Detection and characterization by ³²P-postlabelling of DNA adducts induced by a Fenton-type oxygen radical-generating system

Abstract

Reactive oxygen species can give rise to numerous modifications of DNA. We have investigated the formation of such modifications using the nuclease P1 digestion method of the 32P-postlabelling procedure for the detection of DNA damage. Analysis of DNA that had been treated with a Fenton-type system of copper (or iron) ions and H2O2 resulted in the detection of up to ten discrete 32P-labelled spots, displaying chromatographic characteristics similar to aromatic adducts, on PEI-cellulose TLC. Maximum total levels equivalent to 28 adducts/10(8) nucleotides were achieved after 15 min of treatment with Cu2+/H2O2. The formation of adducts was 1.5 times greater if single-stranded rather than double-stranded DNA was employed, suggesting an intrastrand effect. Experiments with 3'-deoxyribonucleotides demonstrated that the adducts detected did not represent base modifications such as 8-hydroxydeoxyguanosine or thymidine glycols. However, treatment of specific dinucleotides (dApdG and dApdA) was found to produce two major adducts that were chromatographically identical by TLC and HPLC to the two major adducts formed in DNA. It is proposed that these species with aromatic adduct-like characteristics are the result of the intrastrand linking of specific adjacent bases in DNA.

The case for 8,5′-cyclopurine-2′-deoxynucleosides as endogenous DNA lesions that cause neurodegeneration in xeroderma pigmentosum

“…Our results using the 32 P postlabeling assay indicate that the "putative intrastrand purine dimers" (Carmichael et al, 1992, Lloyd et al, 1997 which were proposed as candidate neurodegenerative lesions in XP by (Satoh et al, 1993) are also dinucleotides containing cyclodA. However, because the postlabeling studies were done in different laboratories, and used different reagents and chromatographic solvents, it is not absolutely certain that the same adducts were being studied in both cases.…”

“…These authors used the in vitro NER assay (Wood et al, 1988) to show that exposure of DNA to oxygen radicals generated a class of DNA lesion(s) that are normally repaired by the NER, and proposed that oxidative stress could be the source of the DNA damage that causes neuronal death in XP patients. In considering the possible identities of oxidatively-induced lesions that are repaired by NER, Satoh et al suggested "putative intrastrand purine dimers" detected using the 32 P-postlabeling method (Carmichael et al, 1992; see also Randerath et al, 1991) as well as cyclopurine-deoxynucleosides. The possible role of cyclopurine-deoxynucleosides in XP neurological disease had also been considered by Robbins and colleagues (Dizdaroglu et al, 1987).…”

Tandem double CC-->TT mutations are produced by reactive oxygen species.

“…A primary candidate for an alteration in DNA that could produce the double mutation is the crosslinking of two adjacent cytosines. There is evidence that exposure of dinucleotides to oxidative damage mediated by Cu/H202 results in a crosslink between the two bases, although cytosine crosslinks were not assayed (29). The finding that CC -> TT substitutions are observed after damage to DNA by both reactive oxygen species and UV-irradiation suggests that both types of agents may produce a similar intermediate, leading to a stable mutagenic adduct.…”

Effects of NQO1 deficiency on levels of cyclopurines and other oxidative DNA lesions in liver and kidney of young mice

“…19 Other type II I-compounds may represent base-base 1,20,21 or base-sugar intrastrand crosslinks. 21 Dinucleotides containing cA present as bulky ROSinduced DNA lesions.…”