ABSTRACT
Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/analysis , Cytochrome P-450 Enzyme System/genetics , DNA Adducts , DNA/analysis , DNA/metabolism , Glutathione Transferase/genetics , Lung/chemistry , Polycyclic Compounds/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/analogs & derivatives , Base Sequence , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/analysis , Disease Susceptibility , Genotype , Humans , Molecular Sequence Data , Mutation , Polymorphism, GeneticABSTRACT
Human exposure to reactive oxygen species is unavoidable and has been implicated in the etiology of a number of human diseases. This exposure results in the formation of various modified DNA bases: the promutagenic lesion 8-hydroxydeoxyguanosine (8OHdG), in particular, is a major product. We have developed an assay using ion-pair HPLC and 32P-postlabelling to quantify 8OHdG in human DNA with high specificity and sensitivity. An internal standard is used to account for variations in labelling efficiency. Chemically synthesized 8OHdG 3'-monophosphate and 5'-monophosphate standards were used to optimize the HPLC-32P-postlabelling and TLC separative steps, respectively. The assay was validated using known ratios of 8OHdG to normal nucleotides. The limit of detection is in the range of one 8OHdG residue per 10(6)-10(7) dG residues. Using this procedure, 8OHdG levels of 16-35 8OHdG adducts per 10(5) dG residues have been found in leukocytes isolated from patients who received 180-200 cGy of ionizing radiation. These levels were 2-4-fold greater than those found in an unexposed individual. Since 8OHdG may be formed during DNA extraction and digestion, current procedures for measuring background levels are discussed.