DeCaprio A P, Briggs R G, Jackowski S J, Kim J C
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Toxicol Appl Pharmacol. 1988 Jan;92(1):75-85. doi: 10.1016/0041-008x(88)90229-3.
2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, reacts with protein amines to form alkylpyrrole adducts. Pyrrolylation of neurofilament protein may be the initiating molecular event in 2,5-HD neuropathy. The present study compares the neurotoxic and pyrrole-forming potentials of 2,5-HD with those of perdeuterio-2,5-HD ([D10]-2,5-HD) in the rat. Due to a requirement for C-H bond breaking in the reaction mechanism, the latter derivative was expected to exhibit a primary isotope effect, thus forming the pyrrole at a slower rate. In vitro studies confirmed that [D10]-2,5-HD pyrrolylated protein at only one-third of the initial rate seen with native 2,5-HD. Prolonged incubation resulted in similar pyrrole concentrations with both derivatives. Adult, male Wistar rats were administered daily (5 days/week) ip doses of either 3.5 mmol 2,5-HD or [D10]-2,5-HD/kg/day for 17 days or 2.5 mmol/kg/day for 38 days. At termination, animals administered 2,5-HD and [D10]-2,5-HD exhibited 27 and 8% body weight loss, respectively. Moderate to severe hindlimb paralysis was present in the 2,5-HD groups while only mild effects were seen in [D10]-2,5-HD-dosed rats. Neuropathological changes were prominent in spinal cord sections from 2,5-HD-treated animals, while no effects were present in rats given the deuterated derivative. Pyrrole adduct concentrations in serum and axonal cytoskeletal proteins from 2,5-HD-treated animals were two- to threefold higher than in rats given equimolar doses of [D10]-2,5-HD. Levels of covalent crosslinking of axonal cytoskeletal proteins (assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) appeared to correlate with pyrrole concentrations. Tissue concentrations of each diketone isomer were not significantly different, indicating similar uptake of native and deuterated 2,5-HD. Mass spectrometry revealed rapid back exchange of the terminal (methyl) but not of the internal (methylene) deuteriums of [D10]-2,5-HD in vivo. These findings support an absolute requirement for pyrrole formation in gamma-diketone neurotoxicity.
2,5 -己二酮（2,5 - HD）是正己烷的神经毒性代谢产物，它与蛋白质胺反应形成烷基吡咯加合物。神经丝蛋白的吡咯化可能是2,5 - HD神经病变中的起始分子事件。本研究比较了2,5 - HD与全氘代 - 2,5 -己二酮（[D10]-2,5 - HD）在大鼠体内的神经毒性和形成吡咯的潜力。由于反应机制中需要断裂C - H键，预计后一种衍生物会表现出一级同位素效应，从而以较慢的速率形成吡咯。体外研究证实，[D10]-2,5 - HD使蛋白质吡咯化的初始速率仅为天然2,5 - HD的三分之一。长时间孵育后，两种衍生物的吡咯浓度相似。成年雄性Wistar大鼠每天（每周5天）腹腔注射剂量为3.5 mmol 2,5 - HD或[D10]-2,5 - HD/kg/天，持续17天，或2.5 mmol/kg/天，持续38天。处死时，给予2,5 - HD和[D10]-2,5 - HD的动物体重分别减轻了27%和8%。2,5 - HD组出现中度至重度后肢麻痹，而给予[D10]-2,5 - HD的大鼠仅出现轻微影响。2,5 - HD处理动物的脊髓切片中神经病理变化显著，而给予氘代衍生物的大鼠则无影响。2,5 - HD处理动物的血清和轴突细胞骨架蛋白中的吡咯加合物浓度比给予等摩尔剂量[D10]-2,5 - HD的大鼠高两到三倍。轴突细胞骨架蛋白的共价交联水平（通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳评估）似乎与吡咯浓度相关。每种二酮异构体的组织浓度无显著差异，表明天然和氘代形式的2,5 - HD摄取相似。质谱分析显示，[D10]-2,5 - HD的末端（甲基）而非内部（亚甲基）氘在体内迅速发生回交换。这些发现支持了γ -二酮神经毒性中吡咯形成的绝对必要性。
