NUCLEIC-ACID ALKYLATION BY FREE-RADICAL METABOLITES OF ETHANOL - FORMATION OF 8-(1-HYDROXYETHYL)GUANINE AND 8-(2-HYDROXYETHYL)GUANINE ADDUCTS

Alcohol consumption is associated with an increased risk of several ty pes of malignancy by mechanisms that remain to be elucidated. Most of the ingested ethanol is converted to acetaldehyde but the formation of free radical metabolites such as the l-hydroxyethyl radical has been also demonstrated to occur in vitro and in vivo. Here we tested the po ssibility of ethanol-derived free radicals alkylating nucleic acid and nucleic acid components. Ethanol oxidation by Fenton systems has been extensively used to mimic ethanol metabolism to free radical intermed iates and it was also employed in our studies. Two adducts, 8-(l-hydro xyethyl)guanine and 8-(2-hydroxyethyl)guanine, were isolated in incuba tions containing guanine/ethanol/hydrogen peroxide/iron(II) at pH 1 un der anaerobic conditions. The adducts were produced in comparable yiel ds and were characterized by ultraviolet absorption, mass spectrometry , and proton nuclear magnetic resonance spectroscopy. Both adducts wer e also produced in incubations containing DNA and RNA at pH 4 and 7. U nder these conditions, the obtained yields of 8-(l-hydroxyethyl)guanin e were about 10 times higher than those of 8-(2-hydroxyethyl)guanine. Higher yields of both adducts were obtained at pH 4 than at pH 7 and w ith RNA as compared with DNA. As expected, nucleic acid oxidation prod ucts such as 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydroadenine wer e also produced under the employed experimental conditions. Their yiel ds tended to increase in the presence of ethanol, particularly at pH 4 , suggesting that ethanol can protect oxidized bases from further degr adation. Parallel spin-trapping experiments with alpha-4-pyridyl-1-oxi de N-tert-butylnitrone and 3,5-dibromo-4-nitrosobenzenesulfonic acid c onfirmed that ethanol was oxidized to both the l-hydroxyethyl and 2-hy droxyethyl radicals by hydrogen peroxide/iron(II) at pH 4-7 in the pre sence and in the absence of nucleic acids. The results demonstrate tha t free radical metabolites of ethanol can alkylate nucleic acids in vi tro. Both the l-hydroxyethyl and 2-hydroxyethyl radicals may play a ro le in ethanol-mediated toxicity.