Lu Ding, Sullivan Mathilde M, Phillips Martin B, Peterson Lisa A
Department of Medicinal Chemistry, Masonic Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA.
Chem Res Toxicol. 2009 Jun;22(6):997-1007. doi: 10.1021/tx800377v.
Furan is a liver toxicant and carcinogen in rodents. On the basis of these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450-catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids, and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a monoglutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized as follows: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine, and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from the reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the epsilon-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the alpha- and the epsilon-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the epsilon-amino group of lysine. A GSH-BDA-lysine cross-link was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the epsilon-amino group of lysine; however, small amounts of the alpha-amino reaction product were also observed. Western analysis of liver and hepatocyte protein extracts using anti-GSH antibody indicated that GSH was covalently linked to proteins in tissues or cells exposed to furan. Our data support the hypothesis that GSH-BDA can react with either free lysine or protein lysine groups. These data suggest that there are multiple pathways by which furan can modify cellular nucleophiles. In one pathway, BDA reacts directly with proteins to form cysteine-lysine reaction products. In another, BDA reacts with GSH to form GSH-BDA conjugates, which then react with cellular nucleophiles like free lysine or lysine moieties in proteins. Both pathways will give rise to N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine. Given the abundance of these metabolites in urine of furan-treated rats, these pathways appear to be major pathways of furan biotransformation in vivo.
呋喃是啮齿动物的肝脏毒物和致癌物。基于这些观察结果以及人类接触呋喃的巨大可能性，呋喃已被归类为可能的人类致癌物。呋喃诱发肿瘤的机制尚不清楚。然而，其毒性需要细胞色素P450催化的呋喃氧化。这种氧化的产物顺式-2-丁烯-1,4-二醛（BDA）能与谷胱甘肽、氨基酸和DNA迅速反应，并且在Ames试验菌株TA104中是一种细菌诱变剂。对呋喃尿代谢物的表征有望提供有关活性代谢物结构的信息。最近，已鉴定出几种尿代谢物。我们报告了一种单谷胱甘肽-BDA反应产物，即N-[4-羧基-4-(3-巯基-1H-吡咯-1-基)-1-氧代丁基]-L-半胱氨酰甘氨酸环状硫化物的存在。呋喃的另外三种尿代谢物也被表征如下：R-2-乙酰氨基-6-(2,5-二氢-2-氧代-1H-吡咯-1-基)-1-己酸、N-乙酰-S-[1-(5-乙酰氨基-5-羧基戊基)-1H-吡咯-3-基]-L-半胱氨酸及其亚砜。据推测，这三种代谢物源自降解的蛋白质加合物。然而，并未排除这些代谢物是由BDA与游离赖氨酸和/或半胱氨酸反应产生的可能性。在后一种情况下，可以预测硫醇-BDA与游离赖氨酸的反应不会仅发生在ε-氨基上。BDA与赖氨酸存在时的N-乙酰半胱氨酸或谷胱甘肽的反应表明，赖氨酸的α-氨基和ε-氨基均可被硫醇-BDA修饰。N-乙酰半胱氨酸-BDA-N-乙酰赖氨酸尿代谢物仅通过赖氨酸的ε-氨基相连。谷胱甘肽-BDA-赖氨酸交联物是呋喃的一种重要肝细胞代谢物。在这种情况下，主要产物是与赖氨酸的ε-氨基反应生成的；然而，也观察到少量的α-氨基反应产物。使用抗谷胱甘肽抗体对肝脏和肝细胞蛋白提取物进行的蛋白质印迹分析表明，谷胱甘肽在暴露于呋喃的组织或细胞中与蛋白质共价连接。我们的数据支持谷胱甘肽-BDA可与游离赖氨酸或蛋白质赖氨酸基团反应的假说。这些数据表明，呋喃可以通过多种途径修饰细胞亲核试剂。在一条途径中，BDA直接与蛋白质反应形成半胱氨酸-赖氨酸反应产物。在另一条途径中，BDA与谷胱甘肽反应形成谷胱甘肽-BDA缀合物，然后该缀合物与细胞亲核试剂如游离赖氨酸或蛋白质中的赖氨酸部分反应。两条途径都会产生N-乙酰-S-[1-(5-乙酰氨基-5-羧基戊基)-1H-吡咯-3-基]-L-半胱氨酸。鉴于这些代谢物在呋喃处理的大鼠尿液中的丰度，这些途径似乎是呋喃在体内生物转化的主要途径。
