Toxic Effects Induced By Exposure To Low-dose Carboxylated Black Carbon-lead Complex In ICR Mice

Objective:Black carbon is one of the major components of PM_2.5.Due to its special properties,Black carbon can adsorb many toxic and hazardous substances during long-distance atmospheric transportation,and form a black carbon-pollutant complex after a series of complex changes.The toxic effects and health risks caused by black carbon-pollutant complexes（such as black carbon-heavy metal complex particles）are not clear,because of the diverse sources of PM_2.5 and the relatively complex chemical components.Therefore,in order to further evaluate the in vivo toxicity effect induced by black carbon-heavy metal composite particles,this study simulated the chemical reaction process of black carbon particles adsorbing heavy metals in the atmosphere during gas-particle conversion,and artificially prepared a carboxylated black carbon-lead composite,ICR mice were exposed to low-dose medium and long-term exposure to explore the systemic effects and pathological characteristics of carboxylated black carbon-lead complex particles induced by multiple target organs.It provides a theoretical basis for further revealing the mechanism of carboxylated black carbon-lead complex particles-induced toxicity in vivo.Method:We used the carboxylated black carbon-lead complex particles prepared by our research group in the previous study,and compared it with commercial black carbon and carboxylated black carbon.The three kinds of black carbon materials are characterized by transmission electron microscope（TEM）,inductively coupled plasma atomic emission spectroscopy（ICP-AES）,x-ray photoelectron spectroscopy（XPS）and other chemical analysis techniques.Low-dose（2mg/kg,body weight）particles of black carbon,carboxylated black carbon and carboxylated black carbon-lead complex were used for medium and long-term exposure.The method of exposure is nasal drip.We used malondialdehyde,albumin,superoxide dismutase and other kits to detect the oxidative damage of the lung tissues of mice,and counted the inflammatory cells in the alveolar lavage fluid of mice and the rate of mouse epididymal sperm deformity.The content of inflammatory factors such as tumor necrosis factor-α,interleukin-6,interleukin-8,C-reactive protein in BALF and serum,testosterone levels in serum of mice were detected by ELISA.The H&E staining method was used to examine the pathological damage characteristics of the three black carbon materials on the target organs（lung,liver,kidney,and testis）.Result:The characterization results of three black carbon materials showed that the carboxylated black carbon after the modification of commercial black carbon was successfully prepared and characterized.The study showed that the body weight of the ICR mice was significantly lower than that of the control group at different exposure time points,and the body weight growth rate of the mice was also lower than that of the control group（P<0.05）.The detection of oxidative damage index in mouse alveolar perfusion fluid showed that carboxylated black carbon-lead complex particles compared with the control group could significantly induce oxidative damage in alveoli,and the oxidative damage effect induced by carboxylated black carbon-lead complex particles was significantly higher than that of carboxylated black carbon particles.The detection of inflammatory cells in the alveolar lavage fluid in mice also showed that the number of inflammatory cells in the alveolar lavage fluid increased significantly after the exposure of the three particles,especially in the carboxylated black carbon-lead complex treatment group after 56 days of exposure.The detection of inflammatory cytokines in BALF of mice also found that the levels of inflammatory cytokines（IL-6,IL-8,TNF-α）in mice after exposure to the three particles showed a time-dependent effect,and the carboxylated black carbon-lead complex treatment group was the most significant（P<0.05）.The variation trend of levels of inflammatory factors in peripheral blood was consistent with that in BALF,and both of which were significantly higher in the carboxylated black carbon-lead complex treatment group than in the carboxylated black carbon treatment group（P<0.05）.Meanwhile,BALF induced by carboxylated black carbon-lead complex was significantly correlated with Serum IL-6 and CRP content,which showed that IL-6 and CRP in alveoli entered the blood circulation system to induce an obvious systemic inflammatory response.In addition,the testosterone level of carboxylated black carbon-lead complex particles significantly decreased after short-term exposure（P<0.05）,while the testosterone level of black carbon and carboxylated black carbon particles only reached a significant level after long-term exposure（P<0.05）.Sperm density decreased significantly after short-term exposure of the three particles,but it increased when carboxylated black carbon-lead complex was exposed for more than 28 days.Carboxylated black carbon and carboxylated black carbon-lead complex particles were significantly induced sperm malformation at 14d and 56d,but there was no significant difference between carboxylated black carbon particles and control after exposed at 28d.The pathological results of H&E showed that long-term exposure of three kinds of particles（56 days）could induce lung injury,especially in the carboxylated black carbon-lead complex group.In addition,long-term exposure to carboxylated black carbon-lead complex treatment group can also induce a certain degree of inflammation and pathological damage in the liver,kidney and testes.Conclusion:Low dose and long-term exposure in mice showed that carboxylated black carbon-lead complex particles had a negative effect on the weight gain of mice,and carboxylated black carbon-lead complex particles could significantly induce the inflammatory response and oxidative damage in the lungs.In addition,carboxylated black carbon-lead complex particles can induce systemic toxic effects of the body through blood circulation,leading to inflammatory reactions and pathological damage in the liver,kidney and testes.The carboxylated black carbon-lead complex particles can induce the reproductive system toxicity,in the short term and long term exposure caused by testosterone levels dropped significantly,while the black carbon and carboxylated black carbon particles only after long time exposure,testosterone levels and contrast difference reached significant level.Simultaneously,the carboxylated black carbon-lead complex particles exposure to 28 d,sperm density had a rising trend and at the same time the sperm deformity and the control group had no significant difference.This study indicated that the toxic effect induced by carboxylated black carbon-lead complex particles in vivo may be the result of the synergistic effect of black carbon particles and Pb,especially the reproductive toxicity effect in mice is worthy of further study.