IMMUNOCHEMICAL, P-32 POSTLABELING, AND GC MS DETECTION OF 4-AMINOBIPHENYL-DNA ADDUCTS IN HUMAN PERIPHERAL LUNG IN RELATION TO METABOLIC-ACTIVATION PATHWAYS INVOLVING PULMONARY N-OXIDATION, CONJUGATION, AND PEROXIDATION/

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the hum an urothelium. Since preliminary studies indicated that even higher le vels of ABP-DNA adducts may be present in human peripheral lung, we ut ilized a sensitive immunochemical assay, in combination with P-32-post labeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(g uan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smok ers and ex-smokers. No differences in adduct levels were detected betw een smokers and ex-smokers by immunoassay. In contrast, the P-32-postl abeling method showed statistically significant differences between ad duct levels in smokers and ex-smokers; however, a relatively high back ground of smoking-related adducts chromatograph near the major ABP add ucts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by P-32-postlabeling were 20 - to 60-fold lower than that measured by immunoassay. Since P-32-postl abeling may underestimate and immunochemical assays may overestimate a dduct levels in the lung, selected samples were also evaluated by GC/M S. The immunochemical and GC/MS data were concordant, leading us to co nclude that N-(guan-8-yl)-ABP adducts were not related to smoking stat us. Since ABP-DNA adduct levels in human lung did not correlate with s moking status as measured by immunoassay and GC/MS, the metabolic acti vation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be respons ible for the adduct. The rates of microsomal ABP N-oxidation were belo w the limit of detection, which was consistent with a lack of detectab le cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comp arative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to t his activity. 4-Nitrobiphenyl reductase activity was found in lung mic rosomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP . Lung microsomes also demonstrated high peroxidative activation of AB P, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, an d 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomet hacin or eicosatetraynoic acid, which suggested the primary involvemen t of myeloperoxidase rather than prostaglandin H synthase or lipoxygen ase. This was confirmed by immunoinhibition and immunoprecipitation st udies using solubilized human lung microsomes and antisera specific fo r myeloperoxidase. These data suggest that ABP-DNA adducts in human lu ng result from some environmental exposure to 4-nitrobiphenyl. The bio activation pathways appear to involve: (1) metabolic reduction to N-hy droxy-ABP and subsequent O-acetylation by NAT1 and/or NAT2; and (2) me tabolic reduction to ABP and subsequent peroxidation by myeloperoxidas e. The myeloperoxidase activity appears to be the highest peroxidase a ctivity measured in mammalian tissue and is consistent with the presen ce of neutrophils and polymorphonuclear leukocytes surrounding particu late matter derived from cigarette smoking. (C) 1997 Elsevier Science B.V.