Oxidation of DNA at Guanine by Chromium: Products and Mechanisms
7,8-dihydro-8-oxoguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at the nucleobase guanine. Recent studies have shown that 8-oxoG has a lower reduction potential than the parent guanine and is a hot spot for further oxidation. Spiroiminodihydantoin (Sp) has been identified as one of these further oxidation products. Chromium(VI) is a human carcinogen that, when reduced by a cellular antioxidant such as ascorbate, can oxidize DNA. The major studies involved in this thesis are as follows: When duplex DNA was reacted with Cr(VI) and ascorbate guanine bases were preferentially oxidized. A technique was designed to trap base excision repair enzymes specific for 8-oxoG (hOGG1) and specific for Sp (mNEIL2) to any oxidative lesions formed. Results showed almost exclusive trapping of mNEIL2, suggesting that 8-oxoG was not the major lesion formed. LC-ESI-MS along with a HPLC-ECD was used to identify the specific type of DNA damage. The results showed that Sp was present in concentrations 20 times greater that 8-oxoG. To our knowledge these are the first examples of Sp formation directly from guanine oxidation in dsDNA. The LC-ESI-MS technique was used to analyze purified genomic DNA from two types of DNA repair deficient E. coli; Nei- and MutM-/MutY-. Only the Nei deficient E. coli. demonstrated significant growth inhibition when incubated with chromate. LC-MS analysis of the genomic DNA showed an accumulation of Sp far greater than 8-oxoG. The MutM/MutY deficient E. coli showed no growth inhibition when incubated with chromate and genomic DNA analysis revealed no increase in Sp over wild type, but did reveal a small increase in 8-oxoG formation. These data demonstrate that the Nei BER enzyme is critical for the recognition and repair of the Sp lesion. To our knowledge, these are the first studies to show the formation and biological significance of the Sp lesion in a cellular system. Mechanistic studies were performed for the Cr(V)-salen induced oxidation of 8- oxoG nucleoside by LC-MS with 18O stable isotope labeling. Results revealed that chromium oxidation proceeds through an epoxide intermediate followed by nucleophilic attack of water and a rearrangement into the Sp molecule. This study suggests a new mechanism for the formation of the Sp lesion. Finally studies into the oxidative cross linking of proteins to DNA containing a 8-oxoG lesion showed the formation of a hOGG1-DNA cross link at the 8-oxoG, when the system was treated with Cr(V)-salen.
© Copyright 2006 Peter Grayson Slade