of work: One theory of the mechanism of aging of post-mitotic tissues proposes that an accumulation of oxidative damage to mitochondrial DNA (mt-DNA) leads to impaired biogenesis of mitochondria and thus a failure to maintain cellular energy homeostasis. We are testing several elements of this hypothesis. In this project, we have quantitated the occurrence of 8-OHdeoxyguanosine (8OHdG), an oxidative product of deoxyguanosine, in mt-DNA from liver of rats of various ages, using separation of nucleosides by HPLC and measurement by an electrochemical array detector. We found that 8-OHdG is more prevalent in mt-DNA than in nuclear DNA. Further, there is a significant increase with aging; from 8 per 100000 deoxyguanosine residues at 6 months to 14 at 12 months and 22 at 23 months of age. There was no such age-linked change in nuclear DNA. As previous work using GC/MS for separation/detection of 8-OHdG had shown no increase with aging in mt-DNA. we have taken some pains to validate the HPLC/EC technique. Treatment both of mt-DNA from tissues and of commercial calf thymus DNA with the 8-OHdG-specific formamidopyrimidine glycosylase (fpg enzyme) resulted in stoichiometric removal of the peak attributed to 8-OHdG. Oxidative damage induced by light in the presence of methylene blue was equally recognized by the fpg enzyme and HPLC techniques, encouraging us to use a combination of fpg treatment and Southern blotting to investigate the 8-OHdG content of mt-DNA from very small tissue samples, eg T-lymphocytes sorted by FACS technology, in current and future work. This is important to us since much of our previous work has involved the aging rat heart and the yield of mt-DNA isolated from one rat heart is too small to support the HPLC analysis. We also plan to extend this work to the measurement of 5-OHcytosine as another oxidative product of mt-DNA. We have established the chromatographic and potentiometric characteristics of 5-OHcytosine synthesized by Dr Rebecca Selzer, also of LMG. In a related project which investigates damage to mt-DNA of human T-lymphocytes as a function of donor age, we have demonstrated the existence of the """"""""common"""""""" 5-kb deletion of mt-DNA in cells from old (more than 70 years) donors and have failed to make a full-length PCR product from the mitochondrial genome in these cells, despite the successful synthesis of a full-length product using DNA from young (less than 30 years) donors. Although further work is necessary to characterize the nature of the damage which prevents the mt-DNA acting as a template, these studies with human T-lymphocytes offer the potential of correlating extent of damage to mt-DNA with mitochondrial functioning, on a cell-by-cell basis.
| Tuo, J; Muftuoglu, M; Chen, C et al. (2001) The Cockayne Syndrome group B gene product is involved in general genome base excision repair of 8-hydroxyguanine in DNA. J Biol Chem 276:45772-9 |
