Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging

Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as “comet” assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies.

► Age dependent DNA damage, including the levels of OGG1 and UDG sensitive sites in DNA, was measured at the single cell level in the rat cerebral cortex through “comet” assay.
► DNA damage in terms of SSBs and DSBs increases with age in neurons and astrocytes.
► OGG1 and UDG sensitive sites accumulate in DNA of aging neurons and astrocytes.
► BER enzymes activities like OGG1, UDG and APE1 decline with age in neurons and astrocytes.
► This study shows that a single comet assay procedure, adapted suitably, can be used to assess various DNA damage and repair parameters at a single cell level and is of great utility for population studies.