Apoptosis and Mitochondrial Dysfunction

Nanoparticle-induced cell death can occur by either necrosis or apoptosis, processes that can be distinguished both morphologically and biochemically. Morphologically, apoptosis is characterized by perinuclear partitioning of condensed chromatin and budding of the cell membrane to form apoptotic bodies, whereas necrosis is characterized by cellular swelling (oncosis) and bleb-bing of the cell membrane.109 In vitro studies have demonstrated the ability of nanoparticles, such as dendrimers and carbon nanotubes, to induce apoptosis.110-112 In vitro exposure of macrophage-like mouse RAW 264.7 cells to cationic dendrimers led to apoptosis confirmed by morphological observation and the evidence of DNA cleavage.112 Pretreatment of cells with a general caspase inhibitor (zVAD-fmk) reduced the apoptotic effect of the cationic dendrimer. Apoptosis has also been observed in cultured human embryonic kidney cells (HEK293) and T lymphocytes treated with single walled carbon nanotubes, and in MCF-7 breast cancer cells treated with quantum dots.101,110,113

Apoptosis in mammalian cells can be initiated by four potential pathways: (1) mitochondrial pathway, (2) Death receptor-mediated pathway, (3) ER-mediated pathway, and (4) Granzyme B-mediated pathway.114 Our laboratory has focused on caspase-3 activation in liver and kidney cells as a biomarker of apoptosis, since this a downstream event in all the classical apoptotic signaling pathways and can be measured using a fluorometric protease assay. This assay quantifies caspase-3 activation in vitro by measuring the cleavage of DEVD-7-amino-4-trifluoromethyl coumarin (AFC) to free AFC that emits a yellow-green fluorescence (1max = 505 nm).115 This initial apoptosis screen can then be followed by additional analysis, as cellular morphology studies using nuclear staining techniques to detect perinuclear chromatin, or agarose gel electro-phoresis to detect DNA laddering.116

Evidence supports a role for ROS in generation of the mitochondrial permeability transition via oxidation of thiol components of the permeability transition pore complex.117 As discussed in the preceding sections, nanoparticles have been shown to induce oxidative stress, and thus this ROSmediated pathway for induction of the mitochondrial permeability transition is a plausible apoptotic mechanism for nanomaterials. For instance, ambient ultrafine particulates have been shown to translocate to the mitochondria of RAW 264.7 murine macrophage cells, cause structural damage, and altered mitochondrial permeability.98 A subsequent study demonstrated that mito-chondrial dysfunction and apoptosis in the RAW 264.7 cells could be induced by polar compounds fractionated from ultrafine particles, suggesting that the mitochondrial dysfunction caused by ultrafines was the result of redox cycling of quinone contaminants on the surface of the particle.118 This link between oxidative stress, mitochondrial dysfunction, and apoptosis has also been observed for man-made nanoparticles. For example, metal and quantum dot engineered nanopar-ticles have both been shown to induce oxidative stress, mitochondrial dysfunction, and apoptosis in various in vitro models.101,119 Water-soluble, derivatized fullerenes, which have been shown to accumulate in the mitochondria of HS 68 human fibroblast cells, have also been shown to induce apoptosis in U251 human glioma cells.120,121 While this derivatized fullerene-induced apoptosis in the glioma cell line did not involve oxidative stress, mitochondrial dysfunction was not measured and cannot be ruled out. Mitochondrial dysfunction and apoptosis have also been observed in a human gastric carcinoma cell line exposed to chitosan nanoparticles.122 Taken together, these observations support a role for mitochondrial dysfunction and oxidative stress in nanoparticle-induced apoptosis. Apart from apoptosis, mitochondrial dysfunction has long been associated with necrotic cell death, and represents a potential necrotic mechanism of nanoparticle-induced injury as well.123

Mitochondrial dysfunction can result from several mechanisms in addition to opening of the permeability transition pore complex, including uncoupling of oxidative phosphorylation, damage to mitochondrial DNA, disruption of the electron transport chain, and inhibition of fatty acid b-oxidation.124 Methods used to detect mitochondrial dysfunction include measurement of ATPase activity (via luciferin-luciferase reaction), oxygen consumption (via polarographic technique), morphology (via electron microscopy), and membrane potential (via fluorescent probe analysis).125 Our laboratory measured loss of mitochondrial membrane potential in rat hepatic primaries, and Hep-G2 and LLC-PK1 cell lines, using the 5,5',6,6'-tetrachloro-1,1 ',3,3'-tetraethyl-benzimidazolcarbocyanine iodide (JC-1) assay, which is a convenient assay that does not require mitochondrial isolation or use specialized equipment.126 This fluorescent dye partitions into the mitochondrial matrix as a result of the membrane potential. Concentration of JC-1 in the matrix results in aggregation that fluoresces at 590 nm (red). Upon loss of membrane potential, the dye dissipates from the matrix and can be measured, in its monomer state at emission 527 nm (green). The proportion of green to red fluorescence reflects the degree of mitochondrial membrane depolarization.

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