Combined micropreparative techniques with synchronous fluorescence spectroscopy or 32P-postlabelling assay for...

Methods for the detection of carcinogen-DNA adducts require both sensitivity and specificity. The 32P-postlabelling assay is highly sensitive but lacks adduct specificity. The strategy reported herein combines micropreparative techniques including HPLC and immunoaffinity chromatography (IAC) to enhance chemical specificity. The resultant assays have retained sensitivity for human DNA analysis. Combined HPLC and 32P-postlabelling has detected 7-methyldeoxyguanosine in human lung samples, while combined IAC and 32P-postlabelling has detected O6-methyldeoxyguanosine adducts in stomach tissue. The limits of detection are one adduct in 10(7) and 10(8) unmodified deoxyguanosine (dG), respectively. IAC was combined with 32P-postlabelling to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human lung. The detection limit was one adduct in 10(7) dG. Our laboratory has also employed synchronous fluorescence spectroscopy (SFS) for the detection of adducts formed from benzo[a]pyrene in human lung. Complex fluorescence matrices suggest the presence of other PAH-DNA adducts. Both the SFS assay and the 32P-postlabelling assay were applied to a series of human samples and a high correlation was found for adduct levels. The development of such assays using synthetic standards, internal standards, determination of calibration curves and validation with corroborating methods is required for chemically specific and sensitive adduct detection.