A new perspective on calmodulin-regulated calcium and ROS homeostasis upon carbon black nanoparticle exposure

Toxicological studies propose that exposure to carbon black nanoparticles induces organ injuries and inflammatory responses. Besides, current understanding of the molecular mechanisms implies that carbon black nanoparticles (CBNP) exposure induces the production of reactive oxygen species (ROS) causing inflammation, mitochondrial dysfunction or disturbance in calcium homeostasis. However, the precise mechanisms whereby CBNP exert these effects in the lung are still not fully understood. To gain insight into the possible mechanism of CBNP exerted toxicity, human alveolar epithelial cells (A549) were exposed to different concentrations of CBNP and for different timepoints. The reaction of the cells was monitored by the systematic use of cell-based measurements of calcium and ROS, in the presence and absence of calcium (Ca²⁺) pump inhibitors/chelators and antioxidants. Followed by an in-depth PCR analysis of 84 oxidative stress-related genes. The measurements revealed, as compared to the control, that exposure to CBNP nanoparticles leads to the generation of high ROS levels, as well as a disturbance in calcium homeostasis, which remained primarily unchanged even after 24 h of exposure. Nevertheless, in presence of antioxidants N-acetylcysteine (NAC) and Trolox, ROS formation was considerably reduced without affecting the intracellular calcium concentration. On the other hand, Ca²⁺ pump inhibitors/chelators, BAPTA (1,2-bis(o-amino phenoxy)ethane-N, N, N', N'-tetraacetic acid) and verapamil not only decreased the Ca²⁺ overload, but also further decreased the ROS formation, indicating its role in CBNP-induced oxidative stress. Further, a PCR array analysis of A549 cells in presence and absence of the calmodulin (CaM) antagonist W7, indicated toward nine altered oxidative stress-related genes which further confirmed our cytotoxicity results. Obtained data suggested that CBNP exposure elevates calcium ion concentration, which further contributes to oxidative stress, via the calcium-binding protein CaM. Its inhibition with W7 leads to downregulation in gene expression of nine oxidative stress-related genes, which otherwise, as compared to control, show increased gene expression. The results of the study thus confirm that exposure of lung epithelial cells to CBNP leads to oxidative stress; however, the oxidative stress itself is a result of a disturbance in both calcium and ROS homeostasis, and should be considered while searching for a new strategy for prevention of CBNP-induced lung toxicity.