FORMATION OF DNA-ADDUCTS BY THE FOOD MUTAGEN AMINO-3,4,8-TRIMETHYL-3H-IMIDAZO[4,5-F]QUINOXALINE (4,8-DIMEIQX) IN-VITRO AND IN-VIVO - IDENTIFICATION OF A N-2-(2'-DEOXYGUANOSIN-8-YL)-4,8-DIMEIQX ADDUCT

The covalent binding of the mutagenic N-2-hydroxy metabolite of the fo od mutagen amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiM eIQx) to 2'-deoxynucleosides and DNA was investigated in vitro and in vivo. N-2-Hydroxy-4,8-DiMeIQx reacted to a small extent spontaneously with 2-deoxyguanosine. However acetylation of N-2-hydroxy-4,8-DiMeIQx, with acetic anhydride to form the N-2-acetoxy derivative prior to rea ction with 2-deoxyguanosine resulted in much higher yield of adduct. N -2-Acetoxy-4,8-DiMeIQx did not form adducts with 2'-deoxyadenosine, 2' -deoxycytidine or 2'-deoxythymidine. The adduct formed between the N-2 -OH metabolite of 4,8-DiMeIQx and 2-deoxyguanosine was analysed by mas s spectrometry and NMR spectroscopy and the structure of the adduct wa s shown to be N-2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx. N-2-Acetoxy-4,8 -DiMeIQx reacted with calf thymus DNA and formed a covalently bound 4, 8-DiMeIQx residue, which could not be removed by repeated precipitatio ns or solvent extractions. The 4,8-DiMeIQx-DNA was hydrolysed enzymati cally with nuclease P1/acid phosphatase and HPLC analysis showed that 70% of the bound mutagen was recovered as N-2-(2'-deoxyguanosin-8-yl)- 4,8-DiMeIQx. An additional minor adduct accounting for similar to 15% of the bound mutagen showed UV spectral characteristics similar to N-2 -(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx and is probably an undigested oli gomer. P-32-Postlabelling analysis of calf thymus DNA modified with 4, 8-DiMeIQx in vitro and liver DNA from rats dosed with 50 mg/kg 4,8-DiM eIQx showed a similar adduct pattern. In both samples N-2-(2'-deoxygua nosin-8-yl)-4,8-DiMeIQx accounted for 60-70% of the bound mutagen. Thu s, these results show that 4,8-DiMeIQx similar to other heterocyclic a mines form adducts with C-8 of guanine both in vitro and in vivo via i ts N-2-OH metabolite.