Geeta Vanage

Hypermethylation of estrogen receptor promoter region in adult testis of rats exposed neonatally to bisphenol A

BACKGROUND Bisphenol A (BPA) is an estrogenic endocrine disruptor commonly used in manufacture of polycarbonate plastics and epoxy resins. Due to its ubiquitous presence in the environment, health concerns are increasing. Earlier studies from our group have shown that neonatal exposure of male rats to BPA affected spermatogenesis leading to impairment in fertility during adulthood. Further we also observed an altered gene expression of ERα and ERβ in adult testis upon BPA exposure. Based on these results, we hypothesized that apart from endocrine action, BPA might mediate perturbations in expression of ERs via epigenetic mechanism. OBJECTIVES The present study was undertaken to determine the effect of exposure of neonatal male rats to BPA on DNA methylation profile of estrogen receptor promoter region and on DNA methylation machinery. METHODS In order to test this hypothesis, neonatal male rats were subcutaneously injected with 2.4μg of BPA/day for the first five days of life, i.e., on postnatal days (PND) 1-5, while control group received vehicle (sesame oil). Animals were sacrificed during adulthood (PND-125) and testes were dissected out for analysis. Methylation pattern of promoter region of ERα and ERβ was analyzed in the testis by bisulfite sequencing and expression levels of DNA methyltransferases by quantitative RT-PCR and Western blotting respectively. RESULTS Bisulfite sequencing revealed significant hypermethylation of ERα promoter to varying extents from 40% to 60%, and ERβ promoter region with varying extent from 20% to 65%. Approximately 2-fold increase in Dnmt3a and Dnmt3b expression at transcript and protein level was also observed. CONCLUSION The experimental evidence demonstrated that the neonatal exposure of rats to BPA led to aberrant DNA methylation in testis, indicating methylation mediated epigenetic changes as one of the possible mechanisms of BPA induced adverse effects on spermatogenesis and fertility.

Perinatal exposure of rats to Bisphenol A affects the fertility of male offspring

AIMS The exposure to endocrine disruptor (ED) induces functional and behavioral abnormalities associated with reproduction. Humans are ubiquitously exposed to Bisphenol A (BPA), an ED, as it leaches from polycarbonate plastics into their contents. The aim of the present study was to determine the effect of perinatal exposure of male rats to BPA on fertility parameters and perturbations in the expression of testicular steroid receptors (SRs) in adult F(1) offspring. These effects were studied in adult males of the F(2) and F(3) generations to determine the vertical transmission of BPA exposure. MAIN METHODS Pregnant female rats (F(0)) were gavaged with either BPA (1.2 and 2.4 microg/kg bw), a vehicle control or positive control with Diethylstilbestrol (10 microg/kg bw) during the perinatal period. Adult F(1) males were subjected to fertility assessment by mating with unexposed females. The reproductive functions of the subsequent F(2) and F(3) litters were investigated in a similar manner. Immunohistochemical localization of SRs was carried out in the testes of F(1), F(2) and F(3) generation adult rats. KEY FINDINGS A significant increase in post implantation loss and a decrease in litter size and sperm count and motility were observed in the F(1) male offspring. A reduction in the testicular expression profile of SRs was observed. These effects were very prominent in the subsequent F(2) and F(3) generations. SIGNIFICANCE Perinatal exposure to environmentally relevant doses of BPA affects the male germ line, leading to impairments in the fertility of F(1) male offspring and their subsequent F(2) and F(3) generations.

Neonatal exposure of male rats to Bisphenol A impairs fertility and expression of sertoli cell junctional proteins in the testis

BACKGROUND Sertoli cell junctional proteins (SCJP) (viz. adhesion, gap and tight junctions) are important for spermatogenesis and perturbations in expression of these proteins are associated with impairments in process of sperm production. Bisphenol A (BPA) is an endocrine disrupter that has been associated with impaired spermatogenesis. However the mechanistic basis of impaired spermatogenesis is unknown, whether BPA is a Sertoli cell toxicant has not yet been fully investigated. OBJECTIVES The present study was undertaken to decipher the effects of neonatal exposure of male rats to BPA on fertility and its effect on the testicular expression of SCJP during development. METHODS Neonatal male rats were s.c. injected with BPA at doses ranging from 0.6 to 10 microg/rat (100-1600 microg/kg bw of BPA) on post-natal days (PNDs) 1-5, and controls received vehicle. Diethylstilbestrol (DES) was used as a positive control. Male fertility was assessed during adulthood and the lowest dose of BPA that was most effective at impairing fertility was determined. Immunohistochemical localization for Connexin 43 (Cx-43, gap junctional), Zona Occludin-1 (ZO-1, tight junctions) and N-cadherin (adherens junction) was carried out on testicular tissue sections obtained from PNDs 15, 30, 45 and 90 of rats exposed to lowest dose of BPA that impaired fertility. RESULTS Females mated with male rats that were exposed neonatally to various concentrations of BPA showed a significant increase in post-implantation loss and a decrease in litter size. There were significant changes in sperm count along with hormonal imbalances in the rats exposed neonatally to BPA. The 2.4 microg dose (400 microg/kg bw) of BPA was determined as the lowest dose that was capable of impairing male fertility. A significant reduction in the expression of Cx-43 (PND 45 and 90) and increases in the expression of N-cadherin (PND 45 and 90) and ZO-1 (PND 90) were observed in the testes of rats exposed neonatally to effective dose of BPA. Interestingly, there was an altered expression pattern of Cx43 amongst the sloughed cells in the testes of the experimental rats as compared to controls. CONCLUSION Neonatal exposure of BPA to rats impairs their fertility and has the potential to induce perturbations in SCJP. These perturbations may be one of the contributing factors that lead to impairments in spermatogenesis in the exposed animals and can be used as potential biomarkers to study BPA-induced effects on testes.

