Ozone aging and protein corona adsorption exacerbate inflammatory effects of carbon black on macrophages and induce blood–testis barrier dysfunction in mice

Abstract

Carbon black (CB) is a man-made, pure carbon particle with numerous applications in a variety of commercial and consumer products. Upon inhalation, it may bioaccumulate in various organs, raising serious health concerns. However, biotransformation processes that CB undergoes can alter its chemical and physical properties, thereby affecting its toxicities. When airborne CB is exposed to UV radiation, it undergoes an aging process. Upon entering physiological environments, biomacromolecules, such as proteins, rapidly adsorb onto CB's surface, forming a protein corona that mediates cellular interactions. Our study reveals that ozone aging influences the adsorption of CB in mouse plasma. Exposure to pristine CB and ozone-aged carbon black (CB-O₃) triggers inflammatory responses in J774A. 1 macrophage cell lines and activates the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. Notably, ozone aging and plasma protein corona adsorption enhance CB uptake by J774A. 1 cells, thereby increasing its cytotoxicity. Mechanistically, CB and CB-O₃ exposure induce lysosomal damage and dysfunction, leading to cathepsin B release and activating the NLRP3 inflammasome. Importantly, this activation correlates with a reduction in blood–testis barrier-associated protein expression. In vivo experiments confirm that prolonged exposure to CB and CB-O₃ activates the NLRP3 inflammasome within the testes, leading to a significant compromise of the blood–testis barrier integrity in mice. These findings highlight the importance of considering environmental aging processes and protein corona formation in toxicological evaluations, offering a more comprehensive framework for regulatory policies and protective measures aimed at mitigating the adverse health impacts of CB exposure. Additionally, this study underscores the potential for targeting the NLRP3 inflammasome pathway in therapeutic strategies to alleviate CB-induced inflammatory and reproductive toxicity.