Unraveling Hypothalamus-Pituitary dysregulation: Hypergonadotropism in F1 progeny due to prenatal exposure to hexavalent chromium.

The endocrine disruptor hexavalent chromium [Cr(VI)] is a proven reproductive toxicant. We recently demonstrated that prenatal Cr(VI) exposure causes testicular resistance to gonadotropins, resulting in hypergonadotropic hypoandrogenism in F1 rats. However, the mechanism driving hypergonadotropism in F1 rats exposed to Cr(VI) prenatally remains an enigma. Therefore, we hypothesized that 'Prenatal Cr(VI) exposure may disrupt steroid hormones-mediated negative feedback regulation of the hypothalamic GnRH, and its receptor in the pituitary of F1 rats, leading to hypergonadotropism.' We administered potassium dichromate (50, 100, or 200 mg/L) to pregnant rats through drinking water between days 9 and 14, and their male F1 offspring were euthanized at 60 days of age. Prenatal Cr(VI) exposure in F1 rats resulted in the accumulation of Cr in the hypothalamus and pituitary. Western blot detected decreased hypothalamic GnRH, Kisspeptin1, and its receptor GPR54, along with diminished ERα, AR, aromatase, and 5α reductase, and GnRH regulatory transcription factors Pit-1 and GATA-4 proteins. Immunohistochemical studies revealed increased immunopositivity of GnRH receptor, AR, 5α reductase, ERα, ERß, and aromatase proteins in the pituitary, whereas decreased Kisspeptin1, GPR54, and inhibin ß. Our findings imply that Cr(VI) exposure during the prenatal period disrupts the hypothalamic Kisspeptin-GPR54-Pit-1/GATA4-GnRH network, boosting the pituitary GnRH receptor. We conclude that prenatal exposure to Cr(VI) alters GnRH expression in the hypothalamus and its receptor in the pituitary of F1 progeny through interfering with the negative feedback effect of androgens and estrogens.

High Fat-High Fructose Diet Elicits Hypogonadotropism Culminating in Autophagy-Mediated Defective Differentiation of Ovarian Follicles.

Pituitary gonadotropins directly govern ovarian functions, which are in turn regulated by the ovarian steroid hormones. The precise interplay of gonadotropins and steroid hormones is critical for follicle growth and differentiation. Furthermore, autophagy regulates ovarian follicle differentiation. However, how the high-fat-high fructose (HFD-HF) diet regulates gonadotropins and facilitates autophagy-mediated follicular differentiation in the ovary is obscure. We fed prepubertal rats (PND 25) an HFD-HF diet until PND 90. The results showed diminished adenohypophyseal GnRHR, PR, and aromatase expression, whereas AR, ERα, PRLR, and inhibin were augmented, resulting in gonadotropins decline. Interestingly, autophagy biomarkers, Beclin-1, ATG5, ATG12, LC3-II, and LAMP1 were reduced but SQSTM1/p62 was augmented in the ovaries of HFD-HF-fed rats, causing autolysosome to aggregation. The diet altered T, E2, P4, PRL, and their receptors status in the ovary, disturbed estrous cyclicity, and delayed vaginal opening. Ovarian histomorphology exhibited numerous cystic and atretic follicles, along with disturbed follicular maturation and ovulation. Moreover, the reduction of FSHR; steroidogenic proteins; receptor proteins AR, ERß, PR; and signaling proteins Wnt2 and ß-catenin was also noticed in the ovary, whereas PRLR, inhibin, and pGSK3ß were augmented. In conclusion, exposure to a prepubertal HFD-HF diet leads to hypogonadotropism and the autophagy-mediated defective differentiation of ovarian follicles, abating fertility in adult rats.

A Comprehensive Transcriptomic Analysis of Arsenic-Induced Bladder Carcinogenesis.

Arsenic (sodium arsenite: NaAsO2) is a potent carcinogen and a known risk factor for the onset of bladder carcinogenesis. The molecular mechanisms that govern arsenic-induced bladder carcinogenesis remain unclear. We used a physiological concentration of NaAsO2 (250 nM: 33 µg/L) for the malignant transformation of normal bladder epithelial cells (TRT-HU1), exposed for over 12 months. The increased proliferation and colony-forming abilities of arsenic-exposed cells were seen after arsenic exposure from 4 months onwards. Differential gene expression (DEG) analysis revealed that a total of 1558 and 1943 (padj < 0.05) genes were deregulated in 6-month and 12-month arsenic-exposed TRT-HU1 cells. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that cell proliferation and survival pathways, such as the MAPK, PI3K/AKT, and Hippo signaling pathways, were significantly altered. Pathway analysis revealed that the enrichment of stem cell activators such as ALDH1A1, HNF1b, MAL, NR1H4, and CDH1 (p < 0.001) was significantly induced during the transformation compared to respective vehicle controls. Further, these results were validated by qPCR analysis, which corroborated the transcriptomic analysis. Overall, the results suggested that stem cell activators may play a significant role in facilitating the arsenic-exposed cells to gain a survival advantage, enabling the healthy epithelial cells to reprogram into a cancer stem cell phenotype, leading to malignant transformation.

Prenatal exposure to hexavalent chromium disrupts testicular steroidogenic pathway in peripubertal F1 rats.

