DNA ALKYLATION BY 4,5-DIOXOVALERIC ACID, THE FINAL OXIDATION-PRODUCT OF 5-AMINOLEVULINIC ACID

The heme precursor 6-aminolevulinic acid (ALA) accumulates under patho logical conditions, namely, acute intermittent porphyria (AIP) and tyr osinosis, two diseases that are associated with increased liver cancer incidence. This has been previously linked to an enhanced production of reactive oxygen species generated by a metal-catalyzed ALA oxidatio n process, which was shown to cause DNA single-strand breaks and guani ne oxidation within both isolated and cellular DNA. In the present wor k, we established that the final oxidation product of ALA, 4,5-dioxova leric acid (DOVA), is an efficient alkylating agent of the guanine moi eties within both nucleoside and isolated DNA. Adducts were produced t hrough the formation of a Schiff base involving the N-2-amino group of 2'-deoxyguanosine (dGuo) and the ketone function of DOVA, respectivel y. The modified dGuo nucleosides were characterized, following reducti on into stable secondary amines, by extensive NMR, infrared, and mass spectrometry analyses. A method, based on the use of HPLC with electro chemical detection, was then developed for the sensitive measurement o f the DOVA-dGuo adducts. Using this assay, we showed that the guanine moieties of isolated DNA can undergo the same reaction as the free nuc leoside. The present data provide additional information on the genoto xic potential of ALA and reinforce the hypothesis that AIP may be invo lved in the induction of primary liver cell carcinoma.