Analysis of DNA adducts of some low molecular weight aldehydes : Methods development and application in human biomonitoring

Abstract: Doctoral Thesis present at the Karolinska Institute, 1997 Center for Nutrition and Toxicology Departrnent of Biosciences at NOVUM Karolinska Institute 141 57 Huddinge Malondialdehyde (MA), acetaldehyde (Aa) and methylglyoxal (MG) are ubiquitously present in the environment and endogenously forrned in animals and humans. They have been shown to be genotoxic and to readily react with DNA to form DNA adducts under physiological conditions. The present studies were aimed at developing specific 32p_ postlabelling methods for the analysis of these DNA adducts and to apply them to measure these DNA adducts in vitro (MG, Aa) and in vivo in mice (Aa) and in humans (MA, Aa). Endogenously formed DNA adducts of MA were detected in DNA isolated from total white blood cells and from breast tissue of known unexposed healthy individuals by using the 32P-postlabelling technique. A large interindividual variation in adduct levels was observed. The effects of dietary fatty acid composition on the endogenous formation of DNA adducts of MA were investigated in humans ingesting carefully controlled diets. MA-DNA adduct levels in total white blood cell DNA from subjects given a sunflower oil-based (rich in polyunsaturated fatty acids) diet, which resulted in higher concentrations of polyunsaturated fatty acids in plasma triglycerides, were 3.6-fold higher than the corresponding levels in individuals given a rapeseed oil-based (rich in monounsaturated fatty acids) diet. Aa was shown to readily react with deoxynucleosides in a buffered solution at room temperature and neutral pH. AU the adducts formed were chemically unstable at room temperature and neutral pH. Chemically unstable adducts were also formed by the co- operative reaction of Aa with ethanol. Five stable adducts were obtained in the reaction of Aa with deoxyguanosine (dG) after reduction with NaBH4 and structurally characterized. A 32P- postlabelling assay was developed for the determination of N2-ethyl-3'-dGMP, the major stable adduct obtained in vitro, and was shown to be sensitive enough for the detection of adducts in both calf thymus DNA exposed to Aa in vitro and in liver DNA from mice treated with ethanol. Further, the effect of alcohol drinking on the formation of DNA adducts of Aa was investigated in humans. A large interindividual variation in adduct levels was observed. The average adduct levels in granulocyte and Iymphocyte DNA from alcoholic patients were 13- and 7-fold higher than the corresponding levels in the control subjects. Four stable isomeric reaction products formed between MG and dG or 3'-dGMP were separated by reversed-phase HPLC and structurally characterized. A 32P-postlabelling assay was developed for the determination of MG-3'-dGMP adducts and was shown to be sensitive enough for the detection of adducts in both calf thymus DNA and human Iymphocytes after in vitro exposure to MG. The results presented here suggest that DNA adducts of MA and Aa could serve as important biomarkers to assess the contribution of dietary and life-style factors such as fat intake and alcohol drinking, respectively, to human carcinogenesis. Jia-Long Fang, 1997 ISBN 91-628-2322-1