The culprits of superoxide dismutase inactivation under size-dependent stress of ultrafine carbon black: Superoxide anion, genotoxicity and protein corona

Abstract

As a critical component of atmospheric ultrafine particulates, ultrafine carbon black (UFCB) brings great exposure risk to organisms. At present, the action pathway and activity regulation mechanism of UFCB on functional proteins in vivo are not clear, and the size-dependent effects of UFCB during this process need to be elucidated. Superoxide dismutase (SOD), one of the most applied biomarkers to assess the environmental impact of pollutants, plays crucial roles in resistance to oxidative stress. Here, based on the inactivation of SOD (84.79 %, 86.81 % and 91.70 %) in primary mouse hepatocytes exposed to UFCB (13 nm, 50 nm and 95 nm), oxidative stress, genotoxicity and protein molecular studies were employed to elucidate the inactivation mechanisms. Results showed that inhibition of UFCB-mediated superoxide anion (O2−) contributed to a decrease in SOD activity. Furthermore, the significant increase in 8-hydroxy-2-deoxyguanosine content and the comet tail formation indicated the occurrence of DNA damage, supporting that concomitant aberrant transcriptional and protein translational under gene regulation should be responsible for SOD inactivation. At the molecular level, the constricted backbone, reduced content of α-helix and fluorescence sensitization all demonstrated that the attachment-type binding of SOD on UFCB to form the ‘protein corona’ disrupted protein structure. Enzyme activity assays indicated that SOD backbone tightening and helix decay resulted in decreased activity, which should be another reason for intracellular SOD inactivation. More importantly, the particle sizes of UFCB exert powerful influences on SOD inactivation mechanisms. Smaller UFCB (13 nm) induced more severe O2− inhibition and DNA damage, while UFCB50nm with the best dispersity bound more SOD and induced stronger molecular toxicity, which are their different strengths in stressing SOD inactivation in hepatocytes. Our findings provide novel insights for exploring functional proteins activity and underscore a potentially size-dependent risk of nanoparticles.