Effect Of Bisphenol A On Occurrence And Development Prolactinoma And Its Underlying Mechanisms

BACKGROUNDPituitary tumor is the most common intracranial tumors, accounting for 15% of the total intracranial tumors. The clinical detection rate of pituitary adenomas in the general population is 10-25%. Recent studies showed that the prevalence of prolactinoma was 3-5 times higher than the previously reported population studies in Europe and South America.Prolactinoma represents approximately 66.2% of all pituitary tumours. Endocrine disorders incuding amenorrhea, infertility, abnormal lactation caused by hyperprolactinemia and the central nerve systerm symptom oppressed by tumor has been a serious threat to patients’ physical and mental health. Although bromocriptine, pergolide, cabergoline and other new dopamine receptor agonists can nomalize serum prolactin level in more than 90% of prolactinoma patients, the tumor recurrence rate after drug withdrawal is very high and tumor’s insensitive to dopamine agonists is still very significant.The cause of prolactinoma remains unkown.Recent studies showed that estrogen (estrogen, E2) was involved in the occurrence and development of pituitary tumors. In F344 rat prolactinoma models, estrogen receptor antagonist fulvestrant could inhibit the growth of prolactinoma significantly. Bisphenol A (BPA)is one of the world’s highest production of chemical, widely used in the manufacture of polycarbonate and epoxy.It was demonstrated that BPA had estrogen-like activities in animal models. BPA will accelerate the estrus,cause prostate growth and change the adolescent breast development, permanent change the morphology and function of the female reproductive tract and ovary in the growth process of the mice exposed to different doses of BPA during fetal development. Recent studies showed that BPA could significantly increase the incidence of breast cancer, ovarian cancer, prostate cancer and testicular cancer.The underlying mechanisms is still unknown.It was suggested that BPA could induce the breast cancer and endometrial cancer by acting on the breast and endometrial cells through the ERβ, ERβ or other nongenetic pathways. But up to now, the specific mechanism is unclear. A previous study found that BPA could increase the intracellular Ca2+ levels and lead to hyperprolactinemia. It is unclear that long-term exposure to bisphenol A could lead to the occurrence of prolactinoma and its underlying mechanisms. No clinic studies have been reported to demonstrate the association between the occurrence of prolactinoma and long term exposure to BPA.What are the reasons for the increase of the prolactinoma incidence? Was it related to the increasing environmental pollution, especially widely-used environmental endocrine disruptors BPA? This is the problem we are interested in.It will provide certain theoretical basis for the prevention of prolactinoma, the use of BPA products and the formulation of regulations in our country to reveal the relationship between bisphenol A and prolactinoma, and to explore its potential mechanisms. ObjectiveTo investigate the effects of Bisphenol A(BPA) on prolactin(PRL) release, pituitary cell proliferation, prolactinoma formation in estrogen-sensitive Fischer 344 (F344) rats. To compare with serum and urine BPA concentration of prolactinoma patients and healthy individuals. To investigate the correlation between BPA and prolactinoma and to explore its potential mechanisms. Experiment materials and methods 1. Animal experiment partAccording to a completely random method,60 female F344 rats (4 weeks old) were divided into six groups, each group possess 10 rats. For comparsion, control group also have been gavaged by normal saline. The groups listed as follows:control group, corn oil group, BPA experimental groups and positive control group. The experimental groups were daily fed with BPA 50 mg/kg/d group (named as BPA50 group), BPA 200 mg/kg/d group (named as BPA200 group) and BPA 400 mg/kg/d group (named as BPA400 group). Rats with E2 intraperitoneal injection were set as positive group(2 mg/kg/5d, named as E2 group). In each group after one week adaptability feeding, rats were given different drugs such as BPA (sigma aldrich), daily lavage by weight, and the rats of control group were given for the same volume of saline.After treated six weeks, rats were hocused by fluoride oxide inhalation anesthesia and the blood samples were gotten by angular vein plexus for PRL test. After 12 weeks and before death, in each groups two rats were randomly selected for head MR dynamic enhanced scan to observe the pituitary. The blood samples were collected and the serum were used for the PRL,GH,ACTH,TSH test through the enzyme linked immunosorbent (ELISA) method. The body weight changes of each rat were obtained by measure the weight everyday. After recording the pituitary weight, the pituitary gland was divided into two parts. One part used for paraformaldehyde fixed, subsequently for hematoxylin and eosin (HE)staining and immumohistochemical staining such as prolactin (PRL), proliferating cell nuclear antigen (PCNA), estrogen receptor alpha (ERa)expression. The other part for liquid nitrogen preservation, and western blot method was used to detect the ERa, estrogen receptor beta (ERβ), PCNA, pituitary tumor-transforming gene (PTTG) protein expressed in the pituitary gland tissue. Reverse transcription-ploymerse chain reaction (RT-PCR) method was used to measure the gene expression level of ERa, ERβ, PCNA,PTTG.1. Clinical trials partA total of 120 subjects were enrolled in our study. Participants were divided into prolactinoma group and healthy controls. Tissue samples were collected from March, 2015 to December,2015.The serum and midstream urine were collected for further examination. Fluorescence-high performance liquid chromatography was used to detect plasma and urine BPA concentration.2. Statistical analysisSPSS 10.0 statistical software was used for statistical analysis. Quantitative data were presented as mean ± SD. The Student t-test, one-way analysis of variance, Dunnett’s test, and Fisher’s exact