Study On Synergistic Toxicity And Metabonomics Of Human Respiratory Tract Cells Induced By Trimethylamine And Its Metabolites

Volatile organic amines are widely present in the atmosphere.Due to the characteristics of low olfactory threshold,high emission intensity and high reactivity,they have adverse effects on the ecological environment and public health and thus become the main pollutants in the control of air pollution.Trimethylamine(TMA),a typical organic amine with the largest emission and the most abundant content,is more likely to do harm to human health through inhalation exposure,causing adverse reactions and diseases in the human respiratory system.The respiratory tract is the main target organ for the chemical toxicity of exogenous pollutants,which contains abundant enzymes that enable the respiratory tract to metabolize exogenous volatile pollutants.During this metabolism,the pollutants may be detoxified,but more likely to be activated into the more toxic metabolites,which may cause more severe toxic damage to the human respiratory system.Nevertheless,there are still few studies on the toxic effects and mechanisms of volatile organic amines on human respiratory system,and the metabolic intermediates produced by volatile organic amines in respiratory tract and their metabolic mechanism have not been elucidated.Therefore,in this thesis,the typical volatile organic amine TMA was selected as the target exposure pollutant,and human bronchial epithelial cells16 HBE cultured in vitro were used as the subject of exposure,so as to study the in vitro synergistic toxic effects and metabolomics of human respiratory cells induced by exogenous pollutant TMA and its identified metabolites.The main research contents and results include:(1)Firstly,the acute toxicity of 16 HBE cells induced by TMA exposure at different doses and different time was studied from the perspective of cytotoxic effects.The results showed that within the range of 0-91.35 m M of TMA concentration,the viability of 16 HBE cells decreased from 94.66% ± 3.11% to 5.88 ± 1.48% in a dose-dependent manner,while the production of intracellular reactive oxygen species(ROSs)increased from 27.25% ± 4.17%to 154.82% ± 32.67% in a dose-dependent manner.The excessive production of ROSs led to the imbalance of the redox state in cells,which further induced the activation of the antioxidant enzyme superoxide dismutase(SOD)and inhibited the expression of the antioxidant enzyme catalase(CAT).The aggravation of oxidative stress further resulted in the massive secretion of the pro-inflammatory cytokine Interleukin-6(IL-6)with the highest concentration up to84.12 ± 7.47 pg/m L,which eventually led to the damage of 16 HBE cell membrane as well as the cell necrosis.(2)Furthermore,the metabolic intermediates produced by TMA in 16 HBE cells and their metabolic mechanisms were investigated to reveal the synergistic toxic effects of TMA and their metabolites on 16 HBE cells during exposure.The real-time qualitative and quantitative analysis of TMA and its volatile intermediates were performed by proton transfer reactationtime of flight mass spectrometry(PTR-TOF-MS)and proton transfer reactation-quadrupole mass spectrometry(PTR-QMS).In addition,the main metabolites of TMA in cells and culture media were identified by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS),which combined the non-target detection method of direct injection with the target detection method of amine-containing metabolites labeled with dansyl chloride.As the results,it was found that the reduced content of TMA in the cell headspace and culture medium indicated that TMA may undergo a certain degree of metabolic depletion during cell exposure,and the increased dimethylamine(DMA)was considered as a key metabolite of TMA.In addition to the formation of metabolites DMA and methylamine(MA)by means of demethylation,TMA can also be oxygenated to generate N,Ndimethylformamide(DMF)and N,N'-Bis(2-hydroxyethyl)-1,2-ethanediaminium by Nformylation or hydroxylation,which was considered to be involved in the participation of cytochrome P450(CYP)enzymes.These identified metabolites had higher toxicity than TMA itself,and DMF was also classified as a carcinogen.Therefore,during the TMA exposure,16 HBE cells may produce more toxic metabolites,which synergistically induced intracellular toxic effects together with the parent organic amine.(3)Finally,the potential impact of TMA exposure on the metabolomic perturbation in16 HBE cells was explored in the basis of the non-targeted cell metabolomics using mass spectrometry.Results showed that TMA induced significant changes in the metabolome of16 HBE cells.The statistical results of volcano plots,principal component analysis(PCA)and partial least squares discrimination analysis(PLS-DA)also showed that TMA had a dosedependent effect on the perturbation changes of the metabolome of 16 HBE cells.In addition,a total of 18 main endogenous differential metabolites were screened and identified,and 5main metabolic pathways mediated by these main metabolites were further determined,including sphingosine and sphingosine-1-phosphate metabolism,spermine and spermidine biosynthesis and degradation,serotonin degradation,bile acid biosynthesis,amino sugar and nucleotide sugar metabolism.The metabolomic perturbation induced by TMA may be closely related to the toxicological effects such as cell apoptosis and the activation of oxidative stress,as well as the pathogenesis of neurological disorders,metabolic disorders,tumors and other diseases.In summary,this thesis aims to reveal the possible metabolic pathways and mechanism of TMA after exogenous pollutant TMA acted on human respiratory tract cells 16 HBE,and to comprehensively clarify the potential toxic mechanism of TMA and its metabolites to human respiratory tract by combining the cytotoxic effects and metabolomic studies.Volatile organic amines can induce respiratory cells to generate more toxic metabolites,which may synergistically induce intracellular oxidative damage and cell necrosis together with the parent organic amine.In addition,the metabolomic perturbation induced by TMA in respiratory tract cells may be potentially related to the toxicological effects of TMA and the cellular mechanism of diseases.This research provides valuable insights into the synergistic toxicity effects induced by TMA and its metabolites in human respiratory tract cells.The integration of metabonomics research provides a theoretical basis for the determination of specific molecular mechanisms and major metabolic pathways after the exposure of TMA on human respiratory system,which is of great significance for the study of the respiratory toxicity and environmental health effect of volatile organic amines.In addition to focusing on substances such as organic amines,the synergistic effects and hazards of metabolites and their parent compounds on the environment and human health should be paid more attention to further researches and disease risk assessments.