DETECTION OF O-6-BUTYL- AND O-6-(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 bladder carcinogen in rats. Its organ specificity may depend on several factors, including metabolic activation, DNA alkylation and repair within the target organ. Metabolic activation of BBN, which is asymmetrical, may result in butylating and 4-hydroxybutylating species. To test this view, BBN was administered as a single oral dose of 20 or 120 mg/rat or six doses of 20 mg/rat over 2 weeks. The animals given the single 120 mg dose were killed 3, 6 and 24 h after treatment. Rats given 20 mg or 6X20 mg BBN were killed 24 h after the last dose. DNA from liver and urothelial cells was hydrolyzed and analyzed for O-6-butylguanine (O-6-BuG) and O-6-(4-hydroxybutyl)guanine [O-6-(4-OH-Bu)G] as their pentafluorobenzyl-trimethylsilyl derivatives by high-resolution gas chromatography-negative ion chemical ionization mass spectrometry with selective ion recording after immunoaffinity extraction. Polyclonal antibodies raised against O-6-(4-hydroxybutyl) guanosine [O-6-(4-OH-Bu)GR] were coupled to CNBr-activated Sepharose 4B. This was mixed with a gel coupled to antibodies raised against O-6-BuG, already available in the laboratory, and the mixed gel was used for the one-step sample clean-up, enrichment and extraction of O-6-(4-OH-Bu)G and O-6-BuG from hydrolyzed DNA. O-6-BuG in urothelial DNA of rats given a single dose of 120 mg BBN increased from 0.44 +/- 0.12 mu mol/mol guanine (mean +/- SE)3 h after treatment, to 17.9 +/- 7.23 mu mol/mol guanine at 24 h. O-6-(4-OH-Bu)G in the same tissue was 7.7 +/- 3.19 mu mol/mol guanine 3 h after treatment and 12.2 +/- 7.01 mu mol/mol guanine at 24 h. O-6-BuG and O-6-(4-OH-Bu)G were always lower in the liver than in urothelial cells. Twenty-four hours after a single dose of 20 mg BBN, urothelial O-6-BuG was 5.41 +/- 1.73 mu mol/mol guanine and did not accumulate after six doses of 20 mg/rat BBN, since it was 2.59 +/- 1.23 mu mol/mol guanine 24 h after the last dose. O-6-BuG in liver DNA was detectable after the single dose of 20 mg, but not after 6X20 mg/rat BBN. O-6-(4-OHBu)G was not detected in either the bladder or the liver after 20 mg or after the six doses of BBN. The results indicate that both butylating and 4-hydroxybutylating species are formed in the target organ DNA of rats given the bladder carcinogen BBN, but that O-6-BuG seems to be the lesion most relevant to the carcinogenic effect.