Biodegradation Mechanism Of Triphenyl Phosphate And Toxicity Of Its Metabolites

Organophosphate flame retardants?OPFRs?are a kind of phosphorous flame retardants widely used.In recent years,OPFRs have attracted widespread attention because they are easily released into the environment and have toxic effects,posing a potential threat to the environment and human health.Up to date,only limited studies have focused on the biodegradability and degradation mechanism of OPFRs.In the present study,triphenyl phosphate?TPHP?was used as representative contaminant of OPFRs,and Brevibacillus brevis was selected as the experimental strain.The objective of this work was to investigate the biodegradation characteristics and transformation mechanism of TPHP by B.brevis and the changes of proteomic profile of B.brevis in response to the stress of TPHP.In addition,the cytotoxic effects and mechanism of TPHP and its metabolites were also analyzed.The detailed conclusions are as follows:?1?B.brevis could degrade TPHP effectively and utilize TPHP as the carbon and energy source for growth.The degradation efficiency of TPHP depended on the bacteria dosage and the temperature as well as pH of reaction system.The highest degradation efficiency of 1 mg/L TPHP by B.brevis?2 g/L?reached 92.1%at pH 7 and 30°C after 5 days of incubation,and the pH value of the degradation system also decreased gradually with time during this process.B.brevis could degrade more than 80%of TPHP after 4 days of incubation when the TPHP concentration was relatively low??10 mg/L?.However,the high concentration of TPHP?50mg/L?can destroy the normal morphological structure of B.brevis,causing cell damage,hence resulting in the degradation efficiency of TPHP was only 26.9%.Bioaugmentation with B.brevis could stimulate the TPHP degradation in river water microcosms by reducing the lag time of degradation reaction,and TPHP decreased by approximately 97.9%after 96 h of incubation.These results supported the feasibility of using in situ bioaugmentation as an alternate for treating TPHP-impacted river water.?2?The major metabolites of TPHP were identified using high-resolution liquid chromatography-qualitative quadrupole time-of-flight mass spectrometry?LC-Q-TOF-MS?within 5 days of degradation,including diphenyl phosphate?DPHP?,phenyl phosphate?PHP?,monohydroxylated-TPHP,monohydroxylated-DPHP,monohydroxylated-PHP,dihydroxylated-TPHP and dihydroxylated-DPHP.The degradation pathways of TPHP by B.brevis were proposed based on the detected metabolites,which mainly involved hydrolysis and hydroxylation processes.DPHP and PHP were detected within 5 days of incubation with the maximum concentrations at 77.1 and 2.1?g/L,respectively.In addition,B.brevis could degrade1 mg/L DPHP and PHP,and the highest degradation efficiencies were 14.4%and 23.3%after5 days of incubation,respectively.The activities of superoxide dismutase and catalase along with malondialdehyde content also increased significantly,indicating that TPHP caused a severe oxidative stress on B.brevis.Moreover,the addition of cytochrome P450?CYP?inhibitor piperonyl butoxide?PB?markedly decreased the degradation of TPHP by B.brevis and resulted in down-regulated expression of CYP gene in B.brevis,and the inhibitory effect was enhanced with the increase of PB concentration,indicating that CYP may be involved in the biodegradation process of TPHP.?3?The differentially expressed proteins?DEPs?in B.brevis were identified and quantified by isobaric tags for relative and absolute quantification?iTRAQ?in response to TPHP exposure,of which 102 proteins were up-regulated and 80 proteins were down-regulated.These DEPs were mainly involved in transportation and metabolism,information storage and processing,cellular processes and signaling,etc.The carbohydrate metabolism in B.brevis was activated by TPHP obviously,leading to the up-regulation of protein expression involved in glycolysis.TPHP also provided more energy for cell growth and metabolism by enhancing the amino acid and lipid metabolism in B.brevis.The ribosomal proteins involved in assembling 50S and 30S subunits were up-regulated significantly under TPHP stress,which played an important role in protein synthesis in cells.ATP binding cassette?ABC?transporter proteins and antioxidant stress proteins also showed different expression levels under TPHP stress in response to TPHP exposure.B.brevis maintained the stability of the intracellular environment and enhanced the adaptability of the cell to TPHP by regulating the expression of different functional proteins under environmental stress.?4?TPHP and its metabolites,namely DPHP and PHP,all had different degrees of inhibition on the viability of HepG2 cells,among which TPHP had the strongest inhibition effect and PHP was the weakest.The increased exposure concentration of TPHP can lead to a significant increase in intracellular Ca²⁺content,which destroys the integrity of mitochondrial membrane and promotes reactive oxygen species?ROS?accumulation in cells.TPHP further induced oxidative stress in cells,resulting in decreased mitochondrial membrane potential and inducing apoptosis of HepG2 cells.High concentration??100?mol/L?of TPHP can also cause nuclear damage and chromatin condensation.The induction effect of DPHP and PHP on apoptosis was significantly weaker than that of TPHP.Furthermore,TPHP induced cell cycle arrest at G0/G1 phase in HepG2 cells.,and decreased the proportion of cells at S phase.Compared with DPHP and PHP,TPHP has the strongest toxic effect on HepG2 cells,followed by DPHP,and the toxicity was dose-time dependent.The results showed that the toxicity of TPHP and its metabolites to HepG2 cells were related to the regulation of cell cycle progression and the apoptosis of cells through mitochondria pathway.Overall,biodegradation played a role in the detoxification of TPHP,indicating that bioremediation will be an effective and safe strategy for TPHP contamination treatment.The results in this study revealed the mechanism of microbial degradation and transformation of TPHP at proteinic and toxicological levels,and analyzed the potential toxic effects of TPHP and its metabolites on the environment and human health.This study also provided the data support for developing bioremediation technology for environments contaminated with OPFRs.