DETECTION OF O-6-BUTYL- AND O-6-(4-HYDROXYBUTYL)GUANINE IN UROTHELIALAND HEPATIC DNA OF RATS GIVEN THE BLADDER CARCINOGEN N-NITROSOBUTYL(4-HYDROXYBUTYL)AMINE

N-Nitrosobutyl(4-hydroxybutyl)amine (BBN) is a selective bladder carci nogen in rats. Its organ specificity may depend on several factors, in cluding metabolic activation, DNA alkylation and repair within the tar get organ. Metabolic activation of BBN, which is asymmetrical, may res ult 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 d ose were killed 3, 6 and 24 h after treatment. Rats given 20 mg or 6X2 0 mg BBN were killed 24 h after the last dose. DNA from liver and urot helial 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 pentafluor obenzyl-trimethylsilyl derivatives by high-resolution gas chromatograp hy-negative ion chemical ionization mass spectrometry with selective i on recording after immunoaffinity extraction. Polyclonal antibodies ra ised against O-6-(4-hydroxybutyl) guanosine [O-6-(4-OH-Bu)GR] were cou pled to CNBr-activated Sepharose 4B. This was mixed with a gel coupled to antibodies raised against O-6-BuG, already available in the labora tory, and the mixed gel was used for the one-step sample clean-up, enr ichment and extraction of O-6-(4-OH-Bu)G and O-6-BuG from hydrolyzed D NA. O-6-BuG in urothelial DNA of rats given a single dose of 120 mg BB N increased from 0.44 +/- 0.12 mu mol/mol guanine (mean +/- SE)3 h aft er treatment, to 17.9 +/- 7.23 mu mol/mol guanine at 24 h. O-6-(4-OH-B u)G in the same tissue was 7.7 +/- 3.19 mu mol/mol guanine 3 h after t reatment 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. T wenty-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 th e single dose of 20 mg, but not after 6X20 mg/rat BBN. O-6-(4-OHBu)G w as not detected in either the bladder or the liver after 20 mg or afte r 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 le sion most relevant to the carcinogenic effect.