Oxidative Stress

The generation of free radicals by nanomaterials is well documented.97'98 In most cases, the studied material was of ambient or industrial origin (quartz, carbon black, metal fumes, and diesel exhaust particles). However, engineered nanomaterials, such as fullerenes and polystyrene nanoparticles, have been shown to generate oxidative stress as well.40,99,100 Lovric et al., for example, determined ROS to play an important role in cytotoxicity of quantum dots that have lost their protective coating.101 The unique surface chemistries, large surface area, and redox active or catalytic contaminants (e.g., metals, quinones) of nanoparticles can facilitate ROS generation.102 For example, fullerenes can perform electron transfer (phase-I pathway) or energy transfer (phase-II pathway) reactions with molecular oxygen following photoexcitation,44 resulting in the formation of the superoxide anion radical or singlet oxygen, respectively. The superoxide anion radical can then undergo further reactions, such as dismutation and Fenton chemistry, to generate additional ROS species (e.g., OH), resulting in cellular injury (see Scheme 7.1).103 Evidence of fullerene-induced oxidative stress includes lipid peroxidation in the brains of exposed fish and treated rat liver microsomes.40,104 Additional biomarkers of oxidative stress include a decrease in the reduced glutathione/oxidized glutathione ratio (GSH/GSSG), DNA fragmentation, and protein carbonyls.105

Biomarkers of nanoparticle-induced oxidative stress measured in our laboratory include ROS, lipid peroxidation products, and GSH/GSSG ratio. The fluorescent dichlorodihydroflourescein (DCFH) assay is used for measurement of ROS, such as hydrogen peroxide.106 DCFH-DA is a ROS probe that undergoes intracellular deacetylation, followed by ROS-mediated oxidation to a fluorescent species, with excitation 485 nm and emission 530 nm. DCFH-DA can be used to measure ROS generation in the cytoplasm and cellular organelles, such as the mitochondria. The

2GSH

(1) ROS Generation

GSSG + H2O

GSSG + H2O

Protein/DNA Damage

Lipid Peroxidation

SCHEME 7.1 (1) Photoexcited fullerenes can perform electron-transfer reactions with molecular dioxygen to form the superoxide anion radical (O2_). Superoxide can then undergo superoxide dismutase (SOD)-catalyzed dismutation to hydrogen peroxide (H2O2). H2O2 is a substrate for catalase (CAT)-and glutathione peroxidase (GSHPx)-catalyzed detoxification reactions. (2) The oxidation of glutathione (GSH) to form oxidized glutathione (GSSG) during detoxification of H2O2 can result in a loss of glutathione homeostasis. GSH can be regenerated by glutathione reductase (GR). (3) Alternatively, hydrogen peroxide can undergo transition metal (Fe++)-catalyzed Fenton chemistry to form the highly reactive hydroxyl radical (HO') that is capable of initiating lipid peroxidation and DNA/protein oxidation.

Fullerene

Lipid Peroxidation

Protein/DNA Damage

SCHEME 7.1 (1) Photoexcited fullerenes can perform electron-transfer reactions with molecular dioxygen to form the superoxide anion radical (O2_). Superoxide can then undergo superoxide dismutase (SOD)-catalyzed dismutation to hydrogen peroxide (H2O2). H2O2 is a substrate for catalase (CAT)-and glutathione peroxidase (GSHPx)-catalyzed detoxification reactions. (2) The oxidation of glutathione (GSH) to form oxidized glutathione (GSSG) during detoxification of H2O2 can result in a loss of glutathione homeostasis. GSH can be regenerated by glutathione reductase (GR). (3) Alternatively, hydrogen peroxide can undergo transition metal (Fe++)-catalyzed Fenton chemistry to form the highly reactive hydroxyl radical (HO') that is capable of initiating lipid peroxidation and DNA/protein oxidation.

thiobarbituric acid reactive substances (TBARS) assay is used for measurement of lipid peroxidation products, such as lipid hydroperoxides and aldehydes. A molondialdehyde (MDA) standard curve is used for quantitation. MDA, a lipid peroxidation product, combines with thiobarbituric acid in a 1:2 ratio to form a fluorescent adduct, that is measured at 521 nm (excitation) and 552 nm (emission). TBARS are expressed as MDA equivalents.107 The dithionitrobenzene (DTNB) assay is used for evaluation of glutathione homeostasis. In the DTNB assay, reduced GSH interacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to form the colored product 2-nitro-5-thiobenzoic acid, which is measured at 415 nm, and GSSG. GSSG is then reduced by glutathione reductase to form reduced GSH, which is again measured by the preceding method. Pretreatment with thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate, prevents GSH measurement, resulting in measurement of GSSG alone.

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