We have reported sub-fertility in F1 progeny rats with gestational exposure to hexavalent chromium [Cr(VI)], which had disrupted Sertoli cell (SC) structure and function, and decreased testosterone (T). However, the underlying mechanism for reduced T remains to be understood. We tested the hypothesis "transient prenatal exposure to Cr(VI) affects testicular steroidogenesis by altering hormone receptors and steroidogenic enzyme proteins in Leydig cells (LCs)." Pregnant Wistar rats were given drinking water containing 50, 100, and 200 mg/L potassium dichromate during gestational days 9-14, encompassing fetal differentiation window of the testis from the bipotential gonad. F1 male rats were euthanized on postnatal day 60 (peripubertal rats with adult-type LCs alone). Results showed that prenatal exposure to Cr(VI): (i) increased accumulation of Cr(III) in the testis of F1 rats; (ii) increased serum levels of luteinizing and follicle stimulating hormones (LH and FSH), and 17ß estradiol, and decreased prolactin and T; (iii) decreased steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A1, 3ß- and 17ß-hydroxysteroid dehydrogenases, cytochrome P450 aromatase and 5α reductase proteins, (iv) decreased specific activities of 3ß and 17ß hydroxysteroid dehydrogenases; (v) decreased receptors of LH, androgen and estrogen in LCs; (vi) decreased 5α reductase and receptor proteins of FSH, androgen, and estrogen in SCs. The current study concludes that prenatal exposure to Cr(VI) disrupts testicular steroidogenesis in F1 progeny by repressing hormone receptors and key proteins of the steroidogenic pathway in LCs and SCs.

Prenatal exposure to excess chromium attenuates transcription factors regulating expression of androgen and follicle stimulating hormone receptors in Sertoli cells of prepuberal rats.

We have reported that gestational exposure to hexavalent chromium (CrVI) represses androgen receptor (Ar) and follicle stimulating hormone receptor (Fshr) in Sertoli cells (SCs) of adult rats, while the mechanism underlying remains obscure. We tested the hypothesis "transient gestational exposure to CrVI during the critical embryonic windows of testicular differentiation and growth may have adverse impact on transcription factors controlling the expression of Ar and Fshr in SCs of the F1 progeny". CrVI (K2Cr2O7) was given through drinking water (50 ppm, 100 ppm and 200 ppm), to pregnant rats from gestational day 9-14 (testicular differentiation) and 15 to 21 (prenatal differentiation and proliferation of SC); male progenies were sacrificed on postnatal day 30 (Completion of postnatal SC maturation). A significant increase in free radicals and decrease in enzymatic and non-enzymatic antioxidants were observed in SCs of experimental rats. Real time PCR and western blot data showed decreased expression of Ar, Fshr, Inhibin B, Transferrin, Androgen binding protein, Claudin 11 and Occludin in SCs of experimental rats; concentrations of lactate, pyruvate and retinoic acid also decreased. Serum FSH, luteinizing hormone and estradiol increased, whereas testosterone and prolactin decreased in experimental rats. Western blot detection revealed decreased levels of transcription factors regulating Fshr viz., USF-1, USF-2, SF-1, c-fos, c-jun and GATA 1, and those of Ar viz., Sp-1, ARA54, SRC-1 and CBP in experimental rats, whereas the levels of cyclinD1 and p53, repressors of Ar increased. ChIP assay detected decreased USF-1 and USF-2 binding to Fshr promoter, and binding of Sp-1 to Ar promoter. We conclude that gestational exposure to CrVI affects SC structure and function in F1 progeny by inducing oxidative stress and diminishing the expression of Ar and Fshr through attenuation of their specific transcriptional regulators and their interaction with the respective promoter.

Transient gestational exposure to drinking water containing excess hexavalent chromium modifies insulin signaling in liver and skeletal muscle of rat progeny.

Chromium (Cr), an essential micronutrient potentiates insulin action, whereas excess hexavalent Cr (CrVI) acts as an endocrine disruptor. Pregnant mothers living in areas abutting industries using the metal and chromite ore dumps are exposed to ground water contaminated with Cr. Nevertheless, the impact of prenatal exposure to excess CrVI on insulin signaling in the progeny remains obscure. We tested the hypothesis "transient gestational exposure to drinking water containing excess CrVI may modify insulin signaling during postnatal life". Pregnant Wistar rats were given drinking water containing 50, 100 and 200 ppm CrVI (K2Cr2O7) from gestational day 9-14 encompassing the period of organogenesis; the male progenies were tested at postnatal day 60. Neither fasting blood glucose nor oral glucose tolerance was altered in CrVI treated progeny. Nevertheless, western blot detection pointed out attenuated expression level of insulin receptor (IR), its downstream signaling molecules (IRS-1, pIRS-1Tyr632, Akt and pAktSer473) and organ specific glucose transporters (GLUT2 in liver and GLUT4 in gastrocnemius muscle), along with a significant increase in serum insulin level in male progenies exposed to CrVI. While 14C-2-deoxy glucose uptake increased in the liver, the same decreased in the skeletal muscle whereas, 14C-glucose oxidation recorded a consistent decrease in both tissues of CrVI exposed rats. These findings support our hypothesis and suggest that transient gestational exposure to excess CrVI may affect insulin signaling and glucose oxidation in the progeny, predictably rendering them vulnerable to insulin resistance.