HISTOLOGICAL EFFECTS OF BISPHENOL- A ON REPRODUCTIVE ORGANS OF FEMALE WISTAR

Noopur Dhagga, M.Sc., Priya Gupta, M. Sc and * Srivastava Seema Ph.D, M.Sc. Reproductive Physiology Laboratory, Department of Zoology, University of Rajasthan, Jaipur-302004 (India). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

BisphenolA (BPA) is one of the most widespread endocrine disrupting chemicals used in the manufacturing of plastics. Many evidences report many harmful effects on health. The aim of the present work was to study the effects of bisphenol-A on reproductive organs (ovary and uterus) and hormone level (estrogen and progesterone) and body weight of female wistar albino rats. Adult females were orally administered the dose of BPA (5, 50, 300, 600 and 800 mg/kg bw/week) for 3 months. Animals were sacrificed; body weight measured and the level of estrogen and progesterone with respect to ovary and uterus were examined. At the doses of groups (50, 300, 600 & 800 mg BPA), significant changes were noticed in body weight but at the dose of 5 mg of BPA, no significant decline was observed. Significant changes were observed in hormone level, ovary and uterus of non pregnant females. In cesarean and post term phases, the level of hormone non significantly decreased and the changes in ovary and uterus were not noted. In conclusion, this study emphasized more on histology of reproductive organs and the adverse effects of BPA on females.

…………………………………………………………………………………………………….... Introduction:-
Estrogenic endocrine disruptors (EEDs) are environmental compounds that are able to bind estrogen receptors (ERs) and evoke biological responses. Several authors exert a great effort to demonstrate the estrogenic activity of EED in vitro and in vivo. One of these EED is bisphenol-A (BPA) 1 . BPA is an organic compound with two phenol functional groups. Bisphenol-A is a widely used as industrial plasticizer with weak estrogenicity 2 . BPA can leach from plastic products 3 . The general human population can be exposed BPA mainly via ingestion, inhalation and skin contact [2][3][4][5][7][8] . Heat and either acidic or basic conditions induce hydrolysis of the ester bond linking BPA monomers, leading to release of BPA into food and liquid and making human environmental exposure assured and ubiquitous 4,6 . In human the BPA is absorbed, rapidly metabolized in the liver, mainly BPA glucuronide 9 with a biological half-life of approximately six hours 10 and nearly complete elimination (excreted > 90%) within 24 hr 10,11 .
However, numerous studies demonstrate that BPA at too low concentrations activate nuclear ERs efficiently and also have cellular effects 5,7 . BPA reportedly have the potential to produce widespread adverse effects through their endocrine disrupting activity, such as carcinogenicity, immunotoxicity, neurotoxicity, developmental abnormalities, and so on 5,8 . Many studies in animal models have reported decreased sperm motility, impaired spermatogenesis and decreased fertility in male reproductive system 12,13 . Toxicity of BPA in females acts adversely on reproductive system and subsequently on fertility. Therefore the objective of the current study was to evaluate the body weight Body Weight:-The body weight of all animals measured initially and final body weight obtained at the time of euthanization at the termination of schedule or fatality.
Hormone Analysis:-Following sodiumpentobarbitalanaesthesia blood was collected by cardiac puncture and serum was separated. Following separation of serum, levels of progesterone and estrogen were measured by ELISA kit.

Histopathology:-
Reproductive organs (ovaries, uterus) fixed in 4% paraformaldehyde, dehydrated in ethanol, cleared in Xylene and embedded in paraffin wax. Five micron thick section will be stained with haematoxylin and eosin for light microscopic observation.

Statistical Analysis:-
The mean values was compared using respective standard deviations followed by statistical comparison between control and test groups for evaluation of significant changes in values by Student's t-test and one way analysis of variance (ANOVA) test. P<0.05 was considered as significant.
154 Results:-Body Weight:-A significant decline in body weights was observed, following 50, 300, 600 and 800 mg/kg bw/week of BPA in phase of non pregnant whereas cesarean (5 mg/kg bw/week) and post term (5 mg/kg bw/week)phases had non significant body weight respectively ( Figure 1A-1C) in comparison to control group females. A Non pregnant phase (tested using one way ANNOVA). B Cesarean phase (tested using Student's t test). C Post term phase (tested using Student's t test). *Significant at p<0.05.
Hormone level:-A significant decline in the hormone levels of BPA (50, 300, 600 and 800 mg/kg bw/week) treated female rats was observed. The serum estradiol and progesterone were decreased significantly in non pregnant phase of all groups (Table 1). In cesarean phase, non significantly decrease in estradiol and progesterone level were seen (Table 2) in comparison to control. In case of post term pregnancy phase, estradiol and progesterone were decreased non significantly with the BPA dose (Table 3).

Histopathology of organs:-Ovary:-
The effects of BPA (50, 300, 600 and 800 mg/kg bw/week) impaired the ovarian developing follicles in non pregnant phase. (Figure 2A-E). In cesarean phase, there were naturally atretic phenomenon in primary developing follicles of BPA treated (5 mg/kg bw/week) animals when compared with control group (Figure 3A-B). The developing follicles in post term phase were remain unchanged of BPA treated (5 mg/kg bw/week) animals to the control group ( Figure 4A-B).

