Michael M. Aruldhas

Lactational exposure to hexavalent chromium delays puberty by impairing ovarian development, steroidogenesis and pituitary hormone synthesis in developing Wistar rats.

Hexavalent chromium (Cr-VI) is used in a wide range of industries. Cr-VI from chromate industries and atmospheric emissions contribute to the Cr contamination in the environment. Cr is a reproductive metal toxicant that can traverse the placental barrier and cause a wide range of fetal effects including ovotoxicity. Therefore, the goal of this study was to investigate the basic mechanisms involved in Cr(VI)-induced ovotoxicity, and the protective role of vitamin C on ovarian follicular development and function in Cr(VI)-induced reproductive toxicity using both in vivo and in vitro approaches. Lactating rats received potassium dichromate (200 mg/L) with or without vitamin C (500 mg/L), through drinking water from postpartum days 1-21. During postnatal days (PND) 1-21 the pups received Cr(VI) via the mothers milk. Pups from both control and treatment groups were continued on regular diet and water from PND-21 onwards, and euthanized on PND-21, -45 and -65. Cr(VI) decreased steroidogenesis, GH and PRL, increased FSH and did not alter LH. Cr(VI) delayed puberty, decreased follicle number, and extended estrous cycle. Spontaneously immortalized rat granulosa cells were treated with 12.5 microM (IC(50)) potassium dichromate for 12 and 24 h, with or without vitamin C pre-treatment. Cr(VI) decreased the mRNA expressions of StAR, SF-1, 17beta-HSD-1, 17beta-HSD-2, FSHR, LHR, ER alpha and ER beta. Vitamin C pre-treatment protected ovary and granulosa cells from the deleterious effects of Cr(VI) toxicity, both in vivo and in vitro. Therefore, Cr(VI) toxicity could be a potential risk to the reproductive system in developing females, and vitamin C plays a protective role against Cr(VI)-induced ovotoxicity.

Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo.

Thyroid gland is one of the non-classical target organs for sex steroids. Presence of androgen and estrogen receptors in the neoplastic and non-neoplastic thyroid glands of mammalian species is well documented. The aim of the present study is to elucidate the changes in serum and thyroidal sex steroids, and their receptors in the thyroid gland of rats from immature to adult age under gonadectomized (GDX) and sex steroids replaced conditions. Normal Wistar male and female rats from immature to adult age (day 21, 30, 45, 60 and 160 post-partum (pp)) were used in the present study. One group (I) of rats was GDX at an early age (day 10 pp) and the other group (II) at the adult age (day 120 pp). Group I rats were sacrificed at different experimental periods such as 21, 30, 45 and 60 days pp, and group II rats were sacrificed at day 160 pp. Another group of GDX rats from group I and II were replaced with physiological doses of testosterone or estradiol. Serum and thyroidal concentrations of sex steroids were estimated by RIA method and the concentrations of receptors by radioreceptor assay. Gonadectomy significantly decreased serum and thyroidal testosterone and estradiol and concentrations of androgen receptor (AR) and estrogen receptor (ER) in the thyroid. Replacement of sex steroids to GDX rats restored the normal level of sex steroids, AR and ER. Therefore, it is suggested from the present study that (i). sex steroids up-regulate their own receptors in the thyroid, (ii). sex steroids may influence thyroid growth and the proliferation of thyrocytes by modulating their receptor concentrations in the thyroid.

Developmental profiles of TSH, sex steroids, and their receptors in the thyroid and their relevance to thyroid growth in immature rats

Sex steroids are reported to influence thyroid pathogenesis in human and experimental animals. However, there is no report on this phenomenon during the early developmental period. The mitotic activity of thyrocytes in rats reaches its peak by day 10 postpartum. Thyrocytes actively proliferate in immature rats during the first three postnatal weeks, during which the pre-pubertal rise in serum titers of testosterone and estradiol has been recorded. The aim of the present study was to analyze whether there is a physiological relevance between thyroid growth and sex steroids during the postnatal period. Serum and thyroid tissue hormones (TSH, testosterone, and estradiol) were assayed by liquid phase RIA, and receptors for these hormones were also quantified. The peak rate of thyrocyte proliferation was observed during the second postnatal week in rats. Since the concentrations of sex steroids and their receptors also reached a peak around this period, it is suggested that elevated sex steroids and their receptors in the thyroid might enhance thyrocyte proliferation. A positive correlation between thyroid growth indices and sex steroids and their receptors further strengthens this suggestion. This is a preliminary study, and further experimental study may strengthen this proposal. This is the first report to show the availability of sex steroids and their receptors in the thyroid glands of immature rats under normal conditions.

