Immunochemical, 32P-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 human urothelium. Since preliminary studies indicated that even higher levels of ABP-DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by 32P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N-(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP-DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N-oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the primary involvement of myeloperoxidase rather than prostaglandin H synthase or lipoxygenase. This was confirmed by immunoinhibition and immunoprecipitation studies using solubilized human lung microsomes and antisera specific for myeloperoxidase. These data suggest that ABP-DNA adducts in human lung result from some environmental exposure to 4-nitrobiphenyl. The bioactivation pathways appear to involve: (1) metabolic reduction to N-hydroxy-ABP and subsequent O-acetylation by NAT1 and/or NAT2; and (2) metabolic reduction to ABP and subsequent peroxidation by myeloperoxidase. The myeloperoxidase activity appears to be the highest peroxidase activity measured in mammalian tissue and is consistent with the presence of neutrophils and polymorphonuclear leukocytes surrounding particulate matter derived from cigarette smoking.
Culp
S J
SJ
National Center for Toxicological Research, Jefferson, AR 72079, USA.
Roberts
D W
DW
Talaska
G
G
Lang
N P
NP
Fu
P P
PP
Lay
J O
JO
Teitel
C H
CH
Snawder
J E
JE
Von Tungeln
L S
LS
Kadlubar
F F
FF
D000217:Acyltransferases / Q000378:metabolism; D000611:Aminobiphenyl Compounds / Q000378:metabolism*; D001560:Benzidines / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000378:metabolism; D001711:Biotransformation; D001713:Biphenyl Compounds / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D008401:Gas Chromatography-Mass Spectrometry; D006151:Guanosine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism*; D012907:Smoking; D015238:Sulfotransferases / Q000378:metabolism
D000611:Aminobiphenyl Compounds; D001560:Benzidines; D001713:Biphenyl Compounds; D002273:Carcinogens; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D006151:Guanosine; C006757:4-biphenylamine; C029876:benzidine; D001564:Benzo(a)pyrene; C028578:N-(guanosin-8-yl)-4-aminobiphenyl; D010544:Peroxidases; D009195:Peroxidase; D000217:Acyltransferases; D015238:Sulfotransferases; C040438:4-nitrobiphenyl