Carbon Black Exposure Induces Alterations in Mitochondrial Morphology in Human Lung Cells: A Software-Based Quantitative Analysis

Nanoparticulates of pure carbon, carbon black (CB), are a common atmospheric pollutant in industrialized and heavily populated areas. They are produced primarily via combustion of fossil fuels, and represent a significant health hazard. They are known to worsen asthma and bronchitis when inhaled and to cause inflammation, heart dysfunction, and oxidative stress when incorporated into other organs. The key focal point of this work was to examine markers of stress signaling and cellular dysfunction when human bronchial epithelial cells (16HBE14o-) were exposed to CB particles ranging in size from 70 nm and averaging 130 nm in diameter. BrdU incorporation and DAPI staining studies revealed a 24-hour CB exposure (25 ug/ml and higher) to reduce rates of cell division and to significantly elevate percentages of cells exhibiting apoptotic nuclear morphology. Chronic exposure (24 days) of cells to low doses of CB (5 ug/ml) revealed a significant impact upon both cell division and survival. A central marker for stress signaling in these lung cells was elevation of reactive oxygen species, which rose within 12 hours of CB exposure, consistent with stress signals that induce mitochondrial apoptosis. Sirtuin 1, a stress regulated protein deacetylase in the cytosol, whose levels are known to be destabilized by ROS elevation in stressed cells, displayed no significant change following CB exposure at varied doses. Analysis of mitochondrial dynamics via fluorescence microscopy revealed clear changes in organization and morphology. HBE cells were treated with varying doses of CB, fixed, and analyzed via immunocytochemistry. Mitochondria were labelled using a Tom20 antibody conjugate (Alexa488), while microtubules were assessed with phalloidin (Alexa594). Using software developed in MatLab, mitochondria were analyzed for changes in mitochondrial size and localization. Significant changes were identified with regard to an increase in mitochondrial size, and strong trends were observed in an increased localization preference for the perinuclear region. A discussion of the link between elevated ROS levels and mitochondrial behavior will be discussed