Aging Aging

Detection of Molecular Events 73have been described that allow the quantitative analysis of DNA fragmentationas well as the parallel measurement of other parameters such as cell cycle andantigen expression (10,11). The most commonly used of these methods is theTUNEL assay.The TUNEL assay is based on the addition of biotinylated dUTP nucleotideto 3' hydroxyl termini at DNA strand breaks. This reaction is catalyzed by theenzyme terminal deoxynucleotidyl transferase that repetitively adds deoxyribonucleotideto the 3' hydroxyl termini of DNA. Fluoresceinated avidin is thenemployed in a second step reaction to fluorescently label the DNA strandbreaks, thus allowing the detection of DNA strand breaks by flow cytometry.1.3. Reactive Oxygen SpeciesThe generation of ROS and alterations in the cellular redox state have beenreported to be a common biochemical event in apoptosis (12). Moreover, ROShave been proposed to be putative second messengers in the initiation ofapoptosis. The production of ROS during cytotoxic drug induced apoptosisand inhibition of apoptosis by antioxidants supports this view (12). Oxidativestress is also believed to play a role in Parkinson’s disease and amylotrophiclateral sclerosis disease states, the latter of which has been linked to geneticlesions in a cellular antioxidant pathway (4). Possible sources of intracellularROS include the depletion of cellular antioxidants such as glutathione, disruptionof mitochondrial respiration, and the activation of oxidant-producingenzymes such as NADPH oxidase.The fluorescent probes 2',7'-dichlorofluorescein diacetate (DCFH/DA) anddihydorethidium (DHE) may be used for the measurement of intracellular peroxideand superoxide anion levels, respectively. DCFH/DA is cell permeantand is nonfluorescent until the acetate groups are removed by cellular esteraseactivity and a peroxide group is subsequently encountered. Hydrolyzed, oxidizedDFCH/DA fluoresces at 529 nm (FL-1, log scale; see Subheading 3. andTable 1) (Fig. 1A,B) and is unable to leave the cell, thus allowing the measurementof intracellular peroxides by flow cytometry.DHE is also cell permeant and is oxidised to ethidium by superoxide anion.Once oxidised, ethidium is free to intercalate with DNA in the nucleus whereuponit emits fluorescence at 605 nm (FL-2) (see Fig. 1A,C).1.4. Mitochondrial Transmembrane Potential AlterationsEmerging evidence suggests a central role for mitochondria during apoptosisinduced by a diverse range of stimuli in a number of cell types. Indeed, severalgroups have proposed mitochondria as the central executors of apoptosis(reviewed in ref. 13). Mitochondrial events during apoptosis include release ofcytochrome-c and disruption of the transmembrane potential (∆ψ m ) (13). Dis-