Analysis Of Population Health Effects And Toxicity Effects In Vitro Due To Atmospheric NO₂ Pollution In Guangzhou

Background:Air pollution is a global public health problem.Over the past few decades,there has been an increasing focus on ambient air quality.The composition of air pollutants is complex and air pollution can cause long-term harm to people.People are exposed to air pollutants through the respiratory system as well as through the skin,which in turn leads to an increased risk of organism-related diseases.Studies have shown that high concentrations of air pollutants can lead to a high incidence of pulmonary and cardiovascular diseases in the region,affecting the health of the local population and increasing the burden of disease.NO₂,the current major air pollutant,is a precursor to particulate matter and ozone,and exposure at high concentrations can irritate the human respiratory tract and lead to the appearance of acid rain.NO₂pollution has become increasingly severe because of the high anthropogenic use of fuels and emissions in recent years.The 2022 update of the WHO Air Quality Database introduced for the first time ground-based measurements of annual average concentrations of NO₂,which showed that only 23%of the areas had annual average concentrations of NO₂inhaled by the population that met WHO air quality standards.Cities and regions that use gas and solid fuels for heating,as well as cities and regions with high traffic,have very high levels of NO₂concentrations.Understanding the state of air pollution is a prerequisite and basis for the distribution of medical resources and air pollution prevention and control.NO₂ and other air pollutants can lead to increased incidence of lung diseases and cancer mortality,NO₂can cause adverse health effects on the population as well as can increase the burden of diseases that bring about respiratory and cardiovascular systems,such as asthma and ischemic heart disease,and studies have shown that NO₂exposure is associated with the development of asthma in children and also has a synergistic effect with allergens.And current in vivo and in vitro studies on NO₂have shown that NO₂can alter the morphology of airborne Pseudomonas cells,that NO₂can increase respiratory inflammation in asthmatics,and that mouse models of asthma show prolonged allergen-induced airway reactivity and increased eosinophilic airway inflammation in the presence of NO₂,which can also lead to an inflammatory cellular response.In addition,rodents exposed to NO₂showed cardiovascular problems and an oxidative-antioxidative imbalance in the blood.Ferroptosis is a novel mode of programmed cell death,and recent studies have shown that ferroptosis is prevalent in various physiological and pathological phenomena,such as inflammation,immune response,and in the latest studies on cancer have also tasted the use of ferroptosis mechanisms to induce cancer cell death.Different oxidative and antioxidant pathways combine autophagy and membrane repair,leading to lipid peroxidation and plasma membrane damage during iron death.Initiation of ferroptosis requires inhibition of the antioxidant enzyme SLC7A11-GSH-GPX4 system and accumulation of free iron,lipid peroxidation occurs leading to increased hydrogen peroxide,and in a final step,lipid peroxidation or its secondary products directly or indirectly form pores in the plasma membrane or organelle membrane,ultimately triggering cellular ferroptosis.Objective:In this study,the population health effects of NO₂and in vitro experiments were combined to investigate the population health effects of NO₂on the respiratory system of Guangzhou city,from which the population health effects of NO₂and the lung damage of individuals were investigated.This will provide an experimental basis for the study of NO₂mechanisms,provide a scientific basis for the subsequent development of air quality standards and air pollution prevention and control,and serve as an indicator for the flow and distribution of medical resources in Guangzhou.Methods:Part 1:Analysis of the pollution status of NO₂and other air pollutants and health effects on the population in Guangzhou1.The daily average pollution concentration data of five air pollutants,meteorological data,outpatient clinic visit data and daily death data of residents in Guangzhou City from 2015 to 2019 were collected,and after quality control,the data were analyzed descriptively to explore the pollution characteristics of the five air pollutants separately.Correlation analysis of the five air pollutants and meteorological data was performed using spearman analysis.Stratified analysis of the cause of death data was performed using chi-square test.2.Joint analysis of air pollutant concentrations with outpatient and resident daily mortality data,respectively,was performed using a distributed lagged nonlinear model to explore the analysis of the effects of air pollutants and population health effects.Part 2:Analysis of the toxic effects of atmospheric pollutant NO₂on human lung bronchial epithelial cells1.To establish an in vitro acute NO₂toxicity model:Beas-2B cells were exposed to different concentrations of NO₂for 3 consecutive days using an air-liquid interface exposure system（ALI）for 0.5 h per day,and control cells were exposed to air.Cells were inoculated on 6-well Transwell plates into cell-exposed triplicate tubes with medium injected underneath the triplicate tubes so that the cells were exposed to the medium