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.

A no observed adverse effect level for DNA adduct formation in rat liver with prolonged dosing of the hepatocarcinogen 2-acetylaminofluorene

“…15,17 Nevertheless, assuming that the intensities of observed spots represent minimum values, initially, the dG-C8-AF adduct predominated, as has been reported by others. 15,17 Nevertheless, assuming that the intensities of observed spots represent minimum values, initially, the dG-C8-AF adduct predominated, as has been reported by others.…”

“…[12][13][14][15] Few data are available, however, on the kinetics of formation of AAF adducts (or for that matter adducts of other DNA-reactive hepatocarcinogens) with repeated, extended duration of administration. [12][13][14][15] Few data are available, however, on the kinetics of formation of AAF adducts (or for that matter adducts of other DNA-reactive hepatocarcinogens) with repeated, extended duration of administration.…”

In Vitro Characterization of DNA Adducts Formed by Foundry Air Particulate Matter

“…The main peak of adduct 2 is most probably derived from B[a]P based on co-migration with the anti(±)BPDE-DNA standard ( Figures 3C, 3D). The development of the methodology in analyzing nitro-PAH-DNA adducts in exposed humans by these two techniques still needs to be improved; however, promising results have been reported in analyzing PAH and nitro-PAH adducts from in vivo DNA (16,(24)(25)(26). This study confirms that the 32P-postlabeling method combined with HPLC is an applicable method for the characterization of PAH adducts in in vitro DNA exposed to complex mixtures of PAHs.…”

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

“…Aliquots of the labeling mixture were analyzed by HPLC, which was conducted by a modification of the method of Möller and Zeisig (35). Specifically, adducts were separated on a 5 µ DeltaPak C 18 -100 column (3.9ϫ150 mm; Waters Associates) using a gradient of 100% solvent A for 5 min, followed by a 35 min linear gradient to 100% solvent B and then an isocratic elution with solvent B for 20 min.…”