Study On Combined Toxicity Mode And Potential Mechanism Of NPCB And Heavy Metals To Beas-2B Cells

Objective:As one of the major environmental threat to the national health, Particulate Matter (PM) is becoming greatly concern due to its health damage and potential mechanism. In the previous studies, the composition of PM shows great variation among different seasons and regions, which reduces the comparability of the results and raises the difficulties in thoroughly understanding the toxic effects and potential mechanisms of PM. So far most toxic effects studies for PM are mainly focused on the study of integrated PM or on the individual component, while the possible interactions of combined exposure to main components of PM have not been investigated systematically yet. Considering the important theoretical and application values of comparing the characteristics of PM toxic effect and variations among different particulates, we choose the typical components of PM to make a combined toxicity study. In this study we investigated the mode and mechanism of combined effects of nano-particle carbon black (NPCB) and heavy metals (Pb, Cr, Cd) to BEAS-2B cells, aiming to clarify the main model and mechanism of the combined effects of PM and to provide basic experimental data for risk assessment.Methods:In the first part of this study, we discussed the combined toxic effects of NPCB and heavy metals (Pb/Cr/Cd) to BEAS-2B cells, including cytotoxicity, oxidative damage, cell cycle and apoptosis. After co-exposure of NPCB and heavy metals (Pb, Cd or Cr) for 24h to BEAS-2B cells, cell viability and LDH leakage were detected with Cell Counting Kit-8(CCK-8) and LDH Cytotoxicity Assay Kit; ROS were detected by redox-sensitive fluorescent probe DCFH-DA after combined exposure 3h to BEAS-2B cells; In order to evaluate the antioxidant defense system, the total antioxidant capacity assay kit and SOD activity assay kit are used. The extent of DNA damage were analyzed with Single cell gel electrophoresis (SGGE); use Flow cytometry (PI staining) to detect the cell cycle and Annexin V/PI apoptosis detection kit to detect cell apoptosis after co-exposure. In The second part of this study we evaluated DNA damage pathways in order to make a primary exploration of DNA damage mechanisms of co-exposure to NPCB and heavy metals. Using Western Blot method to test the DNA damage repair-related proteins ATM, p-p53/p53 and yH2AX expression of BEAS-2B cells after co-exposure to NPCB and heavy metals.Results and discussion:The results showed that NPCB and heavy metals (Pb, Cr or Cd) had combined effects on cell viability and LDH leakage, yet the modes of combined effects varies from different metals, doses and toxic endpoints. The combination of low dosage Pb and NPCB shows no interaction in cell viability, but shows antagonism in LDH leakage; while high dosage of Pb and NPCB shows synergistic effect in viability, yet no obvious interaction in LDH leakage. Both the combination of Cd or Cr with NPCB show synergistic effect in cell viability, and low dose of Cd or Cr with NPCB has no synergistic effect while high dose of the two with NPCB shows synergistic.ROS fluorescence intensity of for combined exposure of NPCB with three metals showed that combined exposure to greatly promote ROS generation; results of total antioxidant capacity and SOD activity test showed combined effects in total antioxidant capacity and SOD activity. The combination of Pb or Cr with NPCB show synergistic effect, in total antioxidant capacity, while Cd with NPCB shows no such effect; the combination of Cd or Cr with NPCB show synergistic effect in SOD activity while Pb with NPCB shows no such effect. Co-exposure of three heavy metals and NPCB significantly increased the extent of DNA damage and the combined effect is synergistic mode; as to the combined effects of cell cycle and apoptosis, none of the three heavy metals combined with NPCB shows obvious synergistic effect.The Western Blotting results show that co-exposure of NPCB with either of the three heavy metals can influence cell DNA damage repair pathway, with the expression of p-ATM, p-p53/p53,yH2AX changed, when comparing to the control or single metal exposure group. The results also suggest that ATM-p53-H2AX DNA repair pathway may play an important role in the combined exposure to NPCB and heavy metals, which of course needs further exploration.Conclusion:Combined exposure of NPCB with heavy metals to BEAS-2B cells had synergistic effects, which can induce a significant production of ROS, reduce cell antioxidant defense ability, lead to cellular oxidative damage and even cause DNA damage. Combination mode analysis results of NPCB with different metals vary among different metals, dosage and assessment parameters. DNA damage repairing pathways plays an important role in co-exposure caused cell DNA damage, although the specific mechanisms need to be further tapped.