Aging Aging

144 Clingen, Lowe, and Greenstrand breaks in individual eukaryotic cells. It has been used in vivo and invitro to assess DNA damage and repair induced by various physical factorssuch as ultraviolet (UV) radiation (7,8), ionizing radiation (9,10), chemicalagents (11) as well as physiological factors such as diet (10), cytokines (12),exercise (13), smoking, and aging (14,15). A range of applications will beaddressed including:• Detecting frank DNA strand breaks• Investigating endogenous levels of damage• Formation and repair of oxidative DNA damage using specific DNA repairenzymes• Measuring intrinsic rates of cellular excision repairConsideration is also given to experimental design and interpretation of dataso that meaningful results may be obtained.1.1. Principle of AssayThe Comet assay is a flexible and sensitive procedure for measuring DNAdamage in a range of tissues and cell types. The methods described here havebeen largely based on the protocols of Singh et al. (16) and Collins et al. (17).Methods and applications of the assay have been extensively reviewed (18–22).In the assay, a single-cell suspension of the cells or tissue under study areembedded in low melting point agarose on a frosted microscope slide. Theslide is placed in a high salt lysis mixture that strips away cell membranes andremoves most proteins from chromatin to leave behind supercoiled DNA instructures known as nucleoids (23). When placed in alkaline buffer the supercoiledDNA starts to unwind. During electrophoresis, DNA containing strandbreaks is pulled toward the anode to form a Comet tail while undamaged DNAremains trapped within the nucleus. Comet tail length or tail moment are thendetermined by image analysis. Within a defined dose range there appears to bea linear relationship between the number of strand breaks and measures ofComet length and/or tail moment. The assay specifically measures DNA strandbreaks and normally can detect less than one strand break per 10 7 basepairs.These may represent:• Direct single-stranded breaks produced by exogenous or endogenous mutagenssuch as reactive oxygen species• Alkali labile sites• Breaks formed during the excision step of repair of damaged bases from cellularDNADirect single-stranded breaks are detected using the standard form of theassay. Although it has been postulated that these include alkali-labile sites, it is