| Volatile organic sulfur compounds are a common class of pollutants in the atmospheric environment.Because of their low odor threshold,strong volatility,and high emission concentration,they will adversely affect the ecological environment and human health.Methanethiol,as a typical volatile organic sulfur compound,was widely found in industrial environments such as landfills,waste fermentation plants,and sewage treatment plants.The notable features of methanethiol are foul odor and strong volatility,which will reduce the comfort of the public life environment and damage human health.The human respiratory system,as the target organ for the toxic effects of volatile organic sulfur compounds,is also the first line of defense against the harm of volatile organic sulfur compounds.However,there are few studies on the toxic mechanisms of volatile organic sulfur compounds in the human respiratory system,and the metabolic transformation mechanism of volatile organic sulfur compounds in the human respiratory system has not yet been elucidated.Therefore,in this thesis,methanethiol,a typical representative of volatile organic sulfur compounds,was selected as the target pollutant,and the human bronchial epithelial cell 16 HBE cultured in vitro was used as the exposure subject.Two different exposure modes,immersion exposure and air-liquid interface exposure were used.In this way,the toxic effect mechanisms,metabolic transformation mechanisms,and transcriptomic and metabolomic characterization of exogenous pollutant methanethiol in human respiratory tract cells were carried out.The main research contents and results include:（1）First,in the immersion exposure mode,it was found that within the concentration range of 10 – 50 μM methanethiol,the 16 HBE cell viability decreased by 28.5%,the cell membrane permeability increased by 27.5%,and a significant increase in the number of necrotic cells by using the live-cell imaging and flow cytometry techniques.At the same time,50 μM methanethiol can induce 16 HBE cells to produce 1.6 times more intracellular reactive oxygen species（ROS）than the control group.Further,through q-PCR and Western blot analysis,it was confirmed that ROS activates the tumor necrosis factor（TNF）signaling pathway,which further promotes the formation of RIPK1-RIPK3 necrosomes,eventually leading to the reduction of intracellular mitochondrial membrane potential and the necrosis of16 HBE cells.Moreover,the metabolite of methanethiol in the 16 HBE cells was detected using high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UPLC-QTOFMS）,proton transfer reaction-mass spectrometry（PTR-MS）,and gas chromatography-sulfur chemiluminescence detector（GC-SCD）.It was found that the thiol Smethyltransferase expressed in 16 HBE cells can catalyze the conversion of methanethiol to dimethyl sulfide through Western blot experiments.In addition,methanethiol and its metabolite dimethyl sulfide can synergize the proliferation,and the oxidative stress of 16 HBE cells was further verified by gene silencing of thiol S-methyltransferase.（2）Secondly,the air-liquid interface exposure mode was further chosen to study in the thesis.By using the CULTEX RFS in vitro cell exposure instrument,it was found that the morphology of the 16 HBE cells become more and more shrunken with the increase of the methanethiol during 10 – 50 μM.Low concentrations of methanethiol gas（10 – 30 μM）had little effect on cell viability,but high concentrations of methanethiol（50 μM）significantly reduced cell viability by 70% and resulted in a 60% increase in cell membrane permeability.An increase in intracellular ROS and cell necrosis after methanethiol exposure were also found using cytofluorescence imaging.Compared with the immersion exposure mode,low concentrations of methanethiol（10 – 30 μM）caused less damage to 16 HBE cells,while high concentrations of methanethiol（50 μM）could reduce 16 HBE cell viability,promote oxidative stress and induce cell necrosis to a greater extent.At the same time,the Illumina Hi Seq2500 sequencing platform was used to sequence the transcriptome of 16 HBE cells exposed to methanethiol gas,and 32 differentially expressed genes were found.Gene Ontology（GO）enrichment results showed that methanethiol could affect the stress response of 16 HBE cells to compounds,cell growth,and the binding and regulation of signaling molecules.The KEGG pathway enrichment results showed that methanethiol could affect lipid metabolism and amino acid metabolism in 16 HBE cells.In terms of Disease Ontology（DO）prediction,differential gene enrichment was also found to be significantly related to lung diseases（lung cancer,chronic obstructive pulmonary disease）.All the above results indicated that methanethiol had a negative effect on 16 HBE cells at the m RNA level.（3）Finally,the metabolomic on 16 HBE cells exposed to methanethiol gas in the air-liquid interface exposure mode was performed using UPLC-QTOFMS,and 58 metabolites with significant differences were found.Most of the compounds are nucleic acids,organic acids,fatty acyl groups and organic heterocyclic compounds.Also,it was found that methanethiol may significantly affect the biosynthesis of phosphatidylcholine and the biosynthesis of pantothenic acid and coenzyme A through the enrichment analysis of The Small Molecule Pathway Database（SMPDB）.Further,a joint analysis of transcriptome and metabolome data was performed using O2 PLS and correlation heatmap analysis and we found that there was a close relationship between differentially expressed genes and metabolites after methanethiol exposure on 16 HBE cells.In addition,the disorder of purine metabolism and folic acid biosynthesis pathways were found in the combined effect of the differentially expressed genes and metabolites after methanethiol exposure through the co-expression analysis of KEGG.In summary,this thesis intends to perform in vitro methanethiol exposure to 16 HBE cells under immersion and air-liquid interface modes,aiming to reveal the toxic effects of exogenous pollutants on the human respiratory tract cells under different exposure modes.In addition,the metabolic transformation mechanism of methanethiol in 16 HBE cells was also discussed.At the same time,the potentially hazardous effects of methanethiol on human respiratory cells were comprehensively elucidated by combining with transcriptomics and metabolomics technology.The results showed that methanethiol exposure could induce a decrease in cell viability,resulting in the generation of oxidative stress and the activation of the cell necrosis pathway.At the same time,it was found that the catalytic enzymes in 16 HBE cells can have a certain bioactivation effect on methanethiol,and methanethiol and its metabolites played a synergistic role in the toxic effect on 16 HBE cells.Besides,the exposure mechanism was studied in a way closer to the real human respiration level through the airliquid interface exposure mode,which in a sense indicated that methanethiol-induced perturbation of the transcriptome and metabolome in respiratory tract cells may have certain potential implications to methanethiol-induced toxicological effects and cellular mechanisms of disease generation.The research in this thesis provides constructive insights into the toxic effects of methanethiol and its metabolites mediated respiratory exposure to a certain extent.What’s more,the comprehensive study of transcriptomics and metabolomics under the airliquid interface exposure mode provides a basic theoretical basis for the determination of the molecular mechanism and metabolic pathway of methanethiol acting on the human respiratory system.Our work is of great significance for the study of the respiratory toxicity of volatile organic sulfur compounds and the assessment of environmental health. |
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