32P-post-labelling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct pattern simplification.

Abstract

Food-derived aminoimidazoazarenes have been shown to be mutagenic and carcinogenic and to form covalent DNA adducts. 32P-Post-labelling analysis of DNA modified with these heterocyclic amines (HA), including 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,4-dimethylimidazo [4,5-f]quinoline (MeIQ), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) has resulted in considerable interlaboratory variation in the characteristic patterns of DNA adduct spots, with up to six being detected for each compound. Similar complex patterns were observed when azido-derivatives of HA were photoreacted with calf thymus DNA. When deoxyguanosine 3'-monophosphate was modified with the azido derivatives and analysed using the 32P-post-labelling procedure, one major spot was observed for IQ, 4,8-DiMeIQx, 7,8-DiMeIQx or PhIP and two major spots for MeIQ or MeIQx. In each case, these adducts were chromatographically indistinguishable from the major adducts formed with DNA. No major adduct spots were observed when 3'-phosphate derivatives of deoxyadenosine, deoxycytidine or thymidine were reacted with the azido-derivatives of HA. In an attempt to identify the additional spots, azido derivatives of PhIP or IQ were reacted with the synthetic homopolymer poly(dG).poly(dC), the alternating copolymer poly(dC-dG) or a synthetic oligonucleotide (TTT-GTTTTTTCTTTCCCT): in each case a reduced number of adduct spots were detected. The introduction of an additional nuclease P1 hydrolysis step following the labelling reaction further reduced the number of adduct spots to only one or two major spots. Reversed-phase HPLC analysis showed that the number of peaks of radioactivity was also reduced to one or two, presumably corresponding to the [32P]-5'-monophosphate deoxyguanosine adducts. We suggest that many of the additional spots commonly observed in conventional 32P-post-labelling analysis of HA-modified DNA are adducted oligonucleotides that are partly resistant to hydrolysis by micrococcal nuclease and spleen phosphodiesterase but are susceptible to hydrolysis by nuclease P1.