Boogaard P J, Sumner S C, Bond J A
Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709, USA.
Toxicol Appl Pharmacol. 1996 Feb;136(2):307-16. doi: 10.1006/taap.1996.0037.
1,3-Butadiene (BD) has been classified as a probable human carcinogen based on sufficient evidence of a carcinogenic response in B6C3F1 mice and Sprague-Dawley rats and limited human evidence of carcinogenicity. Mice are much more susceptible to BD-induced carcinogenicity than rats. Previous in vitro studies revealed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. BDE was also readily quantitated in blood and tissues of mice exposed to BD but could not be detected in rats similarly exposed. These findings suggest a key role for BDE in BD-induced carcinogenicity. The purpose of this study was to characterize the glutathione (GSH) conjugation of BDE by liver and lung cytosol from B6C3F1 mice and Sprague-Dawley rats and human liver cytosol from six different individuals in vitro. BDE and glycine-[2-3H]GSH were incubated, at pH 7.4, with cytosol. 13C NMR and mass spectral analysis indicated formation of two isomeric conjugates, S-(1-(hydroxy-methyl)-2,3-epoxypropyl)glutathione and S-(2-hydroxy-3,4-epoxy--butyl)glutathione, which were rapidly hydrolyzed in cytosol to the corresponding trihydroxy conjugates. Total conjugates were quantitated by HPLC. Conjugation of BDE with GSH followed Michaelis-Menten kinetics in human as well as rat and mouse cytosolic fractions. The conjugation rates in mouse and rat liver cytosol were similar (Vmax 162 +/- 16 and 186 +/- 37 nmol/mg protein/min, respectively) and an order of magnitude higher than in human liver cytosol (Vmax 6.4 +/- 1.9 nmol/mg protein/min). the apparent KM values were lower in human (2.1 +/- 1.4 mM) than mouse (6.4 +/- 1.6 mM) or rat (24 +/- 6 mM) liver. Mouse lung cytosol (Vmax 38.5 +/- 2.5 nmol/mg protein/min, KM 1.70 +/- 0.37mM) is also more efficient in GSH conjugation than rat lung cytosol (Vmax 17.1 +/- 3.0 nmol/mg protein/min, KM +/- 1.7 mM). These results suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in hepatic or pulmonary GSH conjugation of BDE. Also, considering the low oxidation rates of BD to BMO and of BMO to BDE in humans as compared to mice, the relatively low capacity of GSH conjugation of BDE in human liver will not necessarily lead to increased BDE blood levels in humans potentially exposed to BD.
基于B6C3F1小鼠和Sprague-Dawley大鼠致癌反应的充分证据以及人类致癌性的有限证据，1,3-丁二烯（BD）已被归类为可能的人类致癌物。小鼠比大鼠对BD诱导的致癌性更敏感。先前的体外研究表明，小鼠肝微粒体从BD形成1,2-环氧-3-丁烯（BMO）以及从BMO形成1,2:3,4-二环氧丁烷（BDE）的速率远高于大鼠或人类微粒体。在暴露于BD的小鼠的血液和组织中也很容易定量检测到BDE，但在同样暴露的大鼠中却检测不到。这些发现表明BDE在BD诱导的致癌性中起关键作用。本研究的目的是在体外表征来自B6C3F1小鼠和Sprague-Dawley大鼠的肝和肺胞质溶胶以及来自六个不同个体的人肝胞质溶胶对BDE的谷胱甘肽（GSH）共轭作用。将BDE和甘氨酸-[2-³H]GSH在pH 7.4下与胞质溶胶一起孵育。¹³C NMR和质谱分析表明形成了两种异构共轭物，即S-(1-(羟甲基)-2,3-环氧丙基)谷胱甘肽和S-(2-羟基-3,4-环氧丁基)谷胱甘肽，它们在胞质溶胶中迅速水解为相应的三羟基共轭物。通过HPLC对总共轭物进行定量。BDE与GSH的共轭作用在人以及大鼠和小鼠的胞质组分中遵循米氏动力学。小鼠和大鼠肝胞质溶胶中的共轭速率相似（Vmax分别为162±16和186±37 nmol/mg蛋白质/分钟），比人肝胞质溶胶中的共轭速率高一个数量级（Vmax为6.4±1.9 nmol/mg蛋白质/分钟）。人肝中的表观KM值（2.1±1.4 mM）低于小鼠（6.4±1.6 mM）或大鼠（24±6 mM）肝中的表观KM值。小鼠肺胞质溶胶（Vmax为38.5±2.5 nmol/mg蛋白质/分钟，KM为1.70±0.37 mM）在GSH共轭方面也比大鼠肺胞质溶胶（Vmax为17.1±3.0 nmol/mg蛋白质/分钟，KM为±1.7 mM）更有效。这些结果表明，吸入暴露于BD后，小鼠体内BDE的血液浓度高于大鼠，这并非由于肝或肺中BDE的GSH共轭作用存在差异。此外，考虑到与小鼠相比，人类中BD氧化为BMO以及BMO氧化为BDE的速率较低，人肝中BDE的GSH共轭能力相对较低不一定会导致潜在暴露于BD的人体内BDE血液水平升高。
