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 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 6 x 20 mg BBN were killed 24 h after the last dose. DNA from liver and urothelial cells was hydrolyzed and analyzed for O6-butylguanine (O6-BuG) and O6-(4-hydroxybutyl)guanine [O6-(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 O6-(4-hydroxybutyl)-guanosine [O6-(4-OH-Bu)GR] were coupled to CNBr-activated Sepharose 4B. This was mixed with a gel coupled to antibodies raised against O6-BuG, already available in the laboratory, and the mixed gel was used for the one-step sample clean-up, enrichment and extraction of O6-(4-OH-Bu)G and O6-BuG from hydrolyzed DNA. O6-BuG in urothelial DNA of rats given a single dose of 120 mg BBN increased from 0.44 +/- 0.12 mumol/mol guanine (mean +/- SE) 3 h after treatment, to 17.9 +/- 7.23 mumol/mol guanine at 24 h. O6-(4-OH-Bu)G in the same tissue was 7.7 +/- 3.19 mumol/mol guanine 3 h after treatment and 12.2 +/- 7.01 mumol/mol guanine at 24 h. O6-BuG and O6-(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 O6-BuG was 5.41 +/- 1.73 mumol/mol guanine and did not accumulate after six doses of 20 mg/rat BBN, since it was 2.59 +/- 1.23 mumol/mol guanine 24 h after the last dose. O6-BuG in liver DNA was detectable after the single dose of 20 mg, but not after 6 x 20 mg/rat BBN. O6-(4-OH-Bu)G was not detected in either the bladder or the liver after 20 mg or after the six doses of BBN.(ABSTRACT TRUNCATED AT 400 WORDS)
Laboratory of Environmental Pharmacology and Toxicology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
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Airoldi
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Magagnotti
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Bonfanti
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Chiappetta
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Lolli
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Medana
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De Gregorio
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Fanelli
D000212:Acyclovir / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D002085:Butylhydroxybutylnitrosamine / Q000378:metabolism* / Q000633:toxicity*; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism*; D004249:DNA Damage; D004797:Enzyme-Linked Immunosorbent Assay; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D001743:Urinary Bladder / Q000033:anatomy & histology / Q000187:drug effects* / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced*
D000906:Antibodies; C039093:9-(4-hydroxybutyl)guanine; D002085:Butylhydroxybutylnitrosamine; D006147:Guanine; C046953:O(6)-n-butylguanine; D004247:DNA; D000212:Acyclovir