Impairment in protein expression profile of testicular steroid receptor coregulators in male rat offspring perinatally exposed to Bisphenol A.

AIMS Steroid hormones and steroid receptors (SRs) play a crucial role in spermatogenesis. Steroid receptor coregulators are the major determinants of SR functioning, and any alteration in their expression is known to be associated with impaired spermatogenesis. Since Bisphenol A (BPA) exposure leads to an impairment of spermatogenesis, we hypothesized that this effect could be associated with the altered expression of steroid receptors and their coregulators in the testes. The present study describes the effect of perinatal exposure of rats to BPA on the expression profile of testicular steroid receptor coregulators in the F(1) generation. These effects were further studied in the F(2) and F(3) generations to determine vertical transmission. MAIN METHODS Pregnant female rats (F(0)) were gavaged daily with BPA (1.2 and 2.4 microg/kg bw) (or vehicles for controls) from gestation day 12 through postnatal day (PND) 21 to obtain the F(1) and subsequent F(2) and F(3) generations. Immunohistochemical localization of steroid receptor coactivator-1 (SRC-1), G-receptor integrating protein-1 (GRIP-1), p300/CBP/cointegrator-associated protein (p/CIP) and nuclear corepressor (NCoR) was carried out in the testes of F(1), F(2) and F(3) generation adult rats. KEY FINDINGS A significant reduction in the expression of SRC-1 and NCoR, with a parallel increase in the expression of p/CIP and GRIP-1, was observed in the testes of rats exposed perinatally to BPA. Surprisingly, a similar pattern was observed in the testes of F(2) and F(3) rats. SIGNIFICANCE Perinatal exposure of male rats to BPA leads to transgenerational perturbations in the expression profile of testicular steroid receptor coregulators.

Clastogenic and mutagenic effects of bisphenol A: An endocrine disruptor

Bisphenol A (BPA) is a well-known endocrine disruptor (ED) which represents a major toxicological and public health concern due to its widespread exposure to humans. BPA has been reported to induce DNA adduct and aneuploidy in rodents. Recent studies in humans depicted its association with recurrent miscarriages and male infertility due to sperm DNA damage indicating that BPA might have genotoxic activity. Hence, the present study was designed to determine genotoxic and mutagenic effects of BPA using in-vivo and in-vitro assays. The adult male and female rats were orally administered with various doses of BPA (2.4 μg, 10 μg, 5mg and 50mg/kgbw) once a day for six consecutive days. Animals were sacrificed, bone marrow and blood samples were collected and subjected to series of genotoxicity assay such as micronucleus, chromosome aberration and single cell gel electrophoresis (SCGE) assay respectively. Mutagenicity was determined using tester strains of Salmonella typhimurium (TA 98, TA 100 and TA 102) in the presence and absence of metabolically active microsomal fractions (S9). Further, we estimated the levels of 8-hydroxydeoxyguanosine, lipid per-oxidation and glutathione activity to decipher the potential genotoxic mechanism of BPA. We observed that BPA exposure caused a significant increase in the frequency of micronucleus (MN) in polychromatic erythrocytes (PCEs), structural chromosome aberrations in bone marrow cells and DNA damage in blood lymphocytes. These effects were observed at various doses tested except 2.4 μg compared to vehicle control. We did not observe the mutagenic response in any of the tester strains tested at different concentrations of BPA. We found an increase in the level of 8-hydroxydeoxyguanosine in the plasma and increase in lipid per-oxidation and decrease in glutathione activity in liver of rats respectively which were exposed to BPA. In conclusion, the data obtained clearly documents that BPA is not mutagenic but exhibit genotoxic activity and oxidative stress could be one of the mechanisms leading to genetic toxicity.

Perinatal exposure of rats to Bisphenol A affects fertility of male offspring--an overview.

Endocrine disruptors (ED) induce both functional and behavioral reproductive abnormalities. Bisphenol A (BPA) is a known ED that leaches from polycarbonate plastics, as such human exposure is common. Maternal BPA exposure has been shown to have negative effects on the fertility of male offspring. Pregnant rats exposed perinatally to environmentally relevant doses of BPA gave birth to offspring with significantly impaired spermatogenesis and fertility. Perinatal exposure had deleterious effects on the male germ line which manifested as impairments in the fertility of F(1) male offspring and subsequent F(2) and F(3) generations. This overview is an attempt to summarize the currently available data in the literature with regards to perinatal BPA exposure and male fertility.

