Detection and characterization of carcinogen—DNA adducts in exfoliated urothelial cells from 4-aminobiphenyl-treated dogs by 32P-postlabelling and subsequent thin-layer and high-pressure liquid chromatography

Abstract

The recent development of sensitive methods to detect carcinogen-DNA adducts offers a useful biochemical approach to human risk assessment. However, a major obstacle to developing a human biomonitoring method for carcinogen-DNA adducts has been the problem of obtaining target tissue DNA samples by non-invasive means. This work describes a method for the isolation of nanogram quantities of DNA from urothelial cells exfoliated into urine and the detection of carcinogen-DNA adducts from that DNA by 32P-postlabelling methods. Urine samples were collected on ice from dogs treated with 4-aminobiphenyl (ABP) over a 2-week period and pooled according to an experimental plan that involved analysis of cumulative 48- or 72-h samples. The pooled samples were sieved and then washed repeatedly with a sucrose buffer to dissolve contaminating triple phosphate (MgNH4PO4), calcium oxalate and uric acid crystals. DNA was isolated using an enzyme-solvent extraction method with the DNA being co-precipitated from ethanol with glycogen. The DNA was hydrolysed and postlabelled with 32P under conditions of excess ATP so that nucleotides were labelled quantitatively. Adducts observed on the resulting thin-layer chromatograms were identical to those obtained from DNA modified in vitro with N-hydroxy-4-aminobiphenyl and from dog bladder urothelial DNA isolated from the ABP-dosed animals at termination of the experiment. Furthermore, a dose-related increase in ABP-DNA adduct formation was demonstrated. Thus, it appears that the carcinogen-DNA adduct levels in the exfoliated bladder cells are reflective of the levels in the intact urothelium once steady-state levels have been achieved. To establish the identity of the major ABP-urothelial DNA adduct in chronically-treated dogs, the predominant 32P-postlabelled adduct was eluted from the thin-layer chromatograms and co-injected on an HPLC system with a synthetic [3H]N-(deoxyguanosin-8-yl)-4-aminobiphenyl-3',5'-bisphosphate standard. Dual-label analysis of 3H and 32P indicated that both eluted from the column in the same fraction, which coincided with the UV absorbance peak of the synthetic marker. Preliminary experiments with exfoliated urothelial cells from human urines indicate that these methods should have general utility for monitoring humans exposed to urinary bladder carcinogens and for investigations of the biochemical mechanisms by which such adducts are formed in the urothelium.