In Vitro And In Vivo Toxic Effects And Inflammatory Responses Induced By Carboxylated Black Carbon-Lead Complex Exposure

Objective:Black carbon（BC）is a key component of atmospheric fine particulate matter(PM_2.5)and it tends to adsorb various pollutants（e g,heavy metals and organics）during atmospheric transport.This adsorption leads to the complexity and uncertainty of the source and chemical composition of PM_2.5,making the toxicologic effects and health risks induced by PM_2.5 difficult to determine.Here,we used carboxylated black carbon,which was prepared by modifying the commercial black carbon.The original black carbon band increases the polarity of the carboxyl group,and then the carboxylated black carbon and lead combine to form a black carbon lead complex.We use these two materials to study the inflammatory and toxic effects caused by in vitro and in vivo.To clarify the toxic effects of carboxylated black carbon and carboxylated black carbon lead complex on cells,an experimental basis for evaluating the toxic effects of PM2.5 was proposed.Method:we used carboxylated black carbon（c-BC）and c-BC-lead complexes（c-BC-Pb）to investigate the in vitro and in vivo toxic effects and inflammatory responses.The physicochemical properties of c-BC and c-BC-Pb complexes were characterized by the transmission electron microscope（TEM）,X-ray photoelectron spectroscopy（XPS）,dynamic light scattering（DLS）,and in ductively coupled plasma–atomic emission spectra（ICP-AES）.In vitro experiments explored the combined toxicity model of carboxylated black carbon and carboxylated black carbon lead complex on BEAS-2B cells,which mainly examined the cell viability,oxidative damage level,apoptosis and inflammatory factor expression.The cytotoxicity test kit（CCK-8）was used to detect Cell viability assay of BEAS-2B cells exposed to c-BC-Pb and c-CB particles at different concentrations for 24 h.The DCFH-DA probe was used to examined the expression of ROS in BEAS-2B cells was induced by black carbon at different concentrations for 1,4,and 6 h;the carboxylated black carbon and carboxylated black carbon lead complex were detected by SOD and lactate deaminase kit（LDH）for 24 h.The effect of BEAS-2B cells on the antioxidant capacity and LDH leakage rate;ARP（aldehyde reaction probe）technology was used to quantitatively evaluate the DNA damage of cells exposed for 24h;AnnexinV/PI flow cytometry was used to detect the exposure of 24h.Apoptosis rate;inflammatory factors IL-6 and TNF-αwere detected by ELISA;the expression levels of inflammatory factors IL-6 and TNF-αwere detected by RT-PCR.In vivo experiments were conducted to study black carbon by exposing carboxylated black carbon and carboxylated black carbon lead complexes to ICR mice by tracheal instillation to study the lung toxicity induced by black carbon particles and the inflammatory response.We was counted by sorting the inflammatory cells in the lung lavage fluid of mice and the examined inflammatory factors IL-6,TNF-αand CRP were detected by ELISA using BALF and serum.We had the content protein measured by SOD,MDA in BALF to detect oxidative damage in the lungs of mice and to verify the inflammatory response of the carboxylated black carbon and carboxylated black carbon lead complex in mice.Result:The synthesis of carboxylated black carbon（c-BC）and carboxylated black carbon lead complex（c-BC-Pb）was confirmed by characterizing the modified black carbon and black carbon lead composite.TEM was used to further detect the cellular uptake of c-BC and c-BC-Pb particles.The results showed that the particles were taken up in the cells and dispersed in the cytoplasm but did not penetrate the nuclei.The results showed that cell viability of BEAS-2B cells was reduced by c-BC-Pb and c-BC particles in dose-dependent manners.The increase in LDH release is consistent with the CCK-8data and cytomorphological evidence,suggesting that c-BC-Pb inhibits cell viability.c-BC-Pb（25 and 50μg/mL）induced late apoptosis or necrosis,which was more pronounced than that in the c-BC-treated cells,suggesting that Pb plays an important role in c-BC-Pb induced apoptosis.with increasing c-BC-Pb and c-BC particles concentrations,c-BC-Pb and c-BC treatment significantly increased the secretion of IL-6in the cell culture supernatan.The presence of c-BC increases the gene expression of inflammatory cytokines（e.g.,IL-6and TNF-α）as the exposure time and dose increase.In vivo studies have showed a significant correlation between the IL-6 and CRP levels in BALF and in serum,suggesting that serum IL-6 and CRP levels may be affected by IL-6 and CRP levels in lung tissues.Cytotoxicity in vitro showed that the exposure of human bronchial epithelial cells（BEAS-2B）to low-dose c-BC-Pb particles significantly induced greater toxicity than that of c-BC,suggesting that lead（Pb）might play an important role in induced cytotoxicity after combined exposure to c-BC-Pb particles.The findings were further confirmed by the results in vivo,which indicated that c-BC-Pb particles significantly induced inflammation and lung injury.Based on the results of this experiment,the differences in toxicity can be attributed to the synergistic effect of Pb on the BC particles,which play a synergistic role in vitro and in vivo in the development of toxicity.Conclusion:Cytotoxicity in vitro showed that the exposure of human bronchial epithelial cells（BEAS-2B）to low-dose c-BC-Pb particles significantly induced greater toxicity than that of c-BC,suggesting that lead（Pb）might play an important role in induced cytotoxicity after combined exposure to c-BC-Pb particles.The findings were further confirmed by the results in vivo,which indicated that c-BC-Pb particles significantly induced inflammation and lung injury.Based on the results of this experiment,the differences in toxicity can be attributed to the synergistic effect of Pb on the BC particles,which play a synergistic role in vitro and in vivo in the development of toxicity.The c-BC-Pb particles model used in this study may be helpful for the evaluation of cytotoxicity induced by different sources of BC particles or BC-heavy metal complexes and provide a new approach for understanding PM_2.5 induced toxicity and health risks.