Cellular Toxicity of C60 Fullerenes in RAW 264.7 Immortalized Macrophages.

Abstract

Cellular Toxicity of C60 Fullerenes in RAW 264.7 Immortalized Macrophages

C60 fullerenes are nanoparticles (~1nm dia.) composed of 60 carbons in a spherical graphene structure. They are hydrophobic, aggregate in aqueous solution and have been associated with cellular toxicity in multiple cell types. The central hypothesis of this thesis is that exposure of peripheral immune cells to C60 fullerenes will result in their uptake into the cytosol, membrane-bound intracellular compartments and cell membrane(s) resulting in cellular stress and subsequent activation of apoptosis. Electron-dense terbium endohedral fullerenes were an excellent model for visualizing C60 in cells. Transmission electron microscopy on RAW 264.7 immortalized murine macrophages showed that C60 was found in endosomes, embedded within lipid membranes and free in the cytosol and nucleoplasm. Endocytosis was found to be mediated by both caveolin and clathrin. Cell death assays determined that at 24 hours there was no statistically significant occurrence of early or late apoptosis or necrosis. Cells exhibited subtle alterations in cytokine and chemokine gene expression profiles. Examination of the glutathione synthesis pathway showed changes consistent with a mild form of oxidative stress. Cellular proliferation decreased upon exposure to 1 and 4 μg/mL fullerenes and metabolic studies showed alterations in nucleotide catabolism and lipid metabolism.

These studies show that exposure of immune cells to C60 fullerenes results in uptake of the nanoparticles and alterations in the normal functions of the cell. The changes in the synthesis of glutathione, nucleotide catabolism, and lipid synthesis do not result in increased cell death at 24 hours; however, alterations to cellular proliferation and mildly altered inflammatory signals suggest that the cell is in the process of trying to resolve damage inflicted by fullerenes. Moreover, at 24 hours, the cells do not show clear signs of mechanisms leading to survival, proliferation or death.