| Objective:In recent years,ovarian cancer has become one of the major causes of womenrelated cancer deaths,and its incidence is on the rise.There are many risk factors for ovarian cancer,for example,family and genetic history,fertility,age,and environmental influence.Recently,with the development of science and technology and the extensive application of chemical products,endocrine disruptors have attracted people’s attention,which can affect the homeostasis of the body’s inside environment and can produce a certain damaging effect on the human body.Nowadays,PAEs and bisphenols are major types of endocrine disruptors that have drawed much more attention.As the main raw materials of plasticizers and plastic products,they are diffusely used in production and our life,and can be disclosed in the environment or human body,and it has become a more dangerous and harmful chemicals.Due to the fact that environmental pollutants exist in multiple mixed states in real life,and the human body is exposed to complex and diverse ways,and the single action mode of each substance may present different toxic effects in contact with this mixed,so the study on the toxic effect of a single pollutant has been unable to meet the practical needs.DEHP and BPA respectively as representative material of PAEs and BPs,is common in the environment,and the interaction is more complex,because of the limitations on population study,cause investigation conclusion differences,and experimental evidence is not sufficient,it is the lack of accurate and reliable data for safety evaluation.The ovary is one of the target organs of the harmful effects of DEHP and BPA.Whether single or combined exposure of DEHP and BPA can influence the occurrence of ovarian tumors and the mechanism of action are still unclear.As a result,this article want to evaluate the affect of phthalate esters and bisphenol compounds on ovarian tumorigenesis and their related mechanisms caused by singly or combined exposure,and to provide theoretical basis and scientific clues for the safety evaluation of EDCs.Methods:1.160 healthy female SD rats at the age of 4 weeks were used to construct the rat ovarian tumor model,which was randomly divided into the carcinogen DMD(diethylnitrosamine DEN,n-methyl nitrosamine MNU and diisopropanol nitrosamine DHPN)administration group(DA group)and sham administration group(DN group).The DA group was divided into four groups: control group,DEHP 150 mg/kg group,BPA 20mg/kg group and DEHP 150 mg/kg +BPA 20 mg/kg group,with 20 rats in each group,and the DN group was also divided into the above four groups.Namely DA group animals according to 100 mg/kg intraperitoneal injection of DEN on the first day,and on the 5,8,11,14 days according to 20 mg intraperitoneal injection of MNU,the first week and the third week according to the 0.1% DHPN by water,animals in DN group given saline injections and daily drinking water.After the forth week adjustment rest,all animals were given DEHP and BPA alone and combination for 30 weeks,and the growth status,body weight of rats in each group were observed and recorded during the experiments.At the end of experiments,all tissues of the rats were retained,the changes of organ coefficients of the rats in each group were analyzed;2.After HE staining,the ovary tissues were scanned under the microscope,and the films were read by the pathologists;3.IHC method was used to determine the expression of tumor marker proliferating nuclear antigen(PCNA)in ovarian tissue of rats in each group,and immunohistochemical results were analyzed by positive cell count;4.RT-PCR method to detect the expression levels of ERα and PTEN m RNA in rat ovary tissue;5.RT-PCR method to detect candidate micro RNAs of rat ovary tissue(mi R-let7 b,mi R-21,mi R-200 c,mi R-200a-3p,mi R-205,mi R-212-3p,mi R-141-3p,mi R-222,mi R-203-3p)expression levels;6.Western blot method was used to detect the protein expression levels of ERα,PTEN,AKT and P-AKT in rat ovary tissue;7.KaplanMeier plotter website conducts survival analysis of some differentially expressed micro RNAs and confirms that the relevant micro RNAs will be find out.Results: 1.During the 30-week feeding process,the survival and death of rats in each group were recorded at any time.the survival curve of rats in the DA group(pretreated with carcinogen DMD)showed a decreasing trend,and the survival curve of rats was significantly different after BPA and DEHP were exposed individually and in combination(P <0.05);2.During the long-term exposure of DEHP and BPA alone and in combination,the growth of the rats in DN group(without DMD pretreatment)was stable,the food intake and body weight increased gradually,the reaction was rapid,the body hair was thick and smooth,the vulva was dry,and the stool was granular.The rats in group DA(pretreated with DMD)grow slowly,eat less,showed low spirits,and wet hair around the vulva,with more loose stool.The death time of the rats in the DMD group was earlier,but there was no statistically significant difference between the survival curve of the control group.However,after the pretreatment with DMD,the rats in the treatment group in DA group all grow slowly.the weight of the rats in the treatment group in DA group showed a significant decrease(P < 0.05).After the exposure,the ovary tissues of each group of rats were collected and weighed,and the organ coefficients of ovary of rats were calculated and analyzed.the changes in the ovary organ coefficients of rats in each treatment