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

Abstract

DNA adducts have been detected in laboratory animals after exposure to carcinogens as well as in human populations with known or suspected risk of developing cancer. Examples are smokers, coke and aluminium workers, urban citizens and roofers. The formation of DNA adducts is an early event in carcinogenesis which can be used for measuring target dose and as a biomarker for genotoxic risk. A method of analyzing 32P-postlabelled DNA adducts on reverse HPLC with on-line detection of 32P has been developed. The method permits direct injection of the 32P-postlabeling mixture into the analytical system without prior purification with background radioactivity on a low level. The method can be used in parallel with TLC analyses of 32P-postlabelled DNA adducts to improve the analytical capacity. The time for analysis of a typical single sample by HPLC and TLC is 30-60 min and 6-24 h respectively. A high (2 M) salt concentration in the HPLC eluent reduces the 32P background considerably. Also the peak tailing was substantially diminished, giving an ability to separate DNA adducts equal to or better than the TLC method. The method has been applied to 2-nitrofluorene (NF), a carcinogenic air pollutant, and N-acetyl-2-aminofluorene (AAF), a model carcinogen which is also a metabolite of NF. A number of DNA adducts are formed in the livers of rats. After oral administration of AAF and NF, DNA adducts in the liver have been characterized as dG-C8-AF and dG-C8-AAF. The major DNA adduct found in both NF- and AAF-administered animals was dG-C8-AF. The described HPLC method can, with minor adjustments, generally be used to analyze 32P-postlabelled DNA adducts.