Testosterone and estradiol differentially regulate TSH-induced thyrocyte proliferation in immature and adult rats.

Though sex steroids are found to influence thyroid pathogenesis in human and in animals, their role in normal thyroid growth and thyrocyte proliferation is not yet understood fully. The present study is addressed to know the effect of testosterone and estradiol on the basal and TSH-induced thyrocyte proliferation in immature and adult rats in vitro. The male and female Wistar rats were gonadectomized (GDX) and one group of GDX rats were supplemented with either testosterone or estradiol. After the experimental period, the rats were sacrificed by decapitation and thyroid glands were removed, washed in Hanks Balanced Salt Solution (HBSS), pH 7.4 and digested with the enzyme mixture containing 0.08% collagenase and 0.12% dispase in HBSS. The isolated follicles were washed thrice with Dulbeccos modified Eagles medium (DMEM) containing 0.5% fetal bovine serum (FBS), and were cultured in Falcons tissue culture flasks containing 5 ml DMEM with FBS (5%) transferrin (5 microg/ml), hydrocortisone (10(-8) M), somatostatin (10 microg/ml), insulin (10 microg/ml) and glycyl-L-histidyl-L-lysine acetate (10 microg/ml). The cells (2.5 x 10(4)) were exposed to various exponential doses of TSH or testosterone (6.25-800 ng/ml) or estradiol (6.25-800 pg/ml). It is suggested from the present study that both TSH and sex steroids enhance thyrocyte proliferation. The mitogenic effect of TSH is greater than that of sex steroids. Sex steroids modulate TSH-induced cell proliferation in a gender-specific manner.

TESTOSTERONE AND ESTRADIOL DIFFERENTIALLY REGULATE THYROID GROWTH IN WISTAR RATS FROM IMMATURE TO ADULT AGE

Experimental and clinical data from cancerous thyroid tissue suggest gender as one of the major factors influencing thyroid growth. However, there is not much information on the effect of sex steroids on the normal development and growth of the thyroid. The aim of the present study was to examine the effect of sex steroids on thyroid growth, serum TSH and TSH receptors in the thyroid, at various stages of sexual maturation. Normal Wistar male and female rats from immature to adult age (day 21, 30, 45, 60 and 160 post partum (PP)) were used in the present study. One group of rats was gonadectomized (GDX) (Group II); another 2 groups of GDX rats (Groups III and IV) were treated with replacement doses (i.e. physiological amounts) of either testosterone or estradiol. Thyroid growth indices (thyroid weight, concentration of DNA, mitotic index and numerical density of thyrocytes), serum TSH and concentration of TSH receptors in the thyroid were assessed. It is concluded from the present study that (i) sex steroids influence thyroid growth by interfering with the secretion of TSH and regulating its receptor number in the thyroid (ii) while testosterone facilitates the mitogenic activity of the thyroid gland of rats irrespective of sex and age, estradiol plays a gender-specific stimulatory role on thyroid growth.

Lactational hexavalent chromium exposure-induced oxidative stress in rat uterus is associated with delayed puberty and impaired gonadotropin levels

Hexavalent chromium (CrVI) is a transition element utilized in many fields of modern industries. CrVI is a reproductive metal toxicant that can traverse the placental barrier and cause a wide range of fetal effects. Therefore, the present study was carried out to determine the CrVI-induced utero-toxicity. In the present study, lactating rats received drinking water containing CrVI (50 mg/L and 200 mg/L) from postnatal days (PND) 1-21. During PND 1-21, the pups received CrVI via the mother’s milk. Pups from both control and treatment groups were continued on regular diet and water from PND-21 onwards and euthanized on PND-45 and -65. Specific activities antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) were estimated. Hydrogen peroxide (H 2O2), lipid peroxidation (LPO) and serum gonadotropins viz. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) were also assayed. Specific activities of SOD, CAT, GPX, GR and GST and serum testosterone and progesterone were significantly decreased, while H2O2 , LPO and serum FSH was increased in 50—parts per million (ppm) and 200 ppm—treated rats in an age-dependent manner. These results suggest that lactational CrVI exposure induces oxidative stress in rat uterus by decreasing antioxidant enzymes, which were associated with delayed puberty and altered steroids and gonadotrophin levels.

