Toxic Effects Of Black Carbon-lead Compounds On Human Alveolar Type Ⅱ Epithelial Cells (A549) And Lung Injury In Mice

PM_2.5.5 has become a major cause of China’s large-scale,high-intensity and persistent“haze”weather.Black Carbon（BC）is a carbon-containing mixture produced by the incomplete combustion of biomass or fossil fuels.Black carbon is one of the main components of atmospheric fine particles(PM_2.5).The emission of black carbon particles in China accounts for about one-quarter of the world’s total,and the total amount of black carbon emissions in mainland China is about 181.1×10⁴t.Black carbon particles form airborne fine particles with a particle size of less than2.5μm and enter the alveoli almost all with breathing,which has a great impact on human health and atmospheric environmental quality.Black carbon in the gas-particle transport process can strongly adsorb toxic chemicals and form secondary black carbon particles in a complex heterogeneous photochemical reaction process,ie black carbon-pollutant Composites,BC-PCs),BC-PCs enter the body’s path and its target of action is complex,its local/systemic single and/or joint toxic effects and its mechanism of action are unclear.Therefore,black carbon is one of the important components of atmospheric fine particulates,and its toxicological effects are similar to those of toxicology of PM_2.5,and there are also obvious differences.In this study,commercial black carbon was used as the experimental material and compounded with typical lead heavy metal lead to prepare black carbon-lead composites.Exposure to human alveolar type II epithelial cells（A549）and lungs of Kunming white mice was performed using black carbon and black carbon-lead complexes.To explore the causes of lung injury in A549 cells and mice induced by oxidative stress and inflammatory injury,and to compare the toxic effects of black carbon and black carbon-lead complexes.Black carbon C824455 was purchased for the high-temperature modification of strong acid and strong oxidants,and the modified black carbon was used as a composite heavy metal lead to prepare black carbon and lead composites.The cells were exposed directly to A549 cells with carbon black and a complex of carbon black and lead at a concentration of 40μg/mL.The cytotoxicity was measured at 24 h,48 h,and 72 h by MTT assay;the lactate dehydrogenase in the cell supernatant was measured.（LDH）activity,intracellular superoxide dismutase（SOD）,catalase（CAT）,glutathione catalase（GSH-Px）,malondialdehyde（MDA）activity and total protein content The content of reactive oxygen species（ROS）produced by the cells was detected by fluorescence staining,and apoptosis was detected by flow cytometry using Annexin V-FITC/PI double staining.For animals,30 6-week-old female Kunming white mice were weighed（25.0±1.3）g and randomly divided into 3 groups with 10 animals in each group.Carbon black and a complex of carbon black and lead（Pb nitrate:carbon black,250 mg/kg）were instilled into the mice with a phosphate buffer solution to inhale mice at a dose of 4 mg/kg body weight.The injection volume was 50 uL.The blank group was treated with phosphate buffer and exposed once.The mice were sacrificed 72 hours later.Pathological sections of the lungs of mice and the physiological and biochemical indicators of lung lavage fluid were detected.The lesions were divided into lactate dehydrogenase（LDH）and malondialdehyde（MDA）levels and interleukin-8（a marker of inflammation）.IL-8),tumor necrosis factor-alpha（TNF-alpha）content,and lung enzyme activity superoxide dismutase（SOD）,alkaline phosphatase（AKP）,acid phosphatase（ACP）.The relative survival rate of A549 cells was decreased in the treatment group after 24 hours exposure,and the black carbon and lead complexes were the lowest.The trends were consistent at 48 hours and 72 hours and 24 hours.The LDH and MDA in the treatment group were significantly higher than those in the control group（P<0.05）,of which the black carbon and lead complexes were the highest;the CAT activity,SOD activity and GSH-Px content in the treated group were significantly lower than the control group（P<0.01）,and the black carbon and lead complexes were the lowest;the active oxygen ROS carbon black treatment In the group higher than the control group,the black carbon and lead complex treatment group was the highest;the black carbon treatment group apoptosis rate was 2.73 times that of the blank group,and the black carbon and lead complex treatment group apoptosis rate was 4 times that of the control group.Through pathological observation and analysis,the inflammatory lesions appeared in the lungs of the poisoned group.LDH and MDA were significantly higher in the exposed group than in the control group（P<0.01）;IL-8 levels were higher in the control group（P<0.05）;TNF-αlevels were significantly higher than in the control group（P<0.05）;The enzyme activities（SOD,AKP,ACP）were also higher than those of the control group.Both black carbon and lead complexes and black carbon cause toxicity to A549cells due to oxidative damage,leading to a decrease in intracellular antioxidant enzymes and induction of higher apoptosis rate.Black carbon and lead complexes are higher than black carbon There is more toxic damage to A549 cells.Exposure of mouse lungs to short-term high doses of black carbon and black carbon and lead complexes causes acute inflammatory lesions.Black carbon and lead compounds are more toxic than black carbon.