Quantitative detection of DNA damage in the neuronal cells of the cerebellum and cerebrum by the analysis of Feulgen hydrolysis curves.
Touch smears of the cerebellum and cerebrum of ageing rats were fixed with methanol, hydrolyzed with 2 N HCl at various temperatures and for various periods, and stained with pararosaniline-Schiff reagent. The hydrolysis curves were determined by fluorescence cytophotometry and were computer fitted to the Bateman function to determine the kinetic parameters, the initial yield of apurinic acid or single-stranded DNA (y0), and the rate constants for depurination or denaturation (k1) and depolymerization (k2). The values for k1 (1/k1 is correlated with the degree of chromatin condensation) and k2 (which reflects the degree of DNA instability) steadily increased with age. The values for y0, which may indicate the degree of DNA denaturation or damage present before acid hydrolysis, also increased with age in both the cerebellum and cerebrum; however, this value was lower in the cerebellum until 15 weeks, with the situation being reversed after 35 weeks, the cross-over time being at about 25 weeks. The values of lnk1 and lnk2 were plotted as the function of the reciprocal of the absolute temperature (T) (Arrhenius plot) for both the cerebellum and cerebrum of 15- and 74-week-old rats, and the activation energies (E) for depurination and depolymerization were calculated from the slopes. In particular, the values of E for k2 decreased much more quickly with age and were smaller in cerebellum. In conclusion, the degree of DNA damage and DNA instability steadily increases in both the cerebellum and cerebrum of ageing rats, and this process is much faster in the cerebellum.
D000375:Aging; D000818:Animals; D001080:Apurinic Acid / Q000032:analysis; D001921:Brain / Q000473:pathology; D002531:Cerebellum / Q000473:pathology; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis; D006868:Hydrolysis; D007700:Kinetics; D009474:Neurons / Q000473:pathology*; D051381:Rats; D012394:Rosaniline Dyes
D004396:Coloring Agents; D004277:DNA, Single-Stranded; C007722:Feulgen stain; D012394:Rosaniline Dyes; D001080:Apurinic Acid; D004247:DNA