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 types 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 1-hydroxyethyl radical has been also demonstrated to occur in vitro and in vivo. Here we tested the possibility 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 intermediates and it was also employed in our studies. Two adducts, 8-(1-hydroxyethyl)guanine and 8-(2-hydroxyethyl)guanine, were isolated in incubations containing guanine/ethanol/hydrogen peroxide/iron(II) at pH 1 under anaerobic conditions. The adducts were produced in comparable yields and were characterized by ultraviolet absorption, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Both adducts were also produced in incubations containing DNA and RNA at pH 4 and 7. Under these conditions, the obtained yields of 8-(1-hydroxyethyl)guanine 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 with RNA as compared with DNA. As expected, nucleic acid oxidation products such as 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydroadenine were also produced under the employed experimental conditions. Their yields tended to increase in the presence of ethanol, particularly at pH 4, suggesting that ethanol can protect oxidized bases from further degradation. Parallel spin-trapping experiments with alpha-4-pyridyl-1-oxide N-tert-butylnitrone and 3,5-dibromo-4-nitrosobenzenesulfonic acid confirmed that ethanol was oxidized to both the 1-hydroxyethyl and 2-hydroxyethyl radicals by hydrogen peroxide/iron(II) at pH 4-7 in the presence and in the absence of nucleic acids. The results demonstrate that free radical metabolites of ethanol can alkylate nucleic acids in vitro. Both the 1-hydroxyethyl and 2-hydroxyethyl radicals may play a role in ethanol-mediated toxicity.
Nakao
L S
LS
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, 05599-970, SP, Brazil.
Augusto
O
O
D000478:Alkylation; D004247:DNA / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry*; D005609:Free Radicals / Q000737:chemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D010084:Oxidation-Reduction; D012313:RNA / Q000737:chemistry*
C114394:8(1-hydroxyethyl)guanine; C114393:8-(2-hydroxyethyl)guanine; D005609:Free Radicals; D000431:Ethanol; D006147:Guanine; D012313:RNA; D004247:DNA