Abstract:Bisphenol-A

To assess the health risks associated with exposure to 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD), we studied the effects of a relatively low dose of TCDD on the male reproductive system of rats, using the experimental protocol of T. A. Mably et al. (1992, Toxicol. Appl. Pharmacol. 114, 97-107, 108-117, 118-126), and searched for the most sensitive and reliable among several indices of TCDD toxicity. Pregnant Holtzman rats were given a single oral dose of 0, 12.5, 50, 200, or 800 ng TCDD/kg body weight on gestational day (GD) 15, and male offspring were sacrificed on postnatal day (PND) 49 or 120. GC-MS analysis of the abdominal fat tissue and testis clearly showed increased amounts of TCDD in these offspring. However, there was no TCDD effect on body weight of offspring. There were no changes on testicular or epididymal weights by TCDD administration, even at the 800-ng/kg dose in rats sacrificed on either PND 49 or 120. In addition, TCDD administration resulted in no changes in daily sperm production or sperm reserve at any of the doses used. However, the weight of the urogenital complex, including the ventral prostate, was significantly reduced at doses of 200 and 800 ng TCDD/kg in rats sacrificed on PND 120. Moreover, the anogenital distance (AGD) of male rats sacrificed on PND 120 showed a significant decrease in the groups receiving doses greater than 50 ng TCDD/kg. TCDD administration resulted in no apparent dose-dependent changes in levels of either serum testosterone or luteinizing hormone. Interestingly, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that, in the ventral prostates of the PND 49 group, TCDD administration resulted in both a dose-dependent increase in 5alpha-reductase type 2 (5alphaR-II) mRNA level and a dose-dependent decrease in androgen receptor (AR) mRNA level. These results suggest that low-dose TCDD administration had a greater effect on the development of the external genital organs and ventral prostate than on development of the testis and other internal genital organs. Moreover, it is highly suggested that the decrease in the size of the ventral prostate by maternal TCDD exposure might be due to decreased responsiveness of the prostate to androgen due to an insufficient expression level of androgen receptor during puberty.

The androgen receptor (AR) is implicated in prostate cancer growth, progression, and angiogenesis. Hypoxia-inducible factor-1 (HIF-1), which transcriptionally regulates hypoxia-inducible angiogenic factors, is up-regulated in prostate cancers compared with adjacent normal tissues. HIF-1 may be involved in prostate cancer as well as the AR, but the involvement of HIF-1 in prostate cancer angiogenesis and progression has not been fully elucidated. In the present study, we found that in prostate cancer LNCaP cells dihydrotestosterone enhanced the expression of GLUT-1, one of the HIF-1 target genes, and also that hypoxia enhanced the expression of prostate-specific antigen (PSA) that is one of the AR target genes and is involved in tumor invasion. Small interfering RNA that specifically inhibits HIF-1 reduced the expression levels of PSA as well as GLUT-1. Reporter gene analysis showed that dihydrotestosterone activated the HIF-1 -mediated gene expression and hypoxia enhanced the AR-induced promoter activity of human PSA gene. Deletion and site-directed mutation of the 5 ¶-flanking region of human PSA gene revealed that the sequence ACGTG between À3951 and À3947 was essential in the response to hypoxia. Furthermore, chromatin immunoprecipitation assay indicated that HIF-1 interacts with the AR on the human PSA gene promoter. These results indicated that in prostate cancers, HIF-1 might cooperate with the AR to activate the expression of several genes related to tumor angiogenesis, invasion, and progression. (Mol Cancer Res 2007;5(4):383 -91)

