Structure And Cellular Effect Of Three Estrogens Including Estrone And Its Metabolites In Animal Derived Food

There are many kinds of estrogen,which can produce a variety of metabolites with similar structure through the body metabolism.This kind of substance has a high degree of stability.The residues in food and environment can be transferred into the human body through the food chain,so as to play its hormone effect.Although moderate estrogen can protect the nervous system and prevent arthritis,excessive intake can lead to an increase in the incidence rate of breast cancer and autoimmune diseases.Therefore,the detection of estrogen in food is more and more important.Due to the diverse structure of estrogen,the current instrumental analysis and immunoassay methods for quantitative analysis of estrogen have limitations,which can only detect the known structure of estrogen in food.In order to achieve a wide range of screening of estrogen,it is necessary to study the structure and effect differences of estrogen and provide data support for the prediction of compounds with estrogen effect,so as to determine the quantitative detection target of estrogen in food.In this paper,typical endogenous estrogens,such as estrone(E1),estradiol(E2),progesterone(PROG)and metabolites,were used as the research objects to study the regulatory effects of different structures of estrogen on cell proliferation and different proteins in cell pathways,and to explore the relationship between estrogen effects and metabolic characteristics based on metabolomics,so as to provide new detection index for the screening of estrogen.The main results are as follows:Studies on the effects of different structures of estrogen in animal derived foods on the proliferative viability of different cell lines.Using gas chromatography-mass spectrometry,several kinds of estrogen were found in milk products,avian eggs and meat products,and the levels of estrogen reached 7.87?44.43?g/L.Cell lines most sensitive to estrogen were screened based on the half maximal effect concentration(EC₅₀).It was found that the proliferation activities of lung non-small cell carcinoma cells(A549),prostate cancer cells(PC-3)as well as osteoblasts(OB)were mostly inhibited by E1,E2,PROG as well as their metabolites,while breast cancer cells(MCF-7)showed significant activity differences between groups.Among all the estrogen,E1 exhibited a proliferation promoting effect on MCF-7 cells with an EC₅₀ value of 1.48 10^-7 mol/L,which was much lower than that of other cell lines(A549:0.72 mol/L,OB:7.55 10^-6 mol/L).While 2-Hydroxyestrone(2-OH-E1)produced by E1 metabolism exhibited significant apoptotic effect on MCF-7 cells with an EC₅₀ value of7.29 10^-12 mol/L,which was lower than that of other cell lines(PC-3:7.34 10^-5 mol/L,OB:1.34 10^-4 mol/L).The relationship between structural differences and cellular effects of estrogens was explored using targeted proteomics and molecular docking.The results showed that the structural changes of E1 in C-2 could affect the regulation of signal molecules and proteins in MCF-7 cells.Similar to E1,16?-hydroxyestrone(16?-OH-E1)could up regulate the expression of cyclin dependent kinase 2(CDK2),mammalian target of rapamycin(m TOR)and nuclear factor activated kappa light chain enhancer protein(NF-?b)in MCF-7 cells(p<0.05),while its C-2 metabolites 2-OH-E1 and 2-methoxyestrone(2-Me O-E1)were opposite to E1,with downregulatory effects(p<0.05).The structural changes at C-2 position were inferred to result in altered binding of small female hormone molecules to estrogen receptor(ER?)residues ARG394,GLU353,LEU387,HIS524 by molecular docking simulation analysis.Metabolomics was utilized to explore the effects of E1 and its metabolites on cellular metabolic levels and to compare their differences in cellular metabolic features.The results showed that multiple amino acid metabolic pathways in MCF-7 cells were significantly affected by E1 and its metabolites.Among them,estrogens had the greatest effect on tryptophan biosynthesis pathway,and the contribution of this pathway in E1,2-OH-E1,2-Me O-E1 and16?-OH-E1 treatment groups were 0.29,1.01,0.50 and 0.50,respectively.Picolinic acid,an intermediate of tryptophan metabolism,has both neuroprotective and immunomodulatory effects,with a significant rise in metabolic levels in response to different estrogens,which can be used as an assessment of the effects of estrogens.However,the level of cis aconitic acid,an intermediate of TCA cycle,was up-regulated by 21.70 times in 2-OH-E1 group compared with the control group.There was no significant difference in E1,2-meo-e1 and 16?-OH-E1 groups compared with the control group.Therefore,it is inferred that 2-OH-E1 can regulate the metabolism of cis aconitic acid by interfering with TCA cycle,so as to exert its inhibitory effect on cell proliferation,signal molecules and protein levels.To sum up,this paper studied the structure-activity relationship between different estrogen hormones and MCF-7 sensitive cells by comparing their different effects on MCF-7 sensitive cells,so as to provide data reference for establishing the prediction model of estrogen effect.By analyzing the characteristics of cell metabolism under the action of estrogen,the metabolic differences that can be used as the evaluation index of estrogen effect were screened,which laid a theoretical foundation for the monitoring of estrogen level in food.