Carmella S G, Kagan S S, Spratt T E, Hecht S S
Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595.
Cancer Res. 1990 Sep 1;50(17):5453-9.
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) forms hemoglobin adducts in rats. Upon mild base treatment, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) is released from this globin. HPB has been suggested as a dosimeter for exposure to and metabolic activation of tobacco-specific nitrosamines. The purpose of this study was 2-fold: (a) to determine whether cysteine adducts of NNK were precursors to HPB, and (b) to determine to what extent cysteine adducts accounted for the material bound to globin that is not released upon mild base hydrolysis. The chemistry of cysteine adduct formation was investigated by reacting N-acetyl-L-cysteine with three model compounds for pyridyloxobutylation by metabolically activated NNK: 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone (1); 4-oxo-4-(3-pyridyl)-1-butylmethanesulfonate (2); and 4-iodo-1-(3-pyridyl)-1-butanone (3). Five adducts were isolated and characterized by their spectral properties and by independent syntheses: two diastereomers of N-acetyl-S-[1-methyl-3-oxo-3-(3-pyridyl)propyl]-L-cysteine (7a,b), N-acetyl-S-[4-oxo-4-(3-pyridyl)-1-butyl]-L-cysteine (9), and two diastereomers of N-acetyl-S-(2-[2-(3-pyridyl)]-2,3,4,5-tetrahydrofuranyl)-L-cystein e (11a,b). Only 11a,b produced HPB upon mild base treatment; however, the chemistry of this adduct did not support its role as a major precursor to HPB released upon base treatment of globin. The formation of adducts in rat hemoglobin was then examined by reacting it with tritium-labeled 1 [( 5-3H]1) or tritium-labeled 4-oxo-4-(3-pyridyl)-1-butyl p-toluenesulfonate [( 5-3H]4). The results demonstrated that the amino acids corresponding to 7a,b were present in hemoglobin reacted with [5-3H]1, accounting for 72% of the bound tritium. Amino acids corresponding to 9 were not detected in this globin. In contrast, hemoglobin reacted with [5-3H]4 contained the amino acid corresponding to 9 (15% of bound tritium), but not those corresponding to 7a,b. These results indicated that the alpha, beta-unsaturated ketone, 1-(3-pyridyl)-2-buten-1-one (5), played a major role in the hemoglobin binding of 1, but not of 4. Cysteine adducts were not detected in globin isolated from rats treated with [5-3H]NNK. The results of this study provide insights into the mechanisms of cysteine adduct formation in vitro by pryidyloxobutylating agents and indicate that these adducts are not formed in NNK-treated rats.
烟草特异性亚硝胺4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)在大鼠体内形成血红蛋白加合物。经过温和的碱处理后，4-羟基-1-(3-吡啶基)-1-丁酮(HPB)从该球蛋白中释放出来。HPB被认为是烟草特异性亚硝胺暴露和代谢活化的剂量计。本研究的目的有两个：(a)确定NNK的半胱氨酸加合物是否是HPB的前体，(b)确定半胱氨酸加合物在多大程度上解释了与球蛋白结合且在温和碱水解后不释放的物质。通过使N-乙酰-L-半胱氨酸与三种经代谢活化的NNK进行吡啶氧基丁基化反应的模型化合物反应，研究了半胱氨酸加合物的形成化学：4-(乙氧羰基亚硝胺基)-1-(3-吡啶基)-1-丁酮(1)；4-氧代-4-(3-吡啶基)-1-丁基甲磺酸盐(2)；以及4-碘-1-(3-吡啶基)-1-丁酮(3)。分离出五种加合物，并通过其光谱性质和独立合成进行表征：N-乙酰-S-[1-甲基-3-氧代-3-(3-吡啶基)丙基]-L-半胱氨酸的两种非对映异构体(7a,b)、N-乙酰-S-[4-氧代-4-(3-吡啶基)-1-丁基]-L-半胱氨酸(9)以及N-乙酰-S-(2-[2-(3-吡啶基)]-2,3,4,5-四氢呋喃基)-L-半胱氨酸的两种非对映异构体(11a,b)。只有11a,b在温和碱处理后产生HPB；然而，该加合物的化学性质不支持其作为球蛋白碱处理后释放的HPB的主要前体的作用。然后通过使大鼠血红蛋白与氚标记的1[(5-3H]1)或氚标记的4-氧代-4-(3-吡啶基)-1-丁基对甲苯磺酸盐[(5-3H]4)反应，研究了大鼠血红蛋白中加合物的形成。结果表明，与[5-3H]1反应的血红蛋白中存在对应于7a,b的氨基酸，占结合氚的72%。在该球蛋白中未检测到对应于9的氨基酸。相比之下，与[5-3H]4反应的血红蛋白含有对应于9的氨基酸(占结合氚的15%)，但不含有对应于7a,b的氨基酸。这些结果表明，α,β-不饱和酮1-(3-吡啶基)-2-丁烯-1-酮(5)在1与血红蛋白的结合中起主要作用，而在4与血红蛋白的结合中不起主要作用。在用[5-3H]NNK处理的大鼠分离的球蛋白中未检测到半胱氨酸加合物。本研究结果为吡啶氧基丁基化剂体外形成半胱氨酸加合物的机制提供了见解，并表明这些加合物在经NNK处理的大鼠中未形成。