Exposure to a relatively low dose of 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD) during mid-gestation induces a reduction of ventral prostate weight in rat offspring. Recently we reported that a single administration of TCDD (12.5-800 ng/kg body weight) to pregnant Holtzman rats on gestational day (GD) 15 caused a decrease in androgen receptor (AR) mRNA level in the ventral prostate during the prepubertal period, and we proposed that this reduction of AR mRNA is one of the most sensitive adverse endpoints due to perinatal exposure to TCDD (S. Ohsako et al., 2001, TOXICOL: Sci. 60, 132-143). In the present study, to investigate the mechanism of a decrease in AR mRNA level, we administered TCDD to rats at other developmental stages and compared possible alterations of the male reproductive system. Pregnant Sprague-Dawley rats were given a single oral dose of 1 microg TCDD/kg body weight on GD 15 or GD 18, or male pups born from untreated dams were subcutaneously given a single dose of 1 microg TCDD/kg body weight on postnatal day 2 (PND 2). Offspring exposed on GD 15, GD 18, and PND 2 were sacrificed on PND 70. TCDD exposure on GD 15 resulted in significant decreases in the urogenital complex and ventral prostate weights and urogenital-glans penis length of male rat offspring, but not on GD 18 and PND 2. Testicular and epididymal weights were also lower than control group only in the TCDD-exposed GD 15 group. Anogenital distance was significantly reduced in the TCDD-exposed GD 15 and GD 18 groups, but not in the TCDD-exposed PND 2 group. Semiquantitative RT-PCR analysis showed that AR mRNA levels were decreased in the TCDD-exposed GD 15 group only, and that the constitutive level of cytochrome P450 1A1 (CYP1A1) mRNA in the ventral prostate was not changed by TCDD in any of the exposed groups. No changes in AR mRNA level were detected in the testis or brain in any of the TCDD-exposed groups. These results suggest the presence of a critical window during development with regard to impairments of male reproductive organs by in utero and lactational exposure to a low dose of TCDD.

No abstract

Adipose differentiation is a complex process associated with coordinated changes in gene expression, cell morphology, and hormone sensitivity.1) Several transcription factors influence these processes, among which peroxisome proliferator activated receptor g (PPARg) and CCAAT/enhancerbinding protein (C/EBP) family have been extensively studied.2,3) C/EBPb and C/EBPd are induced in the early stage of adipocyte differentiation and then mediate the expression of PPARg and C/EBPa. [4][5][6] In contrast to C/EBPd, C/EBPb is able to induce spontaneous differentiation in 3T3-L1 preadipocytes and enhance the adipogenic potential in NIH-3T3 fibroblasts. 5,6) Highly specific for adipose tissues, PPARg plays a critical role in the expression of most adipocyte-specific genes 7) and is able to convert nonadipogenic mesenchymal cells, such as fibroblasts and myoblasts, to adipocytes. 8,9) On the other hand, although developmentally necessary for adipogenesis, 10) C/EBPa is not always expressed during adipose differentiation and is critical in the establishment of insulin sensitivity. 11)Several recent studies have shown that hypoxia-inducible factor (HIF) is also involved in the regulation of adipose differentiation. [12][13][14] HIF is a transcriptional factor that plays a central role in oxygen homeostasis in mammalian cells by stimulating expression of oxygen-regulated genes related to erythropoiesis, angiogenesis, glucose uptake, and glycolysis. 15,16) HIF is a heterodimer composed of HIF-a and -b, both of which belong to basic helix-loop-helix (bHLH)/Per-ARNT-Sim (PAS) domain transcriptional factors.17) The HIFb subunit is identical to the arylhydrocarbon receptor nuclear translocator (ARNT) that also serves as a heterodimeric partner with the arylhydrocarbon receptor (AhR).18) In contrast, it appears that the HIF-a subunit's sole but critical function is to mediate the response to hypoxia. Under normoxic conditions, HIF-a subunits are hydroxylated at two conserved proline (Pro564 and Pro402 in HIF-1a) residues and one asparagine (Asn803 in HIF-1a) residue by HIF-a prolyl hydroxylases (PHD1, PHD2, and PHD3) and asaparagine hydroxylase FIH-1 (factor inhibiting HIF-1), respectively, and the hydroxylated HIF-a subunit proteins are destabilized and transcriptionally suppressed.19) Deprivation of oxygen by exposing cells to hypoxia prevents HIF hydroxylase activity and then stabilizes and transcriptionally activates HIF-a subunits. The first identified isoform of HIF-a, HIF-1a, was originally discovered as a high-affinity DNA-binding protein localized to the 3Ј hypoxia-responsive element (HRE) on the erythropoietin (EPO) gene. Using a subtraction cloning approach, Imagawa et al. found that HIF-1a mRNA is transiently induced in 3T3-L1 preadipocytes upon treatment with the adipogenic hormone cocktail containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine.12) Yun et al. found that HIF-1a represses PPARg2 gene expression and then inhibits adipogenesis through the induction of expression of transcriptional repressor DE...

