Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro

Abstract

Nitric oxide and superoxide anion, both formed in inflamed tissues, react rapidly to form the peroxynitrite anion (ONOO-), a strong oxidant which can initiate reactions characteristic of hydroxyl radical (HO.), nitronium ion (NO2+) and nitrogen dioxide radical (NO2.). Peroxynitrite, therefore, may cause DNA or tissue damage, contributing to the multistage carcinogenesis process. We have studied reactions of various bases, nucleosides or deoxynucleosides with peroxynitrite in vitro. Guanine reacted rapidly with peroxynitrite under physiological conditions and formed several substances, two of which were yellow, a characteristic of nitro and nitroso compounds. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for approximately 80% of all compounds formed) as 8-nitroguanine. Its formation was maximal at approximately pH 8 and increased dose-dependently with peroxynitrite concentration, but was not dependent on guanine concentration. The presence of ferric ions, which has been shown to catalyse nitration of tyrosine, did not affect nitration of guanine. 8-Nitroguanine could act as a specific marker for DNA damage induced by peroxynitrite in inflamed tissues.

Formation of 8‐nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination

“…The acid-hydrolyzed DNA was purified by preparative HPLC and the fractions containing nitroSGua were collected and evaporated to dryness. This concentrated fraction was analyzed by HPLC with UV detection at 396 nm, as previously reported [20]. A yellow peak eluted at the same retention time as a standard nitro8Gua (7.8 min).…”

“…It has been reported that peroxynitrite induces strand breaks in plasmid DNA [18] and oxidative damage in isolated DNA in vitro [19]. We recently found that guanine reacts rapidly with peroxynitrite under physiological conditions to form several substances, two of which are yellow [20]. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for about 80% of all compounds formed) as a novel adduct, 8-nitroguanine (nitroSGua) [20].…”

“…NitroSGua was synthesized by reaction of guanine with peroxynitrite as described previously [20]. 8-Aminoguanine was prepared by acid hydrolysis of 8-aminoguanosine (Sigma Chemical Co., St Louis, MO) [21].…”

“…We recently found that guanine reacts rapidly with peroxynitrite under physiological conditions to form several substances, two of which are yellow [20]. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for about 80% of all compounds formed) as a novel adduct, 8-nitroguanine (nitroSGua) [20]. In order to study the biological significance of this new adduct, it is essential to determine whether nitroSGua is formed in DNA.…”

In vitro free radical scavenging and DNA damage protective property of Coriandrum sativum L. leaves extract

“…Under these conditions, NO will react rapidly with superoxide anion to form ONOO. The relativities of the free radical NO and superoxide anion were found to be relatively low, but their metabolite ONOO -was extremely reactive and directly induced toxic reactions, including SH-group oxidation, protein tyrosine nitration, lipid peroxidation and DNA modifications (Yermilov et al 1995;Babu et al 2001). Coriandrum sativum showed antioxidant activity, with IC 50 values of 815.6 μg/ml and standard BHA with 49.1 μg/ml.…”