Aging Aging

180 Barnett and Barnettconditions such as nutrition, housing, sanitation, and medical and social services,attempts at increasing the average life expectancy and the quality of lifein the elderly can be achieved only by slowing down the molecular processesunderlying aging. To facilitate such intervention, the factors that cause aging atthe cellular level must be identified and understood, including presumably thoseagents that lead to DNA damage and increase the likelihood of mutation.The detection of in vivo somatic mutations has enhanced our knowledge ofthe causes and mechanisms of mutagenesis in somatic cells (23). The ease ofcollection of peripheral blood cells together with the presence of intact phase Iand phase II xenobiotic metabolizing enzyme systems within these cells hasled to the development of a number of bioassays for the detection andquantitation of mutant frequency including the determination of hemoglobinvariants or glycophorin A variants in erythrocytes and the hypoxanthinephosphoribosyl transferase (HPRT) clonal assay in lymphocytes (these and othersare reviewed in ref. 24).HPRT is encoded by a gene that spans 44 kb containing 9 exons and islocated on the long arm of the X chromosome (Xq26). It is a constitutivelyexpressed, nonessential enzyme that functions in the purine salvage pathway toconvert hypoxanthine and guanine to their respective 5' monophosphate nucleosides.In addition, HPRT can utilize a number of base analogs such as 6-thioguanine(6–TG) to produce the corresponding ribonucleoside monophosphates.Cells harboring mutation in the HPRT gene are able to survive in the presenceof 6-TG, whereas wild-type cells will accumulate the highly cytotoxic 6-TGmonophosphateand die. This differential sensitivity to the cytotoxic potentialof 6-TG between HPRT + and HPRT – cells forms the basis of assays that determinethe frequency of HPRT – cells (6-TG resistant) within a mixed HPRT + /HPRT – cell population.The most frequently used method to quantitate background mutant frequencyat the HPRT gene locus is a clonal assay. Essentially, lymphocytes are incubatedin the absence (non-selective conditions) or presence (selective conditions)of 6-TG in 96-well microtiter plates for approx 14 d, after which timewells are scored according to the presence (positive) or absence (negative) ofclones. The zero form of the Poisson distribution (P 0 ) is then used to determinethe cloning efficiency (CE) in the absence and presence of the selection agent,and the mutant frequency (MF/10 6 cells) calculated. Although the clonal assaycan be demanding of time, finances, and resources it has the advantage that theclones can be expanded for determination of mutant status and the mutationalspectrum analyzed (25,26). The remainder of this chapter provides a thoroughdescription of the clonal assay to quantitate HPRT mutant frequency withincultured peripheral blood derived human lymphocytes.Other methods can be used to detect HPRT – lymphocytes. An autoradiograhictechnique, based on the ability of lymphocytes to incorporate [ 3 H]thy-