test were conducted for analyzing data. P< 0.05 was considered to be statistically significant.Results1. general conditions of ratsFor the control group and corn oil group, the development were normal with smooth and uniform body hair. The mental and responsive action were good. BPA treated group rats had the phenomenon of death, with different degree of hair removal, mental state was poor. Compared with BPA50 or BPA200 group, BPA400 group displayed significantly hair removal. The hair removal of E2 group was the most obvious, but E2 group showed good mental state.2. Body weights of rats (g)After treated 12 weeks, the average body weights of BPA group rats were less than the control group. Especially the weights of BPA200, BPA400 group rats were significantly reduced compared with the control group (P< 0.05).3. rat pituitary nuclear magnetic resonanceBPA 50, BPA400 group and E2 group pituitary were significantly large compared with the control group. 4. rat pituitary weights (mg)BPA 50, BPA400 group and E2 group pituitary weights were significantly higher than the control group (P<0.05), the pituitary weights between BPA200 group, control group, corn oil group were not statistically significant (P>0.05).5. rat serum PRL、GH、ACTH、TSH levelsThe PRL levels of BPA50, BAP200, BAP400 and E2 group were higher than the control group (P< 0.05). Among these groups, the PRL level of E2 group was the highest one. After 12 weeks of drug intervention, the levels of PRL in each group were higher than that in the normal control group and corn oil group, the difference was statistically significant (P<0.05). After drug intervention for 12 weeks, the serum GH level of BPA50 group was significantly higher than control group, corn oil group (P< 0.05), while the serum GH level of BPA400 group was significantly lower than control group, corn oil group (P< 0.05).The serum GH、ACTH levels among all groups were not statistically significant (P>0.05).6. The HE staining of rat pituitaryWith indicated treatments for 12 weeks, no obvious abnormal pituitary tissues were found in the control group or corn oil group. However, in BPA50 group, there were four rats(4/10) demonstrated tumor changes in pituitary, there were 3 rats with pituitary tumor were identified in BPA400 group and one rat was identified in BPA200 group, respectively.7. Immunohistochemical results of PRL, PCNA and ERa in the pituitary of rats7.1 The pituitary PRL immunohistochemistry scores of BPA50,200,400 and E2 group were significantly higher than that of control group and corn oil group, the difference was statistically significant (P<0.05).7.2 The PCNA immunohistochemical scores of BPA50, BPA200, BAP400 and E2 group were obviously higher than those of control group and corn oil group, the difference was statistically significant (P< 0.05).7.3 The ERa express in BPA50 group was obviously increased, higher than the control group and the corntrol oil group, the difference was statistically significant (P < 0.05). However, no significant difference was found in BPA200,400 and E2 group compared with control group and the corn oil group.8. The western blot results of rat pituitary PCNA, PTTG, ERa, ERβ8.1 The protein expression levels of PCNA in BPA50,400 and E2 group were obviously higher than that in the control group and the corn oil group, the difference was statistically significant (P< 0.05). However, no significant difference was found in BPA200 group compared with control group and the corn oil group.8.2 The protein expression levels of PTTG in BPA50,400 and E2 group were obviously higher than that in control group and the corn oil group, the difference was statistically significant difference (P< 0.05). Meanwhile, the PTTG protein expression in BPA200 group was no statistical significance difference (P> 0.05).8.3 The protein expression levels of ERa in BPA50 and E2 group were significantly increased, higher than the control group and the corn oil group, the difference was statistically significant (P< 0.05). The ERa expression in BPA200 and 400 groups were no significant difference (P> 0.05).8.4 The protein expression levels of ERβ in BPA50 group and BPA200 group were obviously increased, higher than the control group and the corn oil group, the difference was statistically significant (P< 0.05). Compared with control group and the corn oil group, there was no significant difference in the BPA400 and E2 groups (P> 0.05).9. The PCR results of rat pituitary PCNA, PTTG, ERa, ERβ9.1 The mRNA expression levels of PCNA in BPA50,200,400 and E2 group were significantly higher than the control group and corn oil group, the difference was statistically significant (P<0.05).9.2 The mRNA expression levels of PTTG in BPA50,200,400 and E2 group were significantly higher than the control group and corn oil group, the difference was statistically significant (P<0.05).9.3 The mRNA expression levels of ERa in BPA50,400 and E2 group were obviously higher than the control group and corn oil group, the difference was statistically significant (P< 0.05). But ERa expression in BPA200 was no significant difference (P> 0.05).9.4 The mRNA expression levels of ERβ in BPA50,200 and E2 group were obviously higher than the control group and corn oil group, the difference was statistically significant (P< 0.05). But ERβ expression in BPA400 was no significant difference (P> 0.05).10. The BPA concentrations in serum and urine of the prolactinoma patients and healthy controlsThe serum BPA concentration (ng/mL) of the prolactinoma patients was significantly higher than normal control group (P< 0.05). But in the urine the concentration of BPA (ng/mL) did not see obvious difference.Conclusion1. Certain concentration of BPA could significantly promote the rat pituitary cell proliferation, prolactin secretion, and the formation of prolactinoma.2. Certain concentration of BPA may have impact on the proliferation and secretion of pituitary cell function through the ER α pathway.3. The BPA concentration in the serum of prolactinoma patients was significantly higher than normal control group. BPA may be related to the occurrence of prolactinoma.