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

Abstract

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.

DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis

“…The structures of the various AIA-DNA adducts identified to date are shown in Figure 3. All form a major adduct with the C8 atom of guanine and, for IQ and MeIQx, a minor adduct with the N 2 atom of guanine has also been identified (110,(116)(117)(118)(119)(120)(121)(122)128,129,(134)(135)(136)(137)(138). N-acetoxy derivatives of several AIAs have also been shown to react with other bases in vitro (120,129,138), but such reactions appear to be of no quantitative importance in vivo.…”

“…While the 32 P-labeled adduct patterns of a particular AIA may differ greatly among various laboratories, the major identified DNA adduct for each AIA is similar in all species examined, including bacteria (Salmonella), rats, mice and monkeys (117)(118)(119)(120)(121)(122)128,129,(134)(135)(136)(137)140). Examination of reaction products formed in vitro between N-acetoxy-…”

DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans

“…The strengths and limitations of these methods are highlighted in Table . These two methods have been used to screen for DNA adducts of PAHs, 4‐aminobiphenyl (4‐ABP), 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐ b ]pyridine (PhIP), MDA, and 8‐oxo‐dG as biomarkers for oxidative stress …”

Reversed-phase high-performance liquid chromatographic separation of32P-labeled nucleotide adducts formed by food-derived carcinogenic aminoimidazoazarenes

“…To avoid this high level of radioactivity we separated the adduct fraction from excess radioactivity by conventional ion-exchange TLC; this might reduce the recovery of adducts but adds an additional separation technique compared with separation solely on reversed-phase material. Although HPLC of3Zp-labeled DNA adducts has recently been used in several studies for analysis of covalent modifications of DNA by individual AIA [13,17,[21][22][23], this is the first report of a method enabling separation of 32p-labeled adducts formed by the structurally very similar members of this group of chemical carcinogens. This might prove useful in studies on the combination effects of mixtures of food-derived heterocyclic amines.…”

“…In recent years several variations of the 32p-postlabeling assay have been described that make use of the superior resolving power of ItPLC analysis. Other groups have described methods in which the whole labeling mix is applied to the HPLC system, either directly on to the analytical column [19,22,24] or on to a precolumn, by use of a column switching technique [23,28]. In combination with the labeling procedure employed in this study (ATP-deficient labeling [26]) this would require introduction of 100 #Ci into the apparatus.…”