Institute of Toxicology, Merck Serono, 64293 Darmstadt, Germany.
Toxicology. 2010 Sep 10;275(1-3):36-49. doi: 10.1016/j.tox.2010.05.012. Epub 2010 Jun 8.
The zebrafish Danio rerio embryo test with metabolic activation (mDarT) was developed to assess the teratogenic effects of proteratogens. In this study induced rat liver microsomes (RLM) were used as a mammalian metabolic activation system (MAS), since they contain various cytochrome P450 (CYP) isoforms at high concentrations. Acetaminophen (APAP) is considered not to be teratogenic in vivo, however, in vitro teratogenic effects were observed, e.g. in rat whole embryo culture. The CYP2E1 activation of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) mainly occurs, when the glucuronidation and sulfatation pathways are saturated. In vivo the soft electrophile NAPQI is usually inactivated by hepatic reduced glutathione (GSH), a soft nucleophile. In this study, we investigated the teratogenic and lethal effects of APAP after CYP activation in zebrafish embryos. In the test groups with APAP and metabolic activation 11.7+/-7.6% (2mM), 25.0+/-8.7% (4mM) and 50.0+/-21.8% (6mM) affected embryos were seen, reaching statistical significance at 4mM APAP. When embryos were exposed to 6mM APAP, MAS and 3mM GSH the percentage of affected embryos decreased to 6.7+/-5.8%. In contrast teratogenic and lethal effects of metabolically activated cyclophosphamide (CPA) could not be prevented by GSH addition, because the CPA metabolites are strong electrophiles, which preferentially bind to hard nucleophiles like DNA and RNA. The teratogenic and lethal effects of metabolically activated APAP observed in zebrafish embryos with our mDarT standard protocol could be explained by the lack of GSH as a detoxifying system. By adding GSH it was possible to mimic the situation in mammals and thus avoid teratogenic effects in zebrafish embryos.
斑马鱼胚胎代谢激活试验（mDarT）被开发用于评估原致畸物的致畸作用。在这项研究中，诱导的大鼠肝微粒体（RLM）被用作哺乳动物代谢激活系统（MAS），因为它们以高浓度包含各种细胞色素 P450（CYP）同工酶。乙酰氨基酚（APAP）被认为在体内不是致畸物，但是在体外观察到致畸作用，例如在大鼠全胚胎培养中。APAP 被 CYP2E1 激活为反应性代谢物 N-乙酰-对苯醌亚胺（NAPQI）主要发生在葡萄糖醛酸化和硫酸化途径饱和时。在体内，软亲电 NAPQI 通常被肝还原型谷胱甘肽（GSH）灭活，软亲核试剂。在这项研究中，我们研究了 CYP 激活后 APAP 在斑马鱼胚胎中的致畸和致死作用。在含有 APAP 和代谢激活的实验组中，2mM、4mM 和 6mM 的胚胎分别有 11.7+/-7.6%、25.0+/-8.7%和 50.0+/-21.8%受到影响，在 4mM APAP 时达到统计学意义。当胚胎暴露于 6mM APAP、MAS 和 3mM GSH 时，受影响的胚胎百分比下降至 6.7+/-5.8%。相比之下，代谢激活的环磷酰胺（CPA）的致畸和致死作用不能通过添加 GSH 来预防，因为 CPA 代谢物是强亲电试剂，它们优先与 DNA 和 RNA 等硬亲核试剂结合。用我们的 mDarT 标准方案在斑马鱼胚胎中观察到的代谢激活的 APAP 的致畸和致死作用可以用缺乏 GSH 作为解毒系统来解释。通过添加 GSH，可以模拟哺乳动物的情况，从而避免斑马鱼胚胎的致畸作用。
