The use of benzo[ a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32P-postlabelling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study

Abstract

The 32P-postlabelling assay is one of the most sensitive methods for detection of DNA adducts induced by exposure to genotoxic chemicals. Under optimal conditions, detection limits of one adduct per 10 9–10 10 nucleotides have been reported. This sensitivity now allows monitoring of occupational and even environmental exposure of humans to certain classes of chemicals, mainly polycyclic aromatic hydrocarbons (PAH). Despite its widespread use, 32P-postlabelling is still not a standardized method. Rigorous interlaboratory comparisons are scarce, and those that have been undertaken often show rather different results, both in relative and in absolute values, for the amounts of DNA adducts in the same samples. Furthermore, the optimization of many steps in the procedure has still not been given adequate attention. This paper deals with some technical aspects of detection of PAH–DNA adducts by 32P-postlabelling, in particular with assay calibration and adduct quantification. For this purpose, benzo[ a]pyrene (BP)-modified DNA standards were prepared, the adduct contents of which were determined by use of an independent fluorometric method, viz. synchronous fluorescence spectrophotometry (SFS). These BP–DNA standards are processed along with the test samples throughout the entire 32P-postlabelling procedure, from the enzymic digestion up to and including the determination of radioactivity in adduct spots on the chromatogram. As such, these reference samples can be considered as external standards for inter-assay calibration. This method for adduct quantification was compared with the commonly used relative adduct labelling (RAL) and comparative dAMP labelling, which appeared to give rise to an underestimation of adduct levels. The method was applied in a biomonitoring study among workers in a carbon-electrode manufacturing plant, exposed to PAH. Although DNA adduct levels in peripheral blood lymphocytes of exposed workers, as determined by 32P-postlabelling, were not significantly different from those of controls, a significant difference was seen when smokers and non-smokers were compared.