Abstract

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.

Highlights

  • Scientific and industrial attainments within the last few years have led to discoveries in nanotechnology which were far beyond the imagination of mankind half a century ago (Jeevanandam et al 2018)

  • The results of the present study provide an evidence that the disturbance in calcium homeostasis upon carbon black nanoparticles (CBNP) exposure in lung epithelial cells plays a pivotal role in generation of reactive oxygen species, and over all toxicity caused by CBNP exposure

  • To investigate further an underlying mechanism for the interaction of calcium and reactive oxygen species (ROS) in the mitochondria, we have focused on the role of calmodulin that is known to contribute to cellular dysfunction by promoting defective intracellular ­Ca2+ handling, including mitochondrial ­Ca2+ overload

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Summary

Introduction

Scientific and industrial attainments within the last few years have led to discoveries in nanotechnology which were far beyond the imagination of mankind half a century ago (Jeevanandam et al 2018). Scientists are still discovering the unique properties of daily used materials at exposure to high concentrations of nanoparticles is known to impair lung clearance by macrophages. The overloading of the lung can initiate a severe inflammatory response, which leads to downstream events like lung fibrosis, and upstream events such as oxidative stress (Gustafson et al 2015). Oxidative stress is one of the most known and reported toxicities which the cells encounter when exposed to nanoparticles (Niranjan and Thakur 2017). High ROS levels generated are known further to cause severe cell damage which leads to cell death. Often this switch is mediated by calcium signaling (Huang et al 2017). Increasing evidence suggests that interactions between calcium and ROS are necessary for signaling and proper functioning of cellular signaling networks (Görlach et al 2015; Hempel and Trebak 2017; Yan et al 2006)

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