Studies On Metabolism Of Hydroxylation Polybrominated Diphenyl Ethers And The Effects On Estrogen By Mouse Liver In Vitro

Hydroxylation polybrominated diphenyl ethers(OH-PBDEs)as a kind of metabolic products which have attracted more and more attention due to their higher endocrine disrupting effect.It can significantly affect thyroid hormone levels in the body because of its molecular structure is similar to that of thyroid hormone(T4),which binds to thyroid hormone transfer protein(TTR).Currently,OH-PBDEs have been detected in environmental media,organisms and even in humans.Studies have shown that OH-PBDEs can further form a phase metabolites through biological enzyme catalytic oxidation in vivo.The metabolic rate is affected by the number of bromine atoms and the positions of the hydroxyl,bromine and ether bonds.Because of their toxicity and effects on estrogen,it is necessary to explore the metabolic mechanism of OH-PBDEs metabolized in biological tissue,in order to provide the theoretical basis of metabolic and toxic Effects by OH-PBDEs.In the present research,5'-OH-BDE-99 ? 6'-OH-BDE-99 ? 6-OH-BDE-99 and 6-OH-BDE-137 were target compounds,using rats liver was reaction media and in vitro metabolism model,researching the metabolism and the effects on the four estrogens(E1,E2,E3,EE2)by rats liver in vitro.Metabolites(2-OH-E2)were detected quantitatively by HPLC-MS/MS.The key enzymes responsible for the metabolism of OH-PBDEs were determined with prototypical Cytochrome P450s(CYPs)inhibitors.The main results are as follows:(1)Four kinds of OH-PBDEs can be metabolized,and the metabolic rate followed the order: 6'-OH-PBDE-99> 6-OH-BDE-99> OH-BDE-137 by rats liver S9 in vitro,which indicated that the difference of the substitution position of OH functional group and Br atom affected the metabolism level and the metabolic pathway of the four kinds of OH-PBDEs.In the experiment,OH-PBDEs with different concentrations(1.5,3,6 ?M)had no significant effect on the metabolic rate of E1 and E2,but inhibited the metabolism of E3 obviously.High concentrations of OH-PBDEs inhibited the metabolism of EE2,but there was no significant difference with CK group.(2)The four OH-PBDEs were metabolized by rats liver microsome in vitro,then OH-PBDEs easier to metabolism than PBDEs,which demonstrated that Hydroxyl groups increased biological availability of PBDEs.The same as metabolized by rats liver S9,when OH-PBDEs contain the same degree of bromination,hydroxyl group adjacent to ether bridge increased the metabolic rate.According to ECOD characterization experiments found that four kinds of OH-PBDEs have different effects on microsomal ECOD activity.The overall trend showed that 5'-OH-BDE-99 significantly promoted ECOD activity(p < 0.05)at low concentrations(1.5 ?M)as the concentration of OH-PBDEs increased in the reaction system;The effect of 6-OH-BDE-99 on the activity of ECOD was not obvious with the increasing concentration.ECOD activity decreased with the increasing concentration of 6-OH-BDE-99(p > 0.05).No significant effect on ECOD activity was observed at low concentrations(1.5 ?M),but significantly increased with increasing concentrations of 6-OH-BDE-137(p < 0.05).In addition,the results of the inhibitor experiments showed that CYP2C9 was the key enzyme system for microsomal metabolism of 5'-OH-BDE-99 and 6'-OH-BDE-99,while the CYP1A2 and CYP3A4 subtypes were more involved in 6-OH-BDE-99 and 6-OH-BDE-137,while CYP1A2 contributed the most;CYP2D6 played a secondary role in the metabolism of the four OH-PBDEs.(3)The concentrations(1.5,3,6 ?M)of four OH-PBDEs in the microsomal system showed different effects on the four estrogens.When OH and Br atoms were in the ortho position(6-OH-BDE-99),the metabolism of E1 was accelerated and tends to be gentle with increasing concentration.However,when the OH and Br atoms were in the para position(5'-OH-BDE-99)inhibited the metabolism of E1.In addition,6-OH-BDE-99 could promote the metabolism of E2,but after reaching a certain concentration,E2 was inhibited with increasing concentration.The inhibitory effect of OH and Br on the metabolism of E2 was more obvious when the OH and Br atoms were in the para position,indicating that the difference metabolism of E2 may be due to the influence on the metabolic enzyme activities accused by OH-PBDEs.Four OH-PBDEs rapidly metabolized E3,but had no significant effect on metabolic rate of EE2.