Study On The Mode And Mechanism Of BEAS-2B Toxicity Induced By Co-Exposure Of Low-Dose Carboxylated Black Carbon And Heavy Metal Lead

Objective: The purpose of this study was to investigate the combined toxic effects of low-dose carboxylated black carbon(Carboxylated black carbon)and lead acetate(Pb)on human bronchial epithelial cells(BEAS-2B).The bronchial epithelial cells were exposed to low-dose carboxylated BC(6.25μg/mL)and Pb(4μg/mL)alone or in combination.By observing cell viability,oxidative stress,DNA damage,changes in mitochondrial membrane potential,Joint toxicity indicators,such as apoptosis and cell inflammation,study the combined toxicity.Factor analysis can also be used to determine the potential interaction between carboxylated BC and Pb.Methods: The CCK-8 kit was used to detect the effect of carboxylated black carbon and heavy metal lead exposure for 24 hours on the survival rate of BEAS cells;using catalase,superoxide dismutase,glutathione peroxidase and malondialdehyde The kit detects cell membrane integrity and oxidative stress;DCFH-DA probe is used to detect the ROS level of cells after combined carboxylated black carbon and lead;single cell gel electrophoresis technology is used to detect the degree of cellular DNA damage;The JC-1 probe detects changes in the mitochondrial membrane potential in the cell,which is detected by the mitochondrial membrane potential(MMP)detection kit;and the flow cytometry is used to detect the apoptosis effect induced by black carbon and lead materials.Results: The cell viability induced by carboxylated BC(6.25μg/mL)showed no significant change compared with the control group.Similarly,at a dose of 4 μg/mL,there was no significant change in lead-induced cytotoxicity.Therefore,we chose carboxylated BC(6.25 μg/mL)and lead(4 μg/mL)to study their co-exposure effects and interactions.Compared with the control group,the LDH levels of BEAS-2B cells exposed to carboxylated BC alone or in combination with carboxylated BC and Pb were significantly increased,and the cells exposed to carboxylated BC and Pb had the highest LDH levels.Factor analysisshowed that the increase in LDH content was caused by the synergistic effect of carboxylated BC and Pb.Compared with the single treatment group,the co-exposure of carboxylated BC and Pb can significantly increase the MDA content and reduce the activity of GSH-Px and SOD.This indicates that the combined treatment group can enhance oxidative damage to a greater extent than the single treatment group.Similarly,there are more fluorescence-positive cells in bound or carboxylated BC.Therefore,the co-exposure of carboxylated BC and Pb can produce more intracellular ROS than the carboxylated BC or Pb alone treatment group.The results of cellular DNA damage showed that BEAS-2B cells exposed to carboxylated BC or Pb alone had no significant effect on DNA damage compared with the control group.The exposure of BEAS-2B cells to the combined effects of carboxylated BC and Pb can cause DNA damage,which is manifested by a significant increase in tail length,tail DNA content,olive tail distance and DNA damage rate.The results showed that the combined effect of carboxylated BC and Pb caused more serious DNA damage to BEAS-2B cells.To comprehensively observe apoptosis,flow cytometry was used to detect the apoptosis of carboxylated BC and/or Pb-treated groups in BEAS-2B cells after 24 hours of exposure.The results showed that compared with the control group,the apoptosis rate of BEAS-2B cells exposed to the carboxylated BC or Pb treatment group was significantly increased,while the apoptosis rate of the co-exposed group was significantly higher than that of the single exposure group.In order to reveal the possible mechanism of co-exposure of carboxylated BC and lead to induce apoptosis,mitochondrial membrane permeability(MMP)was detected after 24 hours of carboxylated BC(6.25 μg/mL)and/or Pb(4 μg/mL),The results show that,compared with a single component,carboxylated BC and Pb co-exposure can cause a significant increase in mitochondrial depolarization.In order to further explore the changes produced by apoptosis-related proteins,caspase-3,8,9 activities were detected with caspase activity detection kit,and caspase-3activity was found in BEAS-2B cells exposed to carboxylated BC or Pb alone Significantly enhanced.In addition,we also evaluated the correlation between oxidative stress and apoptosis markers in the co-exposed group.These results indicate that the generation of ROS is closely related to the apoptosis markers of caspase-3 and caspase-9.The BEAS-2B cellswere exposed to carboxylated BC and/or Pb environment for 24 hours,and the levels of inflammatory cytokines IL-6 and TNF-α were measured.The level of inflammatory cytokines in the co-exposed group was significantly higher than that in the carboxylated BC or Pb-treated group alone,suggesting that co-exposure of carboxylated BC and Pb can promote the secretion of inflammatory cytokines than single exposure.The synergistic effect of co-exposure of carboxylated BC and lead was observed in the factorial analysis of IL-6,while the additive effect was found in TNF-α.Conclusion: This study shows that combined exposure of low-dose carboxylated BC and Pb can cause increased oxidative stress,DNA damage,apoptosis,and inflammation in human bronchial epithelial cells.In addition,factorial analysis further proves that synergistic and additive interactions are responsible for the combined toxicity of carboxylated BC and Pb at low doses.Our data also shows that the co-exposure of carboxylated BC and Pb can cause some unexpected toxicity,even exceeding the toxicity of known low-dose single pollutants.This study provides a new perspective for the study of the interaction of atmospheric pollutants and provides a biological basis for human health risk assessment...