Thymine glycols and pyrimidine dimers in brain DNA during post-ischemic reperfusion

Abstract

Free-radical reactions, known to occur in the reperfused brain, damage DNA in vitro. We therefore examined the hypothesis that thymine glycols and thymine dimers, which are known to block transcription and are formed by free radical mechanisms, are formed in brain DNA during reoxygenation following ischemia. Such biochemical lesions could account for the failure of protein synthesis that occurs following an ischemic insult. Large dogs were anesthetized, instrumented, and divided into four groups: (1) non-ischemic controls; (2) 20-min cardiac arrest without resuscitation; (3) 20-min cardiac arrest, resuscitation and 2 h reperfusion; and (4) 20-min cardiac arrest, resuscitation and 8 h reperfusion. Genomic DNA was isolated from the cerebral cortex. Thymine glycols were labeled by reduction with [3H]NaBH4. Pyrimidine dimers were determined by ELISA using antibody prepared against ultraviolet irradiated DNA. The data was evaluated by Kruskal-Wallis ANOVA with alpha = 0.05. The rabbit antibodies detected the thymine dimer content in 10 pg UV irradiated DNA but did not react with normal DNA. Borohydride labeling qualitatively detected thymine glycols generated by treatment of DNA with osmium tetroxide. There was no difference between the DNAs from the experimental groups in the content of thymine glycols or pyrimidine dimers (P = 0.608 and P = 0.219, respectively). We conclude that significant quantities of thymine glycols and thymine dimers are not formed in brain DNA during post-ischemic reperfusion. Therefore, the inhibition of brain protein synthesis during reperfusion, observed by other investigators, is unlikely to be caused by interruption of transcription by these species.