Discussion:-
As the present study demonstrates the effects of BPA exposure on female reproductive organs (ovary and uterus) and hormone level (estrogen and progesterone) with the following body weight, several recent studies have also reported such subtle effects in rodents that were exposed to different doses of BPA.
In the present investigation, we noted the significant difference in body weight of BPA-treated female rats during the period of weekly exposure administration by oral gavage in non pregnant phase and non significant difference in cesarean and full term phase. In support to the present findings, Delclos and his colleagues found that BPA doses ≤ 2,700 μg/kg bw/day did not affect gestational body weight gain of rat 16 . Moreover, doses of BPA (8, 40, 160 and 800 mg/kg bw/day) were given orally or subcutaneously to dams, there was no significant difference found in the body weight 17 . Cabaton et al (2011) also reports no statistical difference in the dam weights by treatment groups of BPA exposed perinatally in CD-1 mice 18 . Early prepubertal exposure to BPA (10 and 100 mg/kg) in mice significantly decreased body weight from postnatal day 18 to 30 but 0.1 and 1 mg/kg of BPA treated groups showed no significant differences in body weight 19. In contrast, a study reported that significant findings were increased body weights of the pups born to BPA-treated females relative to those born to control females in low and high doses of BPA when Sprague-Dawley female rats that were exposed, via their drinking water, to approximately 0.1 mg BPA/kg body weight (bw)/day (low dose) or 1.2 mg BPA/kg bw/day (high dose) from day 6 of pregnancy through the period of lactation 20 .
Estrogen (estradiol, specifically) and progesterone-that are vital to normal reproductive development and fertility. Both hormones are responsible for the development of follicles. We found that different doses of BPA treated animals have morphological changes in ovaries having atretic follicles because of level of estrogen and progesterone decreased in non pregnant females but normal follicles were seen with normal production of hormone in cesarean and full term females. BPA affected early oogenesis (ESR2) disruption in mouse during prenatal 21 and decreased ovulation 22 . In one study, low-dose neonatal BPA exposure decreased numbers of all type of follicles and increased atretic follicles in rats during adulthood 23 . In vitro studies on the effects of BPA have locused on mature ovarian follicles. In murine preantral follicles, BPA (0.003 μM) extended development to antral follicles 24 . BPA exposure also increased cell proliferation, follicular growth indication, in small antral follicles in neonatally exposed lambs and Wistar rats 25,26 . In a recent study conducted by Lee et al. on adult female rats exposed orally to 1 μg/kg body weight (BW) or 100 μg/kg BW for 90 days, caspase-3 activation was significantly increased by BPA exposure, causing an augmentation of follicular atresia and luteal regression 27 . Follicle activation has been confirmed by Chao et al. (2012), who found that upon BPA exposure, mouse ovaries had significantly decreased primordial follicles but increased primary, secondary and antral follicles 28 . A study conducted on antral follicles isolated from 32-day old mice proved that BPA significantly decreased progesterone and estrogen production 29 . Another study reported that low-dose neonatal BPA exposure increased atretic follicles in rats during adulthood 23 , 30 . The studies suggest that low-dose BPA exposure may alter follicle formation, but high-dose BPA may directly inhibit growth, cause atresia, and induce changes in rodent antral follicles.
In the uterus, estrogens stimulate epithelium proliferation in vivo and play a critical role in uterine epithelial growth, morphogenesis, and secretory activity 31 . The morphological changes in the luminal epithelial of uterus could be observed at high doses of BPA treated animals when compared with control group. The thickness of the total epithelium was significantly reduced after exposure of different doses of BPA (50, 300, 600 and 800 mg/kg bw/week) when compared with control group whereas at low dose of BPA (5 mg/kg bw/week), the luminal epithelium was pronounced to be non significant changes to the control group. The supportive study reported that treatment with BPA disrupt uterine epithelium. The thickness of the epithelium was significantly reduced after exposure to 50 mg/kg per day BPA when compared with the control group. The 0.1-mg BPA dose did not cause any difference in the thickness of uterine wall to the control group 32 . In other study, Branham and his collogues stated that the other environmental compounds (DES and EE) induced luminal epithelium hypertrophy and inhibited uterine gland genesis 33 . Further, adult hens that were exposed in ovo on day 4 of incubation to BPA (134 ng/kg) had decreased thickness of their tunica mucosa and density of uterine glandular structures compared with unexposed hens 34 . Other in vivo studies have provided evidence that BPA impairs proliferation in the uterus. In rodents, BPA 160 exposure decreased expression of uterine Esr1 (estrogen receptor α), which may lead to inhibit endometrial proliferation in the uterine epithelium and stroma [35][36][37][38] . In neonatal exposure to low-dose BPA decreased uterine epithelium proliferation in response to hormone treatments 38 . In two other studies of adult rats, perinatal exposure of BPA caused increase in thickness of uterine epithelia 39,40 . Together, these studies indicate that gestational BPA exposure may be potentially deleterious to uterine morphology in adult females.

Conclusions:-
BPA affects the reproductive organs (ovary and uterus) and hormone levels of the female rats which increase with the enhancing dose. This study stresses to find the body weight and histology of organs. We found significant difference in body weight and in hormone of non pregnant phase. In continuation with, there is a degeneration of follicles in ovary and reduction in thickness of luminal epithelia of uterus in non pregnant phase. Whereas body weight, hormone level, follicles in ovary and luminal epithelia of uterus in cesarean and post term phase remain unchanged. The present study concludes that the body weight of animals and level of hormones get affected, ovary, and uterus are altered in non pregnant phase whereas in cesarean and post term phase have unaffected organs with unchanged body weight and hormone levels. Thus, exposure of animal and humans to widely prevalent chemical BPA may lead to insidious alteration in reproductive organs.