³²P-HPLC analysis of DNA adducts formed in vitro and in vivo by 2- amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethyl- 3H-imidazo[4,5-f]quinoxaline, utilizing an improved adduct enrichment procedure

DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-diMeIQx), synthesized in vitro with calf thymus DNA and formed in vivo in the male Wistar rat, were enriched from digested DNA by butanol extraction before ³²P-postlabeling. The recovery after butanol enrichment was 79% and 32% for in vitro modified PhIP- and 4,8-diMeIQx-DNA adducts, respectively. Crude postlabeling mixtures were chromatographically separated by high-performance liquid chromatography with on-line ³²P-detection (³²P-HPLC). The major PhIP- and 4,8-diMeIQx-DNA adducts formed in vitro cochromatographed with the respective pdGp-C8 adduct standard. ³²P-HPLC was also used to separate hydrolysates of in vitro formed PhIP-DNA and 4,8-diMeIQx-DNA that had been ³²P-postlabeled under ATP-deficient conditions. The adduct recovery of the ATP-deficient method relative to the improved butanol enrichment procedure was 29% and 59% for total PhIP-DNA and 4,8-diMeIQx-DNA adducts, respectively. Simplified DNA adduct patterns were obtained when the postlabeling mixtures were incubated with nuclease P1, suggesting incomplete DNA hydrolysis. After nuclease P1 treatment, the major DNA adducts of PhIP and 4,8-diMeIQx formed in vitro cochromatographed with the respective pdG- C8 adduct standard. In vivo PhIP formed what appeared to be multiple DNA adducts; however, after nuclease P1 treatment the PhIP-associated peaks were concentrated into a single peak cochromatographing with pdG-C8-PhIP. 4,8- diMeIQx formed multiple DNA adducts in vivo. Nuclease P1 treatment resulted in two 4,8-diMeIQx related peaks, one cochromatographing with pdG-C8-4,8- diMeIQx. The second peak remains unidentified. The improved workup procedures in combination with the high resolution and reproducibility of the ³²P-HPLC system should be useful for characterization of PhIP- and 4,8-diMeIQx-DNA adducts in DNA modified by complex mixtures.