DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

Abstract

-To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMSinduced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.

Differential colon DNA damage induced by azo food additives between rats and mice

“…The assay was conducted as previously described (Tsuda et al, , 2001Hashimoto et al, 2007). The protocol used was in accordance with the published comet assay genotoxicity guidelines (Tice et al, 2000).…”

Perfluorooctanoic acid (PFOA) but not perfluorooctane sulfonate (PFOS) showed DNA damage in comet assay on Paramecium caudatum

“…This solution was centrifuged at 500 rpm for 3 min at 0°C, and 100 µl of the precipitated paramecia were suspended in 1 ml of 0.25 M sucrose containing 0.6% Triton X-100, and re-centrifuged at 1,500 rpm for 3 min at 0°C to obtain the large nuclei. The procedure for the comet assay is similar to the previous one (Hashimoto et al, 2007). Briefly, 60 µl of the nuclei suspension was mixed with 60 µl of 2% LGT agarose (45°C), and 60 µl of this mixture was layered on a slide pre-coated with 1% GP-42 agarose.…”

Nuclear DNA Damage as a Direct Cause of Aging

“…44 A study of 8-OHdG repair in kidney and liver tissue of young and aged rats showed a significantly lower BER in the older rats. 45 A study of the DNA glycosylase enzyme required for BER of 8-OHdG in lymphocytes of 78 healthy humans ranging from newborn to 91 years of age showed a significant linear decline to less than half newborn values, with very high individual variation.…”