Microbial Degradation Mechanisms Of Polybrominated Diphenyl Ethers And Toxicity Of Its Metabolites

Polybrominated diphenyl ethers(PBDEs),a class of widely used brominated flame retardants,has caused pollution that poses significant risks to human health and ecological environment.2,2',4,4'-tetra brominated diphenyl ether(BDE-47)was used as representative contaminant of PBDEs.A bacterial strain named Pseudomonas aeruginosa,isolated with a highly effective degradation fuction for BDE-47 from our microorganisms library,was selected as the experimental strain in this study.The degradation characteristics as well as metbolites generation and cytotoxicity of BDE-47 metabolites were studied.The molecular biological mechanism of microbial degradation and transformation of BDE-47 was interpreted from transcriptome sequencing technology.The paper also illuminated the interaction of BDE-47 and Cr(VI)co-exsited pollutants and P.aeruginosa cells,as well as characteristics and mechanism involed in BDE-47 degradation and Cr(VI)removal.BDE-47 can be degraded efficiently by P.aeruginosa,and the addition of a certain amout of carbon source and surfactant could enhance its degradation.The degradation of BDE-47 by P.aeruginosa was maily through the biological action of intracellular enzymes,and the metabolites include two bebrominated metabolites(BDE-28 and BDE-7),four hydroxylated metabolits(6-OH-BDE-47,5-OH-BDE-47,2-OH-BDE-28 and 4-OH-BDE-17),and two brominated phenols(2,4-DBP and 4-BP).P.aeruginosa also exihibited exceptional ability to degrade intermediate metabolies(BDE-28,BDE-7 and 2,4-DBP).Transcriptome sequencing reaveled that 991 genes were up-regulated,and 923 genes were down-regulated in P.aeruginosa during the degradation of BDE-47(FDR?0.001,|log2Ratio|?1).Extracellular membrane adhesion protein,lipoplysaccharides(LPS),ABC transporters,penicillin bingding proteins(PBP),and porin may be involed in the adsorption and transportation of BDE-47.Coenzyme A ligase,dehalogenase,P450 monooxygenase,salicylic acid hydroxylase and homodimelene 1,2-dioxygenase may be involed in BDE-47 reductive debromination,oxidative hydroxylation,ring cleavage and mineralization catabolic process.P.aeruginosa could immediately adjust and respond to BDE-47 exposure through up-regualted expression of antoxidation enzyme,DNA repair protein,and efflux pump protein.The efflux pump mechanism during the degradation/transformation of BDE-47 by P.aeruginosa was characterized by using molecular biology experiments including real-time fluorescence quantitative PCR and RNA interference technology.Extracellular BDE-47 entered into cells through some channels on cell membranes,and parts of them were degraded by intracellular enzymes,and parts of them were discharged into extracellular compartments through theefflux pumps on the cell membrane,so that the intracellular accumulation of BDE-47 was maintained at a lower level.It not only ensured the effective degradation of PBDEs,but also enabled the microbial cells to maintain high activity when exposure to high concentrations of PBDE.The cytotoxic effects of BDE-47 and its eight metabolites on hepatoblastoma cell line-HepG2 cells were investigated in this study.Results revealed that BDE-47 and all its metabolites inhibited cell viability in both a dose-and time-dependent manner.BDE-47 and its metabolites caused cell death and cell apotosis by inducing ROS production,affecting SOD and GSH activity,disrupting cell cycle and indung DNA damage.In,general,brominated phenol products(2,4-DBP and 4-BP)posed the highest toxic effects on HepG2,followed by hydroxylated products(6-OH-BDE-47,5-OH-BDE-47,2-OH-BDE-28,and4-OH-BDE-17),and BDE-47 and its debromination products were comparatively less toxic to HepG2 cells.Pretreatment with low dose of BDE-47,BDE-28 and BDE-7 produced a certain amount ROS that stimulates the cellular antioxidantion system and other defense systems against xenobiotic pollutants,increased SOD and GSH activity,lowered the oxidative stress and activated the DNA repair system,which would increase the cell viability,reduce cell death and cell apotosis when exposure to high dose of pollutants.P.aeruginosa was available for the removal of toxic Cr(VI)and degradation of BDE-47 simultaneously.The removal process of Cr(VI)by P.aeruginosa was through the following two steps.Cr(VI)was first reduced to Cr(III)outside the cell,and then Cr(III)was adsorbed on the cell membrane or entered in the cell.Low concentration of BDE-47 can be used as carbon source to increase cell growth and extracellular enzymes,which could inmprove the Cr(VI)removal rate.However,high concentration of BDE-47 would inhibit the growth of P.aeruginosa when co-contaminated with Cr(VI),thus inhibiting its removal.Low concentration of Cr(VI)facilitated the secretion of rhamnolipid from the strain,altered cell surface hydrophobicity and cell membrane permeability,and promoted intracellular BDE-47 accumulation,thus improving BDE-47 biotransformation.In addition,the stimulation of intracellular enzyme synthesis by Cr(VI)contributed to more BDE-47 elimination in the cells.The achievements in this study provide theretical basis for research on PBDEs microbial degradation,degradation mechanisms,and cell toxicity of PBDEs metabolites,and the effect of co-existed heavy metal on PBDEs adsorption,degradation and transformation on the cellular interface has practical significance.