Effect Of Bis(2-ethyl)Hexyl Phthalate Combined With Bisphenol Aexperimental Study On The Occurrence Of Breast Tumors In Rats

Objective Breast cancer is one of the most important malignant tumors that threaten women's health.In the past 20 years,the incidence and mortality of breast cancer in Chinese women have increased rapidly,with 11.19% of the global incidence,second only to the United States,and the prevention and control situation is very grim.The occurrence and development of breast cancer are the result of environmental and genetic interactions.Its etiology is more complicated.In recent years,endocrine disrupting chemicals(EDCs)have attracted much attention because they affect the homeostasis of the body's environment and can produce certain damaging effects.Low-dose exposure to endocrine disruptors increases the susceptibility to breast cancer during low-grade breast tissue differentiation and critical periods of breast development.At present,bisphenols(BPs)and phthalates(PAEs)compounds are two types of EDCs that have attracted much attention.As the main raw material and plasticizer of plastic products,they are widely used in daily production and Both the environment and the human body can be detected,and it has become a more serious chemical pollutant.Considering the existence of multiple mixed environmental pollutants in real life,and the diversification of human exposure pathways,and the mixed exposure mode may show different toxic effects when each substance acts separately,the research on the toxic effects of single pollutants has been Can't meet actual needs.Di(2-ethyl)hexyl phthalate(DEHP)and bisphenol A(BPA)are ubiquitous in the environment,and their interactions are complex.Due to the limitations of population research,the survey results vary widely,and the experiment The evidence is insufficient.As one of the target organs of DEHP and BPA,whether the mammary gland alone or in combination will affect the occurrence and development of breast cancer and its mechanism remains unclear.Therefore,exploring the relevant mechanisms of breast tumorigenesis caused by endocrine disruptors alone or in combination is the key issue to be addressed in this study.Methods 1.Establishment of breast tumor model: 160 healthy 4-week-old female SD rats were randomly divided into 8 groups(n = 20).It is divided into two parts according to whether to administer carcinogens;the carcinogen pretreatment group uses three kinds of carcinogens such as diethylnitrosamine(DEN)to construct a carcinogenic model of breast tumors,and simultaneously gives DEHP 150 mg / kg and BPA 20 mg / kg separately And the joint gavage was poisoned to death after 30 weeks.2.HE staining was used to observe the pathological changes of rat breast tissue;immunohistochemistry was used to detect the expression of PCNA in breast tissue.3.RT-qPCR method to detect gene expression levels of ER?,CREB,HDAC6 and c-Myc.4.Western blot method to detect the protein expression level of ER?,CREB,HDAC6,c-Myc.Results:1.HE staining detected the pathological changes of rat mammary glands.The results showed that the long-term exposure of DEHP and BPA caused the normal structure of rat mammary glands to basically disappear,the proliferation of mammary ductal epithelial cells,and the number of lobes increased,suggesting that long-term exposure of endocrine disruptors may induce mammary gland hyperplasia,And mammary gland hyperplasia in rats in the combined exposure group of DEHP and BPA was more serious.2.After pretreatment with carcinogen DMD,the combined exposure of DEHP and BPA significantly reduced the incubation period of breast tumors in rats and increased the incidence of breast tumors.The pathological results showed that the mammary tumor tissues of the rats in the DEHP and BPA alone exposure groups were papillary.In the two exposure groups,the degree of differentiation of the mammary tumors of the rats was low,and the papillary arrangement was fused into a mesh.It is suggested that in the rat tumor model,the combined exposure of DEHP and BPA has a synergistic effect on the susceptibility of breast tumors.The expression of tumor marker PCNA was detected by immunohistochemistry.The PCNA in the control group was basically negative,and the positive rate of PCNA in mammary gland tissue of rats exposed to DEHP and BPA was significantly increased.After pretreatment with carcinogens,the positive rate of PCNA increased significantly,and in the DEHP + BPA exposure group,the positive rate of PCNA was much higher than that of DEHP and BPA alone exposure groups.3.Realtime-qPCR and Western blot were used to detect the expression of ER? and downstream transcription factor CREB in the breast tissue of each treatment group.The results showed that in the treatment group without DMD,the combined exposure of DEHP and BPA resulted in the mRNA and protein of ER? in the breast tissue of the rats.Both expressions increased(P <0.05).After DMD pretreatment,the single and combined exposure of DEHP and BPA led to a significant increase in ER? mRNA and protein expression in rat mammary gland tissue(P <0.05),and compared with the DMD alone treatment group,the combined exposure of DEHP and BPA increased significantly ER? mRNA expression level(P <0.05).At the same time,the phosphorylation level of CREB protein in the mammary gland tissue of rats exposed to BPA alone and the combination of DEHP and BPA was significantly higher than that of the control group(P <0.05).After DMD pretreatment,DEHP and BPA alone and combined exposure induced rats The phosphorylation level of CREB protein in breast tissue was significantly increased(P <0.05).4.The expression of HDAC6 and c-Myc in the mammary gland tissue of rats in each treatment group was detected by Realtime-qPCR and Western blot.The results showed that in the non-DMD treatment group,the expression of HDAC6 mRNA in the mammary gland tissue of the rats exposed to DEHP and BPA was significant Higher than the control group(P <0.05),and no significant changes were observed in the protein expression level of HDAC6.After pretreatment with carcinogens,the expression levels of HDAC6 mRNA and protein in the mammary gland tissues of the DEHP and BPA alone and combined exposure groups were significantly higher than those of the control group(P <0.05),and the combined exposure of DEHP and BPA could cause mammary glands in rats The mRNA and protein expression of c-Myc increased(P <0.05).After DMD pretreatment,the exposure of DEHP and BPA alone and in combination induced a significant increase in the mRNA and protein expression of c-Myc in the mammary gland of rats(P <0.05).Compared with the DMD treatment group alone,the combined exposure of DEHP and BPA significantly increased the mRNA expression levels of HDAC6 and c-Myc(P <0.05).Conclusions:1.Long-term joint exposure of bis(2-ethyl)hexyl phthalate(DEHP)and bisphenol A(BPA)may interfere with mammary gland development and induce mammary gland hyperplasia.Exposure of DEHP to BPA leads to increased incidence of breast tumors,shortens the incubation period of breast tumors,and increases the susceptibility of breast tumors caused by DMD.2.DEHP cooperates with BPA exposure to activate downstream signal transduction pathway via estrogen receptor,increase HDAC6 expression,and then up-regulate the oncogene c-Myc,promote the occurrence of breast tumors in rats,and increase the susceptibility of breast tumors caused by carcinogens.