Curcumin-Loaded Hydrogel Nanoparticles: Application in Anti-Malarial Therapy and Toxicological Evaluation

The present investigation involved preparation of hydrogel nanoparticles using a combination of hydroxyl propyl methyl cellulose and polyvinyl pyrrolidone. The objective was to exploit the size and hydrophilic nature of the formulated nanocarriers to enhance absorption and prolong the rapid clearance of curcumin due to possible evasion of the reticulo-endothelial system. Reproducible nanoparticles of size around 100 nm, a fairly narrow distribution and encapsulation efficiency of 72%, were produced by the solvent emulsion-evaporation technique. This optimized system was further subjected to freeze-drying. The freeze-dried product was readily reconstituted with distilled water. The reconstituted product exhibited a size and distribution similar to that before freeze-drying, drug content of greater than 99% and presence of amorphous drug when analyzed by differential scanning calorimetry (DSC) which may result in possible improved absorption of curcumin. In vivo anti-malarial studies revealed significant superior action of nanoparticles over curcumin control suggesting the possibility of the formulation being employed as an adjunct anti-malarial therapy along with the standard therapy. Acute and subacute toxicity studies confirmed the oral safety of the formulation. A battery of genotoxicity studies was conducted to evaluate the nongenotoxic potential of the developed formulation thus indicating the possibility of the formulation being employed for prolonged duration.

Toxicological evaluation of pH-sensitive nanoparticles of curcumin: Acute, sub-acute and genotoxicity studies

Research in nanotoxicology is still in nascent stages. This hampers the design of appropriate regulatory policies for these beneficial nano-drug delivery systems thus affecting their routine employment as therapeutics. Establishing the entire toxicological profile is thus indispensable for proving the human safety of nanocarriers, which was the primary objective of the current investigation. The developed curcumin loaded polymeric nanoparticles of Eudragit S100 were subjected to various toxicological evaluations which included acute-toxicity study, sub-acute-toxicity study (28 days) and various genotoxicity studies like in vivo Micronucleus assay, in vivo Chromosomal Aberration assay and in vivo Comet assay. The formulation was found to be non-toxic at the dose equivalent to 2000 mg/kg of body weight of curcumin in the acute-toxicity study. Sub-acute-toxicity study proved the safety of the formulation for prolonged administration at the commonly used therapeutic dose of 100mg/kg of body weight of curcumin and at twice the therapeutic dose. Genotoxicity studies proved the cellular safety of the developed formulation at the therapeutic dose, and even at doses equivalent to thrice the therapeutic dose. Thus the developed curcumin loaded polymeric nanoparticles of Eudragit S100 were found to be safe for oral administration for a short as well as a prolonged duration.

Mutagenic effect of Bisphenol A on adult rat male germ cells and their fertility.

The present study investigates the effects of Bisphenol A on the induction of dominant lethal mutation and male reproductive functions. The male rats were gavaged with BPA (10 μg, and 5.0 mg/kg/bw) over a period of six days and control group with vehicle. Each male was cohabited with untreated females sequentially over the period of eight weeks. The mated females were sacrificed on 15th day of gestation. The results revealed a significant increase in dominant lethal mutation rate during fourth and sixth week of mating intervals at 5.0mg/kgbw dose of BPA. These findings demonstrate that mid-spermatids and spermatocytes are more sensitive to BPA exposure. The male rats sacrificed at the end of mating study showed an increase in the sperm DNA damage, and decrease in motility at higher dose. However, significant reductions in sperm production effects were observed at both lower and higher doses of BPA. These preliminary results indicate that BPA may be a weak male germ cell mutagen.

Histopathological and ultra structural effects of nanoparticles on rat testis following 90 days (Chronic study) of repeated oral administration

BackgroundNanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and biomedical. However, little is known about the toxicity in reproductive organs of animal model following exposure to nanoparticles.ObjectiveThis study therefore, tried to examine the effects of nanoparticles with a diameter range of 5-20 nm on the histology of the testis of wistar rats and correlate it with Transmission Electron Microscopy results.Materials and methodsSixteen wistar rats were randomly divided into two groups of 8 rats each. Each group received the following via gavage technique for 90 days: Control Group (Group-1)-tap water; Experimental group (Group 2) - nanoparticles (20ug/kg/day). After ninety days (chronic study), rats were sacrificed and testis tissues was processed for histology and transmission electron microscopic study.ResultsThere was significant difference between the observations of group-1 and group 2. The changes observed in the testis were disarray of the spermatogenic cells and disorientation of the testis. These changes were observed to have been disappearing from normal histological features. Detailed structural damages were observed with TEM analysis, such as depletion of germ cells, germinal cells necrosis, especially in spermatogonia and Leydig cells had an abnormal fibroblast-like appearance, abnormal space between neighboring sertoli cells, mitochondria, lost cristae and vacuolated (none energized) with those animals exposed to nanoparticles.ConclusionIt seems that nanoparticles have acute and significant effects on spermatogenesis and number of spermatogenic cells. More experimental investigations are necessary to elucidate better conclusion regarding the safety of nanoparticles on male reproduction system.