RETRACTED: Ameliorative effect of vitamin C on hexavalent chromium-induced delay in sexual maturation and oxidative stress in developing Wistar rat ovary and uterus:

At the request of the Editor and the Publisher, the following article: Samuel JB, Stanley JA, Vengatesh G, Princess RA, Muthusami S, Roopha DP, Suthagar E, Kumar KM, Sebastian MS, Aruldhas MM. (2012) Ameliorative effect of vitamin C on hexavalent chromium-induced delay in sexual maturation and oxidative stress in developing Wistar rat ovary and uterus, Toxicology and Industrial Health 28(8): 720-733. DOI: 10.1177/0748233711422728 http://tih.sagepub.com/content/28/8/720.abstract has been retracted from Toxicology and Industrial Health due to redundant publication by the same group of authors in another journal: Samuel JB, Stanley JA, Roopha DP, Vengatesh G, Anbalagan J, Banu SK, Aruldhas MM. (2011) Lactational hexavalent chromium exposure-induced oxidative stress in rat uterus is associated with delayed puberty and impaired gonadotropin levels, Human & Experimental Toxicology 30(2):91-101. DOI: 10.1177/0960327110364638 http://het.sagepub.com/content/30/2/91.abstract The two papers have used the same data and reporting with the Toxicology and Industrial Health manuscript inserting Vitamin C to differ the reporting. Additionally, the Toxicology and Industrial Health manuscript does not provide citations to original work by another author.

Persistent hexavalent chromium exposure impaired the pubertal development and ovarian histoarchitecture in wistar rat offspring

Hexavalent chromium (CrVI) is a highly toxic metal and a major environmental pollutant. Several studies indicate that CrVI exposure adversely affects reproductive function. We reported that maternal Cr exposure resulted in Cr accumulation in the reproductive organs of female offsprings. CrVI can cross the placental barrier and also can be passed through breastfeeding. The present investigation aimed to determine the persistent (in utero through puberal period) CrVI exposure‐induced toxic effects on the reproductive functions of mother and the offspring. Induction of oxidative stress is one of the plausible mechanisms behind Cr‐induced cellular deteriorations. Mother rats exposed to CrVI showed reduced reproductive outcome, while the offsprings showed higher accumulation of Cr in ovary, altered steroid, and peptide hormones. Specific activities of antioxidant enzymes were decreased and associated with increased levels of H2O2, and lipid peroxidation. CrVI exposure also damaged the ovarian histoarchitecture in various age groups studied. CrVI exposure also delayed the sexual maturation. Results from the present investigation suggest that CrVI exposure from in utero through puberal period significantly damaged the pubertal development through altered antioxidants, anemia, and altered hormone levels. These changes were associated with damaged ovarian histoarchitecture and extended estrous cycle in developing Wistar rats.

Is gender difference in postnatal thyroid growth associated with specific expression patterns of androgen and estrogen receptors

Variations in sex steroids bioavailability were linked to the gender difference in the growth of thyroid glands of neonatal rats. In the present study we tested androgen receptor (AR) and estrogen receptor (ER) concentrations by ligand binding assay, and expression of their genes by RT-PCR and Western blot in the thyroid glands of neonatal rats. AR concentration remained elevated from postnatal day (PND) 10 onwards in males, whereas it decreased by PND 20 in females. AR mRNA and protein expressions were higher in males than females, which increased by PND 10, decreased after PND 15 and reached the nadir by PND 20. ER concentration increased by PND 10 and decreased thereafter in both sex. ERα mRNA expression diminished by PND 15 in both sex; while ERβ mRNA decreased by PND 15 to reach the nadir by PND 20 in males, it was augmented by PND 10 in females to reach the peak by PND 15 and diminished by PND 20. ERα protein expression increased by PND 10 and remained elevated till PND 20 in both sex. ERβ protein expression in males increased by PND 10 and decreased by PND 20, while it remained static up to PND 15 and decreased in females. Testosterone stimulated [(3)H]-thymidine uptake and the expression of IGF-1 and NIS genes in thyrocytes of both sex in vitro, while estradiol stimulated them in females but not in males. We conclude that androgens influence the growth and differentiation of thyrocytes through augmented expression of AR, IGF-1 and NIS in either sex, whereas estrogen imparts the gender difference, which may be at a level beyond the expression of ERs.

Gestational and Neonatal-Onset Hypothyroidism Alters Androgen Receptor Status in Rat Prostate Glands at Adulthood

Infertility associated with congenital and early childhood hypothyroidism is an important reproductive health problem in men. Nevertheless, the exact mechanism underlying hypothyroidism‐induced changes in the prostate gland, an androgen‐dependent organ that contributes a significant portion of the seminal plasma remains obscure. The present study tested the hypothesis “transient gestational‐ or neonatal‐onset hypothyroidism may have duration dependent and lobe specific effect on androgen receptor (AR) status in the prostate glands of adult rats.”