on one side and to the gas on the other.Different concentrations of NO₂were made by mixing 50 ppm NO₂and air at different flow rates.The cells were maintained at 37°C using a cell warming system.2.Cell viability and apoptosis assay:The CCK-8 method was used to detect the effect of NO₂exposure on the cell viability of Beas-2B cells.The effect of NO₂exposure on the apoptosis rate of Beas-2B cells was detected using an apoptosis kit to evaluate the effect of NO₂on cell growth status.3.Cellular oxidative damage assay:the effect of NO₂exposure on ROS and MDA content and cellular SOD activity of Beas-2B cells was detected using ROS,MDA and SOD kits to evaluate the oxidative damage produced by NO₂on cells;4.Cellular inflammatory response assay:q RT-PCR assay was used to detect the effect of NO₂exposure on the expression of IL-6,IL-8,TNF,IL-1A,IL-1B,CP-1 in Beas-2B cells;ELISA assay was used to detect the protein expression of cellular IL-6to evaluate the extent of cellular inflammatory response caused by NO₂.5.Cytogenotoxicity assay:The comet assay was used to detect the effect of NO₂on DNA damage in Beas-2B cells to explore the genotoxicity of NO₂.Part 3:Study on the mechanism of human lung bronchial epithelial cell damage caused by NO₂atmospheric pollutant1.Transcriptome sequencing analysis:Full-length transcriptome sequencing was used to study Beas-2B cells after NO₂exposure,followed by KEGG pathway enrichment analysis of differentially expressed genes in order to investigate the mechanism pathway of NO₂-induced damage in Beas-2B cells.2.Mitochondrial morphology observation:The effect of NO₂exposure on mitochondrial morphology in Beas-2B cells was observed using transmission electron microscopy.3.Cellular iron ion level assay:The effect of NO₂exposure on the iron ion content in Beas-2B cells was examined using an iron ion kit.4.Ferroptosis-related gene assay:q RT-PCR assay was used to verify the expression of ferroptosis-related genes in Beas-2B cells after NO₂exposure in order to analyze the molecular mechanism of ferroptosis caused by NO₂.Results:1.The annual average NO₂concentration in Guangzhou from 2015 to 2019 is 45μg/m³,which exceeds China’s air quality standard（40μg/m³）.2.NO₂can lead to an increased relative risk of respiratory visits with an RR of1.01（1.00,1.01）,mainly leading to an increased relative risk of visits for acute upper and lower respiratory tract infections,and the cumulative relative risk is enhanced as the lag days increase;SO₂,O₃PM_2.5,and PM₁₀all resulted in increased relative risks for respiratory visits.3.NO₂increased the cumulative relative risk of cancer death,and women were more sensitive than men,with RRs of 1.02（1.01,1.03）,1.02（1.01,1.03）,respectively;NO₂led to an increased cumulative relative risk of lung cancer death,and the effect was more significant in men,with RRs of 1.03（1.01,1.05）;SO₂,O₃PM_2.5,and PM₁₀increased the cumulative relative risk of cancer death and lung cancer death.4.NO₂caused a decrease in cell viability（P<0.05）and an increase in apoptosis（P<0.01）in Beas-2B cells.5.NO₂caused oxidative damage in Beas-2B cells with an increase in oxidation product reactive oxygen species（ROS）content（P<0.01）,malondialdehyde（MDA）content（P<0.05）and a decrease in antioxidant enzyme（SOD）activity（P<0.01）.6.NO₂induced an inflammatory response in Beas-2B cells with increased m RNA expression of inflammatory factors IL-6（P<0.05）,IL-8（P<0.05）,TNF（P<0.05）,IL-1A（P<0.05）,IL-1B（P<0.01）and inflammatory vesicle CP-1（P<0.01）,as well as an increase in IL-6 protein expression was increased（P<0.01）.7.NO₂leads to DNA damage in Beas-2B cells with significant increases in cell tail DNA%,tail length and Oliver tail moment（P<0.05）.8.KEGG pathway analysis showed that genes were mainly enriched in ferroptosis.9.NO₂causes smaller mitochondrial damage,increased mitochondrial membrane density,and reduced mitochondrial cristae in Beas-2B cells.10.NO₂exposure caused an increase in intracellular iron ion content in Beas-2B cells,suggesting the occurrence of iron ion aggregation（P<0.01）.11.NO₂caused an increase in the ferroptosis-related gene expression levels of STEAP3 and ACSL4 genes（P<0.05）and a decrease in the expression levels of FTH1,GPX4 and SLC7A11 genes（P<0.05）in Beas-2B cells.12.Knockdown of STEAP3 decreased ROS,MDA content and increased SOD activity in NO₂-exposed Beas-2B cells,suggesting that knockdown of STEAP3attenuated NO₂-induced oxidative damage in Beas-2B cells.13.Knockdown of STEAP3 reduced the iron ion content in NO₂-exposed Beas-2B cells,suggesting that knockdown of STEAP3 attenuated NO₂-induced ferroptosis in Beas-2B cells.Conclusions:1.The annual average NO₂concentration and 24-hour maximum daily average concentration in Guangzhou exceed the national air quality standards,suggesting that NO₂pollution is relatively serious in Guangzhou.2.Increased concentrations of NO₂and other air pollutants in Guangzhou can lead to increased risk of respiratory outpatient visits and increased risk of cancer and lung cancer deaths,suggesting that NO₂can cause health effects on Guangzhou residents.3.NO₂can cause oxidative damage,acute inflammatory response and DNA damage in human lung bronchial epithelial cells Beas-2B cells,suggesting that NO₂is cytotoxic.4.NO₂induced mitochondrial damage and iron ion accumulation in human lung bronchial epithelial Beas-2B cells,suggesting that NO₂-induced cytotoxicity may be associated with ferroptosis and STEAP3 may be involved in cellular ferroptosis.