Fantel A G, Juchau M R, Tracy J W, Burroughs C J, Person R E
Department of Pediatrics, School of Medicine, University of Washington, Seattle 98195.
Teratology. 1989 Jan;39(1):63-74. doi: 10.1002/tera.1420390108.
Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.
反应性中间体与胚胎大分子的共价结合是硝唑咪（NDZ）胚胎毒性的主要机制。研究了胚胎硫醇含量和氧合作用对以下方面的影响：1）畸形发生率；2）还原代谢；3）NDZ还原生物转化产生的代谢产物与胚胎大分子的共价结合。将结果与NDZ的非致畸类似物4'-甲基硝唑咪（MNDZ）的研究结果进行了比较。第10天的大鼠胚胎在体外分别用L-丁硫氨酸-S，R-亚砜亚胺（BSO）或N-乙酰半胱氨酸（NAC）预处理5小时，以调节其谷胱甘肽（GSH）含量。BSO降低了GSH水平，但NAC无效。预处理后，胚胎在初始氧浓度为5%的[14C]NDZ或[14C]MNDZ存在下再培养15小时。在培养期结束时（第11天，上午），检查那些有活跃心跳和卵黄循环的胚胎是否有不对称畸形。从培养基中对药物代谢产物进行多次萃取，并进行定量高效液相色谱（HPLC）分析。用三氯乙酸（TCA）萃取胚胎匀浆，以估计放射性标记母体化合物/代谢产物的共价结合。对其他胚胎进行放射自显影分析。降低胚胎GSH组织水平的BSO预处理显著增加了NDZ向1-硫代氨基甲酰基-2-咪唑烷酮（TCI）的转化（通过NDZ的还原代谢产生）以及共价结合的标记物，但未能增加胚胎毒性。相比之下，NAC对胚胎GSH水平、TCI生成或共价结合没有显著影响。由于NDZ向TCI的代谢速率和共价结合都可能独立于畸形发生率而变化，我们得出结论，它们不是NDZ和相关硝基杂环化合物胚胎毒性的关键机制因素。
