780 (ICI), an E2 antagonist. In addition, the tissue localization of CaBP-9k was determined by immunohistochemistry. CaBP-9k was localized in the cytoplasm of a specific cell type (acidophils) in the anterior lobe of the pituitary gland and highly expressed in the intermediate lobe. Exposure to E2 increased the number of cells that stained positive for CaBP-9k. To determine which ER subtype is involved in CaBP-9k regulation in the pituitary, the immature rats were treated with propyl pyrazole triol (PPT, an ERalpha-selective ligand) or diarylpropionitrile (DPN, an ERbeta-selective ligand) for 3 days. Pituitary CaBP-9k expression was mainly mediated by PPT in immature male rats, whereas no significant alteration of pituitary CaBP-9k gene expression was observed after DPN treatment. In addition, the estrogenicity of PPT in the induction of CaBP-9k expression was completely blocked by an estrogen antagonist, ICI, indicating that pituitary CaBP-9k expression is solely induced by ERalpha. Taken together, these results suggest that pituitary CaBP-9k is induced by E2 in male rats and its expression is predominantly regulated by ERalpha, but not ERbeta.

Induction of Uterine Calbindin-D9k Through an Estrogen Receptor-Dependent Pathway Following Single Injection with Xenobiotic Agents in Immature Rats

Various environmental chemicals, both natural and synthetic, are believed to act as endocrine disruptors (EDs) in mammals. In this study, a new in vivo model of immature rats was used to explore the induction of calbindin-D9k (CaBP-9k) following a single injection of EDs. In a time-dependent experiment, immature rats at postnatal day 16 were treated with high doses (600 mg/kg body weight [BW]) of 4-tert-octyphenol (OP), p-nonylphenol (NP), or bisphenol A (BPA), and euthanized at different time points (3, 6, 12, 24, or 48 h). For a dose-dependent study, immature rats were given different doses (200, 400, or 600 mg/kg BW) and euthanized at 24 h after injection. After treatment with these EDs, the effects on CaBP-9k mRNA and protein were examined by Northern and Western blot analyses, respectively. An anti-estrogen, ICI 182,780, was employed to examine the potential involvement of estrogen receptor (ER) in the induction of estrogen receptor-mediated physiologic responses in vivo. A single treatment with each of the chemicals, at 600 mg/kg BW, resulted in a significant increase in the expression of CaBP-9k mRNA and protein 24 h after injection. In addition, treatment with OP, NP, or BPA resulted in a positive uterotrophic response. Cotreatment with the ER antagonist ICI 182,780 completely prevented the ED-induced uterine weight gain. Taken together, these results demonstrate that a single injection of OP, NP, or BPA results in an increase of CaBP-9k mRNA and protein via an ER-dependent pathway in the uterus of immature rats. This new model may be important to elucidate the mechanism of action of xenoestrogens on estrogen-sensitive tissue.

Membrane-impermeable estrogen is involved in regulation of calbindin-D9k expression via non-genomic pathways in a rat pituitary cell line, GH3 cells.

Estrogen (E2) has been shown to regulate various functions for many pituitary hormones. Recently, the potential roles of non-genomic pathways in E2-induced actions have been proposed in the previous studies, however, the effects of E2 remain to be elucidated in regard to non-genomic induction of cytosolic protein calbindin-D9k (CaBP-9k). To gain a better understanding of the molecular events underlying E2-induced expression of CaBP-9k, rat pituitary tumor cells (GH3 cells) were treated with E-BSA (membrane impermeable E2-conjugated with BSA). Non-genomic induction of CaBP-9k by E-BSA was determined using RT-PCR and western blot analysis. The significant increase in CaBP-9k mRNA level was observed as early as 15 min following treatment with a high concentration of E-BSA (10(-6)M), whereas rapid and significant induction of CaBP-9k protein was noted at 5, 15 and 30 min after E-BSA exposure (p<0.05). In order to determine the potential involvement of different signaling pathways, several inhibitors were employed, i.e., ICI 182,780 for the estrogen receptor (ER) pathway, pertussis toxin (PTX) for the G-protein-coupled signaling pathway, U0126 (U) for the ERK (extracellular regulated kinase) and wortmannin (W) for the Akt (protein kinase B). Co-treatment with ICI 182, 780 and PTX reversed an E-BSA-induced increase in CaBP-9k mRNA and protein. Although neither U nor W alone attenuated E-BSA-induced effects, these inhibitors together abolished E-BSA-induced CaBP-9k expression, suggesting their involvement in its regulation. Taken together, these results demonstrate the involvement of various signaling pathways in E2-induced regulation of CaBP-9k. In addition, ER and G-protein-coupled signaling pathways may play central roles in the non-genomic activities of E2 and that downstream signaling via ERK and Akt are required to evoke ER-mediated induction of CaBP-9k in vitro.

Depletion of follicles accelerated by combined exposure to phthalates and 4-vinylcyclohexene diepoxide, leading to premature ovarian failure in rats

Humans are at daily risk by simultaneous exposures to a broad spectrum of man-made chemicals in the commercial products. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to premature ovarian failure (POF) in mammals. We investigate the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalate, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) on POF. Combination exposure with VCD and phthalate significantly reduced the numbers of primary follicles. The expressions of Amh and Sohlh2 were significantly decreased in the combination groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of follicle-stimulating hormone were significantly increased in combination groups. Taken together, exposure to phthalates promotes the depletion of follicular follicles and consequently increases the risk of premature menopause, and combined exposure of phthalates and VCD to early menopausal women is likely to aggravate the POF syndrome.