Effects Of Environmental Endocrine Disruptor Bromophenol On The Occurrence And Progression Of Prostate Cancer By Inhibiting In Vivo Metabolic Enzymes

Background:Prostate cancer is one of the most common malignant tumors of the genitourinary system of elderly men.According to the data published by GLOBOCAN in 2020,there will be more than 1.4 million new cases of prostate cancer worldwide in 2020,and up to375,000 people will die from prostate cancer.The morbidity and death rate rank second and fifth respectively in male malignant tumors worldwide.The incidence of prostate cancer has obvious regional difference,and the incidence in developed countries such as Northern Europe,Western Europe,North America,and New Zealand is significantly higher than that in developing countries such as Asia.Although the incidence and mortality of prostate cancer in China are lower than those in Europe and the United States,the number of new cases of prostate cancer in China will be about 138,000 in2020,and the death toll will be about 16,000.The morbidity and mortality rank second and seventh in male cancers in China,respectively,which is a serious threat to the life and health of the majority of elderly men.Prostate cancer is an endocrine hormone secretion-dependent tumor.Exogenous factors lead to the disruption of the balance of endocrine hormones(androgens/estrogens)in the body,which is an important factor in the pathogenesis and progression of prostate cancer.The metabolism of prostate cancer-related endocrine hormones is through phase I metabolism,where the main involved enzymes are CYP450 enzymes.The combination of phase II metabolism promotes excretion in the body,where the main involved enzymes are glucuronyl transferase,sulfotransferase and glutathione thiol transferase,etc.The balance of hormone level production and metabolism in the body is the premise to maintain normal life activities.When the balance of production and metabolism in the body is broken,it may lead to the occurrence and progression of diseases.The relationship between environmental pollution and the occurrence and progression of diseases has always been a hotspot of people’s attention and research.Since the Wingspread meeting in 1991,the first scientific meeting to recognize the harmful effects of hormone-like chemicals in humans and wildlife,a growing body of research has linked environmental endocrine disruptors(EDCs)to thyroid,prostate,breast and intrauterine cancers.The occurrence and progression of hormone-related tumors such as membranous carcinoma are related.The main pathogenic mechanisms of EDCs include the following: 1.EDCs mimic normal hormone functions and compete for binding to hormone-specific nuclear receptors;2.EDCs have the active functions of multiple hormones,and a single EDC can disrupt multiple endocrine systems at the same time;3.EDCs interfere with the synthesis and metabolism of normal hormones;4.EDCs can affect the epigenetics of target organs and intracellular non-coding RNAs affect tissue growth and development;5.EDCs can alter epithelial-stromal interactions and biomechanics affect tissue growth and development.The environmental endocrine disruptors bromophenols(BPs)are the most widely exposed and common industrial raw materials,and are commonly used in herbicides,brominated flame retardants,plastic rubber and wood preservatives.Studies have shown that BPs are related to the occurrence and progression of thyroid cancer,breast cancer,ovarian cancer,etc.The main mechanism is to inhibit the formation of thyroxine and affect the function of ER receptors.Whether it can affect the occurrence and progression of prostate cancer is still unclear.We know that the metabolism of prostate cancer-related hormones such as androgens or estrogens in the body requires the participation of phase I and phase II metabolic enzymes.Based on the above research background,we put forward the hypothesis that the environmental endocrine disruptor bromophenol affects the occurrence and progression of prostate cancer by inhibiting in vivo metabolic enzymes.Methods:1.Construct an in vitro incubation system.Eight common BPs were selected as inhibitors,9 CYP enzymes,5 UGTs enzymes and 4 human recombinant SULTs enzymes were selected as experimental objects,and high performance liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to detect the product generation of CYPs,UGTs and SULTs under the inhibition of BPs.2.Carry out the preliminary screening experiment of BPs on the activity of CYPs,UGTs and SULTs