Detection of O6-butyl- and O6-(4-hydroxybutyl)guanine in urothelial and hepatic DNA of rats given the bladder carcinogen N-nitrosobutyl(4-hydroxybutyl)amine

N-Nitrosobutyl(4-hydroxybutyl)amine (BBN) is a selective bladdercarcinogen in rats. Its organ specificity may depend on severalfactors, including metabolic activation, DNA alkylation andrepair within the target organ. Metabolic activation of BBN,which is asymmetrical, may result in butylating and 4-hydroxybutylatingspecies. To test this view, BBN was administered as a singleoral dose of 20 or 120 mg/rat or six doses of 20 mg/rat over2 weeks. The animals given the single 120 mg dose were killed3, 6 and 24 h after treatment Rats given 20 mg or 6x20 mg BBNwere killed 24 h after the last dose. DNA from liver and urothelialcells was hydrolyzed and analyzed for O⁶- butylguanine (O⁶-BuG)and O⁶-hydroxybutyOguanine [O⁶-(4-OH-Bu)G] as their pentafluorobenzyl-trimethylsilylderivatives by high-resolution gas chromatography—negativeion chemical ionization mass spectrometry with selective ionrecording after immunoaffinity extraction. Polyclonal antibodiesraised against O⁶-(4-hydroxybutyl)- guanosine [O⁶-(4-OH-Bu)GR]were coupled to CNBractivated Sepharose 4B. This was mixed witha gel coupled to antibodies raised against O⁶-BuG, already availablein the laboratory, and the mixed gel was used for the one-stepsample clean-up, enrichment and extraction of O⁶-(4-OH-Bu)Gand O⁶-BuG from hydrolyzed DNA. O⁶- BuG In urothelial DNA ofrats given a single dose of 120 mg BBN increased from 0.44 ±0.12 (µmol/mol guanine (mean ±SE) 3 h after treatment,to 17.9 ± 7.23 µmol/mol guanine at 24 h. O⁶-(4-OH-Bu)Gin the same tissue was 7.7 ± 3.19 (µmol/mol guanine3 h after treatment and 12.2 ± 7.01 µmol/mol guanineat 24 h. O⁶-BuG and O6-(4- OH-Bu)G were always lower in theliver than in urothelialcells. Twenty-four hours after a singledose of 20 mg BBN, urothelial O⁶-BuG was 5.41 ± 1.73(µmol/mol guanine anddid not accumulate after six dosesof 20 mg/rat BBN, since it was 2.59 ± 1.23 (µmol/molguanine 24 h after the last dose. O⁶-BuG in liver DNA was detectableafter the single dose of 20 mg, but not after 6x20 mg/rat BBN.O⁶-(4-OHBu) G was not detected in either the bladder or theliver after 20 mg or after the six doses of BBN. The resultsindicate that both butylating and 4-hydroxybutylating speciesare formed in the target organ DNA of rats given the bladdercarcinogen BBN, but that O⁶-BuG seems to be the lesion mostrelevant to the carcinogenic effect