A Physiologically Based Approach to Study the Toxicological Effects of Bisphenol S, an Endocrine Disruptor, on Reproductive System of Male Rats

Abstract

Background: Bisphenols are widely used in manufacturing polycarbonate plastic, plastic toys and food containers. One of these bisphenols, bisphenol A (BPA) is the major component of baby bottles, dental sealants, epoxy resins and food packaging has been well documented for its hazardous and estrogenic mode of action. In many countries, BPA has been banned in some applications and has been replaced by bisphenol S (BPS). However, nearly similar estrogenic potential of BPS to BPA has been reported in different studies. It has been detected in food, receipt papers and in water. Humans are prone to its exposure through dermal exposure and dietary food intake. An increase in the concentrations of BPS has been detected from 2010 to 2014 in human body fluids, suggesting the possibility of increased exposure to BPS in daily life. BPS has shown estrogenic mode of action, oxidative stress inducing potential, obesogenic effect as well as anti-androgenic effects in vitro. In the in vivo studies, BPS exhibited antiandrogenic effect in male zebra fish, decreased gonadosomatic index in female fish and affected endocrine functions. Similarly, BPS concentrations have been linked to the oxidative stress in the humans, however, in mammalian reproductive system; data regarding BPS toxicity is limited. In present studies, we first assessed in vitro effect of BPS on testicular tissues and sperm. Secondly, we aimed to evaluate whether sub-chronic and chronic exposure of BPS can modulate reproductive functions of male rats in vivo. Lastly, the potential effect of BPS on gonadal development of male rats exposed during pre-natal and neonatal periods were evaluated. Materials and methods: Stock solution of BPS was prepared by dissolving 0.5 g in 5 mL of ethanol and was further diluted with media/water to the final concentration of less than 0.5% ethanol in the media/water. In order to attain the first goal, testicular tissues and sperm from adult rats were incubated with media alone or in combination with different concentrations (0.5, 1, 10 and 100 μg/L) of BPS for two hours in 5% CO2 and 95% air at 37 0C. Testicular testosterone concentration was determined through Enzyme Linked Immuno Sorbant assay (ELISA), while testicular tissue and sperm oxidative stress was determined through estimation of antioxidant enzymes activity, lipid peroxidation (LPO) and generation of reactive oxygen species. To achieve the second goal, adult male rats were treated with different concentrations (1 - 50 μg/kg/day) of BPS through oral gavage for 28 days. Secondly, male rats (22 days old) were provided with water containing different concentrations of BPS (0.5, 5 and 50 μg/L) for a period of 48 weeks in water. Animals were killed by cervical dislocation; blood was collected 27 and was used for determination of different hormones in the plasma while testicular and epididymis tissues were used for histology, oxidative stress determination and evaluation of different parameters of sperm. In the next set of experiments, effect of BPS on the development of male gonads was assessed by exposing the animals to different concentrations of BPS during pre-natal and neonatal period of development. In the pre-natal exposure study, pregnant female rats were provided with water containing 0 and 0.5 - 50 μg/L of BPS. The pups born were assessed for alteration in the early sexual development and reproductive functions during adult hood. In the neonatal exposure study, male pups were subcutaneously injected with different concentrations of BPS (0.5 - 50 mg/kg.day in 50 μL castor oil) from post-natal day 1 to 10 and were followed in the adulthood for alterations in the reproductive functions. Plasma hormones levels, histological changes and alteration in the sperm parameters were determined and compared with the control. Results: In the in vitro exposure study, oxidative stress in the testis and sperm was enhanced along with the increase in antioxidant enzymes activity. At the highest concentration, BPS induced formation of reactive oxygen species, caused lipid peroxidation, affected superoxide dismutase levels, and increased DNA fragmentation in the rat spermatozoa. Similarly, BPS induced reduction in the testosterone secretion by the testis at highest concentrations tested. On the other hand, in the in vivo sub-chronic and chronic exposure study, testicular reactive oxygen species (ROS) and lipid peroxidation (LPO) were significant increase in the higher doses tested while antioxidant enzymes activity were significantly reduced. Sub-chronic exposure for 28 days reduced plasma and intra-testicular testosterone, plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations were suppressed in the groups treated with higher doses of BPS. Testicular morphology revealed thin seminiferous epithelium in the treated groups than control. In the epididymis, area of the tubular epithelium showed significant reduction and empty lumen were observed in the groups treated with higher concentrations of BPS. Chronic exposure of male rats to different concentrations of BPS showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organs weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production (DSP) and number of sperm in epididymis were reduced. Plasma testosterone, LH and FSH concentrations were reduced and estradiol levels were high in 50 μg/L exposed groups. Histological observations included significant reduction in the epithelial height of the testis along with disrupted spermatogenesis. Other prominent observations were empty lumen of the seminiferous tubules and caput region of the epididymis. 28 Pre-natal exposure to different concentrations of BPS induced no significant alteration in early sexual development as well as in adulthood of male rats. Body weight gain, Ano-genital distance (AGD), nipple retention (NR) and organs weight exhibited no change. However, exposure to different concentrations of BPS in the neonatal stage resulted significant decrease in GSI and relative organs weights in BPS 5 and BPS 50 mg/kg.day treated groups as compared to the control. Slight delay in puberty onset in the highest dose treated group was noted but this delay was not different than control. Hormonal analysis showed a significant reduction in testosterone and significant elevated levels of estradiol was observed in the treated groups compared to the control. Histological alterations included reduction in epithelial height and diameter of the seminiferous tubules and spermatids arrest. Reduction in the DSP and sperm number in the caput was observed in the highest dose treated group than control. Conclusion: In conclusion, BPS can not only induce oxidative stress in the sperm and testis in vitro but can also exert its toxicological and endocrine disrupting potential in vivo. Beside its effect in adult animals, exposure to higher concentrations to BPS during neonatal stage of development imparts adverse alterations in the reproductive tissues of male rats. BPS exposure exert its effects by inhibiting cell defense system, inducing ROS and LPO which results in adverse structural and functional changes in the reproductive system. These changes are more prominent and long lasting when the animals are exposed in early stages of life especially during the neonatal stage of development. These affects seems to be accompanied by gonadotropin suppression, estrogenic and antiandrogenic effects of BPS, which ends up with the deleterious alterations in the testis of rats that can lead into infertility.