group were not statistically significant;3.HE staining to verify the pathological changes of the rat ovary,the results showed that the ovary structure of the DN group was basically normal,and the granular cells were arranged regularly and tightly,while the normal structure of the ovary of the DA group basically disappeared,and After DEHP and BPA single and combined exposure,the rat granulosa cells are arranged loosely and disordered,atretic follicles increase,some tumor cells are arranged in papillary and cauliflower-like arrangements,and the degree of differentiation of ovarian tumors is low,suggesting that in rat ovarian tumor models,DEHP and BPA are combined Exposure has a synergistic effect on its occurrence.DEHP and BPA in the DA group alone and combined exposure significantly increased the incidence of ovarian tumors in rats(P<0.05);4.Detection of rat ovary tissue The expression of PCNA by IHC,which is a tumor marker,was basically negative in the control group,and the combined exposure of DEHP and BPA in the DN group increased the number of PCNA-positive cells in rat ovarian tissue.The number of PCNA-positive cells in the DA group was significantly increased,and after DEHP and BPA exposure alone and in combination,the number of PCNA-positive cells was much higher than that in the control group,and the difference was statistically significant(P<0.05);5.RT-PCR and western blot showed that the combination of DEHP and BPA in the DN group resulted in an increase in the protein expression level of ERα in the ovary tissue and m RNA expression level in the DN group.There was no significant increase or decrease trend.In the DA group,DEHP and BPA alone and combined exposure caused an increase in the protein expression level of ERα in rat ovary tissue and there was a significant difference(P <0.05),but its m RNA expression level didn’t show a significant increase or decrease trend,similarly;6.The expression of PTEN in the ovary tissue of rats in each treatment group was disclosed by RT-PCR and Western blot.The results showed that in the DN group,BPA and DEHP alone and in the combined exposure group were exposed to PTEN m RNA and the protein expression was lower than the control group,and the protein expression of DEHP and BPA combined exposure group was significantly different(P <0.05).In the DA group,BPA alone and the combined exposure of the two induced the decrease of m RNA and protein expression of PTEN in rat ovarian tissue,and the difference was significant(P<0.05);7.The micro RNA expression of each treatment group in the rat ovary tissue were detected by RT-PCR,the results showed that the expression levels of mi R-200 c and mi R-222 showed an increasing trend,In the DA group,the group which was given carcinogen alone,and the DEHP and BPA alone and combined exposure group.,and their expression level was significantly different(P<0.05).At the same time,the expression of mi R-200 c and mi R-222 in the ovarian tissue of the DEHP and BPA combined exposure group was significantly different(P<0.05);the expression levels of mi R-let7 b and mi R-21 showed an upward trend.In the DA group,the expression levels of DEHP and BPA were significantly up-regulation(P<0.05).meanwhile,compared with the DMD group,the expression of mi R-let7 b and mi R-21 in the ovary tissue of the DEHP and BPA was significantly different(P<0.05);the expression level of mi R-200a-3p showed a decreasing trend.In the DA group,the expression levels of DEHP and BPA were significantly different from those of the control group after exposure to DEHP and BPA alone and in combination(P<0.05);the expression level of mi R-203-3p showed an irregular trend;the expression level of mi R-205 and mi R-141-3p showed a decreasing trend,but the overall expression level showed an irregular trend;the expression level of mi R-212-3p showed a decreasing trend;8.Survival analysis of mi R-let7 b,mi R-200 c,mi R-222,mi R-21 was confirmed by the Kaplan-Meier plotter website.The results showed that compared with the low expression group,the has-mir-222 high expression group had a worse prognosis,and the difference was statistically significant(P=0.028);The prognosis of the has-let-7b(P=0.092)and has-mir-200c(P =0.21)high expression group was poor,and the difference was not statistically significant;the high expression group of has-mir-21 had a good prognosis,but the difference was not statistically significant(P=0.25);9.In ovarian tumor tissue,The level of mi R-222 expression is positively correlated with the expression level of ERα(P<0.05);the expression level of mi R-222 is negatively correlated with the expression level of PTEN(P<0.05).Conclusions: 1.Long-term combined exposure of bisphenol A and bis(2-ethylhexyl)phthalate will affect the normal growth and development of rat ovarian follicles and granulosa cells,but the direct carcinogenic effects of the two inducing ovarian tumors have not been observed.2.Long-term combined exposure of bisphenol A and bis(2-ethylhexyl)phthalate can synergistically promote the occurrence of ovarian tumors in rats caused by carcinogens,and both act as growth promoters to increase the incidence of ovarian tumors.3.After long-term combined exposure of bisphenol A and bis(2-ethyl)hexyl phthalate,it may promote the increase of mi R-222 expression level via ERα,thereby inhibiting PTEN and activating the downstream PI3K/AKT signaling pathway,thus increasing the susceptibility of ovarian tumors in rats. |
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