DNA adduct formation after oral administration of 2-nitrofluorene and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC

DNA adducts have been detected in laboratory animals after exposureto carcinogens as well as in human populations with known orsuspected risk of developing cancer. Examples are smokers, cokeand aluminium workers, urban citizens and roofers. The formationof DNA adducts is an early event in carcinogenesis which canbe used for measuring target dose and as a biomarker for genotoxkrisk. A method of analyzing ³²P-postlabeled DNA adducts on reverseHPLC with on-line detection of ³²P has been developed. The methodpermits direct injection of the ³²P-postlabeling mixture intothe analytical system without prior purification with backgroundradioactivity on a low level. The method can be used in parallelwith TLC analyses of ³²P-postlabeled DNA adducts to improvethe analytical capacity. The time for analysis of a typicalsingle sample by HPLC and TLC is 30–60 min and 6–24h respectively. A high (2 M) salt concentration in the HPLCeluent reduces the ³²P background considerably. Also the peaktailing was substantially diminished, giving an ability to separateDNA adducts equal to or better than the TLC method. The methodhas been applied to 2-nitrofluorene (NF), a carcinogenic airpollutant, and N-acetyl-2-aminofluorene (AAF), a model carcinogenwhich is also a metabolite of NF. A number of DNA adducts areformed in the livers of rats. After oral administration of AAFand NF, DNA adducts in the liver have been characterized asdG-C₈-AF and dG-C₈-AAF. The major DNA adduct found in both NF-and AAF-administered animals was dG-C₈-AF. The described HPLCmethod can, with minor adjustments, generally be used to analyze³²P-postlabeled DNA adducts.