Airoldi L, Magagnotti C, Bonfanti M, Chiappetta L, Lolli M, Medana C, De Gregorio G, Fanelli R
Laboratory of Environmental Pharmacology and Toxicology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Carcinogenesis. 1994 Oct;15(10):2297-301. doi: 10.1093/carcin/15.10.2297.
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)
N-亚硝基丁基（4-羟基丁基）胺（BBN）是大鼠体内一种选择性膀胱致癌物。其器官特异性可能取决于多种因素，包括代谢活化、靶器官内的DNA烷基化和修复。不对称的BBN的代谢活化可能产生丁基化和4-羟基丁基化产物。为验证这一观点，给大鼠单次口服20或120 mg/只的BBN，或在2周内分6次给予20 mg/只的BBN。给予单次120 mg剂量的动物在治疗后3、6和24小时处死。给予20 mg或6×20 mg BBN的大鼠在最后一剂后24小时处死。将肝脏和尿路上皮细胞的DNA水解，并通过高分辨率气相色谱-负离子化学电离质谱法，在免疫亲和萃取后进行选择性离子记录，分析作为其五氟苄基-三甲基硅烷基衍生物的O6-丁基鸟嘌呤（O6-BuG）和O6-（4-羟基丁基）鸟嘌呤[O6-（4-OH-Bu）G]。针对O6-（4-羟基丁基）鸟苷[O6-（4-OH-Bu）GR]产生的多克隆抗体与溴化氰活化的琼脂糖4B偶联。将其与实验室已有的与针对O6-BuG产生的抗体偶联的凝胶混合，混合凝胶用于从水解的DNA中一步净化、富集和提取O6-（4-OH-Bu）G和O6-BuG。给予单次120 mg BBN的大鼠尿路上皮DNA中的O6-BuG在治疗后3小时从0.44±0.12 μmol/mol鸟嘌呤（平均值±标准误）增加到24小时时的17.9±7.23 μmol/mol鸟嘌呤。同一组织中O6-（4-OH-Bu）G在治疗后3小时为7.7±3.19 μmol/mol鸟嘌呤，24小时时为12.2±7.01 μmol/mol鸟嘌呤。肝脏中的O6-BuG和O6-（4-OH-Bu）G始终低于尿路上皮细胞。单次给予20 mg BBN后24小时，尿路上皮O6-BuG为5.41±1.73 μmol/mol鸟嘌呤，在给予6次20 mg/只的BBN后未积累，因为在最后一剂后24小时为2.59±1.23 μmol/mol鸟嘌呤。单次给予20 mg后肝脏DNA中可检测到O6-BuG，但给予6×20 mg/只的BBN后未检测到。给予20 mg或6次BBN后，膀胱或肝脏中均未检测到O6-（4-OH-Bu）G。（摘要截断于400字）
