Conclusion

Because there are no specific genotoxic agents able to produce only 8-oxoG in cellular DNA and because the antibodies raised against this lesion are not specific enough, we chose a shuttle vector assay allowing us to construct a plasmid DNA containing only one GO lesion at a given position. This assay is the only one at this time to allow the study of the repair and mutagenic potency of unique oxidative damage in human cells. This technology, which is equally applicable for other types of oxidative base damage, has been used in the past mostly for DNA lesions repaired by NER.16 Results obtained with shuttle vectors in repair and mutagenesis analyses are highly reproducible and when used in NER studies have always been validated by genomic DNA data when available.17

Our results using a shuttle vector containing a single 8-oxoG showed for the first time the existence of transcription-coupled repair of this lesion in human cells.5 The observed deficiency in this pathway in CS and XP/CS cells correlates with progressive neurological disorders observed in these patients. It is plausible that in nondividing brain cells the accumulation of unrepaired oxidative lesions on transcribed strands leads to apoptosis and therefore to progressive neurological deterioration. We have found that cells homozygous for mutations in the BRCA1 or BRCA2 gene, the major genes predisposing to breast and ovarian cancers, are also deficient in the TCR of 8-oxoG.18 Several studies have reported a high level of oxidative stress in breast tissue that may be linked to estrogen metabolism and hormonal status. It is possible that the absence of repair of oxidative lesions in breast cells already mutant for the BRCA1 or BRCA2 gene will produce a high level of point mutations, thus contributing to malignancy.

The use of shuttle vectors carrying single lesions in defined locations as described in this chapter should allow monitoring of the status of repair of oxidative damage for cells isolated from numerous human diseases and eventually correlation of a given level of repair with a given type of malignancy.

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