Influence Of Bisphenol A On Developing Rat Estrogen-receptor-alpha And Some Cytokines: A Two Generational-study

Object:1.To observe the influence of bisphenol A on the concentration of immune globulin G,immune globulin M,after the exposure of bisphenol A.2.To observe the influence of bisphenol A on the expression of estrogen receptor alpha when the rats of F₀ generation were administered bisphenol A.3 To observe the effect of bisphenol A on the expression of IL-2,IL-12,TNF-α, IFN-γin spleens and the expression of Fas,FasL in thymus,when the rats of F₀ generation were administered bisphenol A..This is important to supple for the research of environmental endocrine disruptors on the immune function.Methods:1.Treatment of ratsFisher 344 rats were four weeks old and were divided into control and 4,40 and 400 mg·kg^-1BPA groups.Rats were allowed to acclimatize for 1 week prior to the treatment.Bisphenol A was given orally to the three treatment groups daily at 4,40, 400 mg/kg respectively.Controls were treated with the same volume corn oil.When the rats were nine weeks old,vaginal smears were examined daily after female rats were placed with males.Sperm-positive smear was used to determine gestational day (GD0).After positive detection,pregnant rats were placed individually.Twenty pregnant rats and twenty male rats were divided into four groups in random and the sex ratio of each group was 1:1.Treatments were prolonged for the offspring were thirty days old.Ten offspring were selected randomly from each group.2.General conditionsTo weigh the rats once a week and observe their general conditions such as clothing hair,excrement and urine,action and mental station et al.3.Histological parameters The rats were sacrificed by cervical dislocation after the last treatment.The surplus portions of all organs,were fixed in Bouin's Solution and buffered formalin(4%), dehydrated and processed for paraffin wax embedding.The sections were cut at 5um and stained with hematoxiylin and eosin(H&E).The sections of the paraffin blocks were monitored and compared.4.Determination of antibodyTo observe the concentration of IgG,IgM by enzyme-linked immunosorbent assay(ELISA),after the exposure of bisphenol A.5.Western BlottingMicrosomes from the spleens and thymus of rats treated with bisphenol A were prepared by differential centrifugation as described previously.The relative concentration of estrogen receptor alpha was evaluated by separated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE,10%gel)and Western Blotting.6.Real- time RT-PCRTotal RNA was prepared from spleens and thymus using Total RNA Kit following the instruction.The relative quantity of IL-2,IL-12,IFN-γ,TNF-α,Fas,and Fasl were gained by Rea-time RT-PCR,and GAPDH was the housekeep gene.Result1.The change of weight and organ index in F₀ generationIn F₀ generation,one female rat died during the exposure at 400mg/kg level.Our results showed that rats exposed to 400mg/kg bisphenol A caused significant decrease in body weights(P＜0.05).There were no significant changes in spleen weights and spleen index(P＞0.05).2.The change of weight and organ index in F₁ generationIn F₁ generation,the terminal body weights and the spleen weights were significantly decreased in the high dose group(P＜0.05).There were no weight changes in the low and middle dose groups,but the spleen index increased significantly.In the middle and high dose groups,not any significantly changes were observed in the liver weights,but the liver index increased greatly(P＜0.05).Both thymus and thymus index had not any change contrasted to the control(P＞0.05).3.The change of sex ratio in F₁ generationThere were more female rats than males in F₁ generation.But there were no significant changes in the number of offspring and the sex ratios contrast to the control(P＞0.05).4.The change in Histology(1)The histological change in F₀ generationIn F₀ generation,white pulp of spleens reduced and splenic corpuscle contracted in 400mg/kg group.There was not any histology change in other groups.No abnormalities or treatment related changes were observed in any of the thymus sections of the treatment groups when compared with the control.In F₀ generations, not any histology change was observed in livers at low and middle dose.But in high dose,granular degeneration was visible in hepatocyte.It was observed that depauperation were occurred in acinus renis at 400mg/kg in F₀ generation.(2)The histological change in F₀ generationIn F₁ generations,not any histology change was observed in livers at low and middle dose.But in high dose,granular degeneration was visible in hepatocyte.It was observed that depauperation were occurred in acinus renis at 400mg/kg in F₁ generation.No abnormalities or treatment related changes were observed in any of the thymus and spleens sections of the treatment groups.5.The change of IgG,IgM(1)The change of IgG,IgM in F₀ generationCompare to the control,the concentration of IgG,IgM in high dose group increased significantly in both female and male rats(P＜0.05),but no significant change was observed in low and middle dose groups(P＞0.05).(2)The change of IgG,IgM in F₀ generationCompare to the control,there was no significant change in female rats in all treatment groups(P＞0.05).Whereas,the concentration of IgG,IgM in high dose group of male decreased significantly in(P＜0.05),but no significant change was observed in low and middle dose groups(P＞0.05).6.The change of ER-α(1)The change of ER-αin spleenIn F₀ generation,the expression of ER-αreduced in male rats but increased in female rats in the high dose group(P＜0.05).There were no any changes in the other groups.Similar findings were found in the middle and high dose groups in F₁ generation.(2)The change of ER-αin thymusIn F₀ generation,the expression of ER-αincreased in female rats in the middle and high dose group(P＜0.05).There were no any changes in the low dose groups and male rats.In F₁ generation,the expression of ER-αincreased in female rats in the middle and high dose group(P＜0.05),but reduced in male rats in the middle and high dose group.There were no any changes in the low dose groups in both female and male rats.7.The expression of IL-2,IL-12,IFN-γ,TNF-α,Fas,FasL(1)The change of gene in F₀ generationThe expression of IL-2 IL-12 IFN-γand TNF-αmRNA down-regulated greatly in all the treatment groups compared to the control group(P＜0.05).The expression of Fas decrease in all treatment groups but the expression of FasL up-regulated in middle and high dose groups after it decrease in low dose group(P＜0.05).(2)The change of gene in F₁ generationThe expression of IL-2 IL-12 IFN-γand TNF-αmRNA down-regulated greatly in all the treatment groups compared to the control group(P＜0.05).The expression of Fas decrease in all treatment groups but the expression of FasL up-regulated in middle and high dose groups after it decrease in low dose group(P＜0.05)...