Copper Oxide Nanoparticles Induce Oxidative DNA Damage and Cell Death via Copper Ion-Mediated P38 MAPK Activation in Vascular Endothelial Cells

BACKGROUND: Inhaled nanoparticles can cross pulmonary air–blood barrier into circulation and cause vascular endothelial injury and progression of cardiovascular disease. However, the molecular mechanism underlying the vascular toxicity of copper oxide nanoparticles (CuONPs) remains unclear. We have recently demonstrated that the release of copper ions and the accumulation of superoxide anions contributed to CuONPs-induced cell death in human umbilical vein endothelial cells (HUVECs). Herein, we further demonstrate the mechanism underlying copper ions-induced cell death in HUVECs. METHODS AND RESULTS: CuONPs were suspended in culture medium and vigorously vortexed for several seconds before exposure. After treatment with CuONPs, HUVECs were collected, and cell function assays were conducted to elucidate cellular processes including cell viability, oxidative stress, DNA damage and cell signaling pathways. We demonstrated that CuONPs uptake induced DNA damage in HUVECs as evidenced by γH2AX foci formation and increased phosphorylation levels of ATR, ATM, p53 and H2AX. Meanwhile, we showed that CuONPs exposure induced oxidative stress, indicated by the increase of cellular levels of superoxide anions, the upregulation of protein levels of heme oxygenase-1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), the elevation of the levels of malondialdehyde (MDA), but the reduction of glutathione to glutathione disulfide ratio. We also found that antioxidant N-acetyl-L-cysteine (NAC) could ameliorate CuONPs-induced oxidative stress and cell death. Interestingly, we demonstrated that p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated in CuONPs-treated HUVECs, while p38α MAPK knockdown by siRNA significantly rescued HUVECs from CuONPs-induced DNA damage and cell death. Importantly, we showed that copper ions chelator tetrathiomolybdate (TTM) could alleviate CuONPs-induced oxidative stress, DNA damage, p38 MAPK pathway activation and cell death in HUVECs. CONCLUSION: We demonstrated that CuONPs induced oxidative DNA damage and cell death via copper ions-mediated p38 MAPK activation in HUVECs, suggesting that the release of copper ions was the upstream activator for CuONPs-induced vascular endothelial toxicity, and the copper ions chelator TTM can alleviate CuONPs-associated cardiovascular disease.