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.

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.

Male reproductive toxicity of CrVI: In-utero exposure to CrVI at the critical window of testis differentiation represses the expression of Sertoli cell tight junction proteins and hormone receptors in adult F1 progeny rats.

The effect of gestational exposure to CrVI (occupational/environmental pollutant and target to Sertoli cells(SC)) was tested in a rat model during the testicular differentiation from the bipotential gonad may interrupt spermatogenesis by disrupting SC tight junctions(TJ) and it's proteins and hormone receptors. Pregnant Wistar rats were exposed to 50/100/200ppm CrVI through drinking water during embryonic days 9-14. On Postnatal day 120, testes were subjected to ion exchange chromatographic analysis and revealed increased level of CrIII in SCs and germ cells, serum and testicular interstitial fluid(TIF). Microscopic analyses showed seminiferous tubules atrophy and disruption of SC TJ, which also recorded decreased testosterone in TIF. mRNA and Protein expression analyses attested decreased level of Fshr, Ar, occludin and claudin-11 in SCs. Immunofluorescent detection revealed weak signal of TJ proteins. Taken together, we concluded that gestational exposure to CrVI interferes with the expression of SC TJ proteins due to attenuated expression of hormone receptors.

In vivo spermatotoxic effect of chromium as reflected in the epididymal epithelial principal cells, basal cells, and intraepithelial macrophages of a nonhuman primate (Macaca radiata Geoffroy).

OBJECTIVE: To understand, through a simulation experiment in a nonhuman primate model, the potential in vivo spermatotoxic toxic effect of hexavalent chromium (CrVI) in men who are occupationally or environmentally exposed to it. DESIGN: Controlled laboratory study. SETTING: Research laboratory in a department of endocrinology in a university in India. ANIMAL(S): Male bonnet monkey, Macaca radiata Geoffroy. INTERVENTION(S): Monkeys were exposed ad libitum to 100, 200, and 400 ppm CrVI, dissolved in drinking water, for a chronic period of 180 days. MAIN OUTCOME MEASURE(S): Examination of epididymis with a transmission electron microscope and assessment of the effect of CrVI in terms of accumulation of sperm-derived lipofuscin (LF) material in the principal cells, basal cells, and intraepithelial macrophages of the epithelium. RESULT(S): The abundance of basal cells and intraepithelial macrophages and the content of LF material in these cell types increased. The principal cells phagocytosed from the lumen the dead sperm resulting from CrVI exposure and processed them partially into LF material, which was acquired by the basal cells and intraepithelial macrophages and processed further. The LF material-laden basal cells and intraepithelial macrophages appeared to leave the epithelium, accompanied by recruitment of fresh basal cells and intraepithelial macrophages. CONCLUSION(S): Occupational or environmental exposure to CrVI, as would occur in the tannery, soap, and other industries in developing and underdeveloped countries, can be toxic in vivo to spermatozoa.

Reproductive toxicity of chromium in adult bonnet monkeys (Macaca radiata Geoffrey). Reversible oxidative stress in the semen.

The present study was designed to test the hypothesis that oxidative stress mediates chromium-induced reproductive toxicity. Monthly semen samples were collected from adult monkeys (Macaca radiata), which were exposed to varying doses (50, 100, 200 and 400 ppm) of chromium (as potassium dichromate) for 6 months through drinking water. Chromium treatment decreased sperm count, sperm forward motility and the specific activities of antioxidant enzymes, superoxide dismutase and catalase, and the concentration of reduced glutathione in both seminal plasma and sperm in a dose- and duration-dependent manner. On the other hand, the quantum of hydrogen peroxide in the seminal plasma/sperm from monkeys exposed to chromium increased with increasing dose and duration of chromium exposure. All these changes were reversed after 6 months of chromium-free exposure period. Simultaneous supplementation of vitamin C (0.5 g/L; 1.0 g/L; 2.0 g/L) prevented the development of chromium-induced oxidative stress. Data support the hypothesis and show that chronic chromium exposure induces a reversible oxidative stress in the seminal plasma and sperm by creating an imbalance between reactive oxygen species and antioxidant system, leading to sperm death and reduced motility of live sperm.