Ultrafine carbon black (CB) nanoparticles constitute a major component of air pollution. These particles are capable of distribution from the lungs and accumulate in diverse tissues with complex toxicological affects upon cells and organ systems. Accumulated nanoparticles can activate elevated reactive oxygen species, stress signaling, and apoptosis. In this study, cell lines from varied organ systems were assessed for nanoparticle accumulation, and for stress signaling from the endoplasmic reticulum (ER), a key regulator of cellular apoptosis. In this study, ultrafine CB particulates were generated using sonication, and shown by atomic force microscopy to display sizes in a 20-50nm range. To examine the capacity for CB to accumulate and activate stress signaling from the endoplasmic reticulum, RAW264.7 mouse macrophages and SVEC4 mouse endothelial cells were used. The accumulation of CB was evaluated over a 48-hour period. Incorporated particulates were purified from cell lysates via centrifugation, and then measured with absorbance readings (A595). CB was hypothesized to enter all cells in a dose and time dependent manner. In RAW264.7 macrophages, particles accumulated gradually over time, with significant accumulation detected after 2 hours of exposure with CB doses of 50, 100, and 200 ug/ml with a peak absorbance reading of 0.9 Au/ 2x105 cells. In SVEC4 endothelial cells, particles gradually accumulated up to 24 hours before decreasing with intracellular carbon black incorporation peaking at 0.4 Au/ 2x105 cells; significant accumulation was detected at 24 hours of exposure with a dose of 100 ug/ml, and after 6 hours of exposure with a dose of 200 ug/ml. Stress signaling from the ER results in predictable changes in gene expression for several genes and pre-mRNA splicing of Xbp1. To assess ER signaling in the presence of CB, cells were treated for a 24-hour period. Assessment of Unfolded Protein Response (UPR) branch activation was determined using qRT-PCR to assess levels of gene expression in RAW and SVEC4 cells at low and high concentrations of carbon black. The level of ER stress was hypothesized to be dependent on cell type. Analysis revealed RAW264.7 cells to display higher levels of ER stress signaling than SVEC4 cells, consistent with the observed differences in nanoparticle accumulation in these cell lines. An additional measure of CB toxicity was provided through the assessment of ER and mitochondrial morphology. To label organelles, cells were transfected with plasmids carrying engineered fluorescent proteins targeted to the ER (red fluorescent protein) and the mitochondria (green fluorescent protein). SVEC4 endothelial cells were transfected and treated with carbon back for 24 hours. Fluorescent microscopy data revealed CB-induced changes in both organelles, altering morphology and distribution. These results demonstrate CB-induced cellular dysfunction and suggest that certain cell types may be more susceptible to air pollution.