We show that highly pure populations of human Schwann cells can be derived rapidly and in a straightforward way, without the need for genetic manipulation, from human epidermal neural crest stem cells [hEPI-NCSC(s)] present in the bulge of hair follicles. These human Schwann cells promise to be a useful tool for cell-based therapies, disease modelling and drug discovery. Schwann cells are glia that support axons of peripheral nerves and are direct descendants of the embryonic neural crest. Peripheral nerves are damaged in various conditions, including through trauma or tumour-related surgery, and Schwann cells are required for their repair and regeneration. Schwann cells also promise to be useful for treating spinal cord injuries. Ex vivo expansion of hEPI-NCSC isolated from hair bulge explants, manipulating the WNT, sonic hedgehog and TGFβ signalling pathways, and exposure of the cells to pertinent growth factors led to the expression of the Schwann cell markers SOX10, KROX20 (EGR2), p75NTR (NGFR), MBP and S100B by day 4 in virtually all cells, and maturation was completed by 2 weeks of differentiation. Gene expression profiling demonstrated expression of transcripts for neurotrophic and angiogenic factors, as well as JUN, all of which are essential for nerve regeneration. Co-culture of hEPI-NCSC-derived human Schwann cells with rodent dorsal root ganglia showed interaction of the Schwann cells with axons, providing evidence of Schwann cell functionality. We conclude that hEPI-NCSCs are a biologically relevant source for generating large and highly pure populations of human Schwann cells.

Abstract. The effect of vinclozolin (VCZ), used as a fungicide and known to have anti-androgenic effects on spermatogenesis and gene expression in the male rat testis was investigated. In Experiment 1, VCZ (100 mg/kg/day) or flutamide (FM, 25 mg/kg/day) was orally administered to male Holzman rats for six days. 8 days after the last administration (D8), a drastic increase in intratesticular testosterone was detected in FM (4.2-fold over control) but not in VCZ treated animals, whereas on D36 post-administration, both groups showed similar levels. Significant decreases in daily sperm production were seen in both VCZ and FM-treated rats on D36. Semiquantitative RT-PCR analysis with testicular and pituitary mRNAs on D8 revealed that LHβ and FSHβ mRNAs were increased in the pituitary by VCZ, as well as by FM. Among the four testicular steroidogenic enzyme genes, cytochrome P450 side chain cleavage (P450scc) and cytochrome P450 17α/C17-20 lyase (P450c17) mRNAs were significantly increased, whereas 17β-hydroxysteroid dehydrogenase type III (17βHSD) mRNA was not changed. A significant increase in 3β-hydroxysteroid dehydrogenase type I (3βHSD) and a decrease in androgen receptor (AR) mRNA were observed only in FM treated rats. Immunohistochemistry demonstrated intense staining of P450scc in the interstitial cells of VCZtreated testis on D8. In Experiment 2, hormone levels were measured at 1, 3, 6, 12 and 24 hours after VCZ (100 mg/kg) administration to Sprague-Dawley rats. Serum LH level remained constant for the first 3 hours and started to increase at 6 hrs. In contrast, serum and intratesticular testosterone levels increased 2-fold at 1 hr and maintained the level until 24 hrs. P450c17 mRNA level was 2-fold increased at all periods, whereas no obvious changes were detected in the other steroidogenic enzyme genes. Although not statistically significant, AR mRNA level increased 2-fold, 3 hrs after VCZ administration. These results indicate that VCZ affects the pituitary in a similar manner as FM, but functions differently on testicular gene expression.