Fluorescence detection of benzo[a]pyrene-DNA adducts in human lung

Abstract

Improved techniques are described for the specific identification of benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts in human tissues. Immunoaffinity chromatography, synchronous fluorescence spectroscopy and second-derivative synchronous fluorescence spectroscopy have previously been used to detect BPDE-DNA adducts in human placenta. Here we report how these methods, together with HPLC and the generation of complete fluorescence excitation—emission matrices, have been used to identify unequivocally BPDE-DNA adducts in samples of human lung. BPDE nucleotide adducts were isolated with immunoaffinity chromatography columns bearing antibodies raised against the (±)anti-7,8-diol-9,10-epoxide-deoxyguanosine adduct of betizo[a]pyrene. These adducts were hydrolyzed to tetrahydrotetrols and the hydrolysis products subjected to HPLC. The major product isolated by HPLC, benzo[a]-pyrene-7,10/8,9-tetrahydrotetrol, was determined by fluorescence spectroscopy. Using this method, levels of BPDE-DNA adducts in the range of 1–40 in 10⁸ nucleotides were measured in 6 out of 25 samples, with a lower detection limit of one adduct in 10⁸ nucleotides. The data may also indicate that adduct levels show regional variation in different parts of the same lung.