subtypes,and calculate the half inhibitory concentration IC50 and the inhibition kinetic parameter Ki by enzyme kinetics test.The in vivo-in vitro extrapolation method(IVIVE)was used to evaluate the exposure rate(AUCi/AUC)of BPs exposure to human hazard,and the concentration threshold of BPs exposure to inhibit the metabolic activity of metabolic enzymes in vivo was calculated.3.Model the three-dimensional structure of BPs molecules and CYPs,UGTs and SULTs proteins,and use the molecular docking method to simulate the optimal spatial binding of BPs and CYPs,UGTs and SULTs protein configurations.Hydrogen bonding between molecules and hydrophobic interactions of amino acid residues were observed,and free binding energies were estimated using the Lachma Genetic Algorithm(LGA).The mechanism of BPs inhibiting the activity of metabolic enzymes was discussed from the perspective of molecular structure.4.Collect clinical prostate cancer and prostatic hyperplasia serum samples,and use LC-MS/MS technology to detect the difference among the levels of serum testosterone,androstenedione,dehydroepiandrosterone,dehydroepiandrosterone sulfate,estrone,estradiol,estriol,17-OH progesterone and nine sex hormones.5.Prostate cancer cell lines LNCap and 22RV1 cell lines were co-cultured with different hormones and bromophenol to explore the effect of bromophenol on hormone metabolism.Results:1.All eight bromophenol compounds have inhibitory effects on CYP450 subtype enzymes.Among them,CYP1A2,CYP2B6,CYP2C8,CYP2C9,and CYP2C19 can be inhibited by a variety of bromophenols,and the inhibitory ability of bromophenol is not strengthened with the increase of the number of bromine substituents.2,4,6-Tribromophenol,which is the most abundant in the human body,was selected as a representative for the inhibition kinetic constant study,including inhibition type and kinetic constant determination.Based on IVIVE result,the threshold value of2,4,6-tribromophenol is determined to be 6.1 μM.From the molecular docking results,the BPs can bind to the active site of amino acid residue of the specific domain of CYP2C9.2.All eight bromophenol compounds have inhibitory activities on the four SULTs isoforms,among which SULT1A1 and SULT1B1 are the strongest inhibitory isoforms.2-Bromophenol substituted bromophenols have particularly strong inhibitory effects on the four SULTs isoforms.2,4,6-Tribromophenol,which is the most abundant in the human body,was selected as a representative for the inhibition kinetic constant study,including inhibition type and kinetic constant determination.According to IVIVE result,the threshold value of 2,4,6-tribromophenol was1.628 μM.From the molecular docking results,the inhibitory effect of 3,5-dibromophenol with more hydrogen bonds was stronger than that of 3-bromophenol with less hydrogen bonds.3.All eight bromophenol compounds have inhibitory effects on the five UGTs subtype enzymes.2,4-Dibromophenol,2,5-dibromophenol and 3,5-dibromophenol can inhibit UGT1A6,UGT1A9,UGT2B7 to a greater extent,where the inhibition degree of enzyme activity is above 80%.The IC50 test results show that the above three compounds have the strongest inhibition on UGT1A9,with IC50 values of 12.8μM,13.2μM,and 9.0μM,respectively.4.The LC-MS/MS technique was used to detect and analyze the serum sex hormones of patients with prostate cancer and benign prostatic hyperplasia.It was found that there was a significant difference in dehydroepiandrosterone between the two groups(p<0.05).No significant difference was found from the rest 8 sex hormones between two groups.5.In the hormone-sensitive cell line LNCa P of prostate cancer cells,compared with the control cell culture medium with DHEA added alone,it was observed that the residual DHEA content was increased(p=0.017)when DHEA and 2,4,6-bromophenol were added simultaneously after 24 hours of culture.Such phenomenon was not observed in the castration-resistant prostate cancer cell line 22RV1(p=0.916).Conclusions:As persistent environmental pollutants,bromophenol compounds have certain toxic effects on human body.This study confirmed that the environmental endocrine disruptors bromophenol compounds have strong in vivo inhibitory effects on phase I metabolizing enzymes CYPs and phase II metabolizing enzymes UGTs and SULTs,and can inhibit the metabolism of prostate cancer-related endocrine hormone dehydroepiandrosterone at the cellular level.The above results provide theoretical basis for the regulation of bromophenol compounds as environmental interferences.