Assessing developmental toxicity and non-CYP mediated biotransformation of two anti-epileptics and their human metabolites in zebrafish embryos and larvae

IF 2.9 Q2 TOXICOLOGY
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Abstract

Zebrafish embryo-based assays are a promising alternative for animal testing to screen new compounds for developmental toxicity. However, recent studies in zebrafish embryos showed an immature intrinsic cytochrome P450 (CYP)-mediated biotransformation capacity, as most CYPs were only active at the end of the organogenesis period. Data on other phase I enzymes involved in the biotransformation of xenobiotics in zebrafish embryos is limited. This information is pivotal for proteratogens needing bioactivation to exert their teratogenic potential. Therefore, this study aimed to investigate whether carbamazepine (CBZ) and levetiracetam (LTC), two anti-epileptic drugs that require bioactivation to exert their teratogenic potential, are biotransformed into non-CYP mediated metabolites in the zebrafish embryo and whether one or more of these metabolites cause developmental toxicity in this species. In the first step, zebrafish embryos were exposed to LTC and CBZ and their non-CYP mediated human metabolites, etiracetam carboxylic acid (ECA) and 9-acridine carboxaldehyde (9ACA), acridine (AI), and acridone (AO), respectively, from 5.25 to 120 hpf and morphologically evaluated. Next, the uptake of all compounds and the formation of the metabolites were assessed using LC-MS methods. As LTC and ECA were, respectively, poorly or not taken up by zebrafish larvae during the exposure experiments, we could not determine if LTC and ECA are teratogenic. However, biotransformation of LTC into ECA was observed at 24 hpf and 120 hpf, which indicates that the special type of B-esterase is already active at 24 hpf. CBZ and its three metabolites were teratogenic, as a significant increase in malformed embryos was observed for all of them. All three metabolites were more potent teratogens than CBZ, with AI being the most potent, followed by 9ACA and AO. The myeloperoxidase (MPO) homologue is already active at 24 hpf, as CBZ was biotransformed into 9ACA and AO in 24 hpf zebrafish embryos, and into 9ACA in 120 hpf larvae. Moreover, 9ACA was also found to be biotransformed into AI and AO, and AI into AO. As such, one or more of these metabolites probably contribute to the teratogenic effects observed in zebrafish larvae after exposure to CBZ.

Abstract Image

评估两种抗癫痫药及其人类代谢物在斑马鱼胚胎和幼体中的发育毒性和非 CYP 介导的生物转化
以斑马鱼胚胎为基础的试验是一种很有前途的动物试验替代方法,可用于筛选新化合物的发育毒性。然而,最近在斑马鱼胚胎中进行的研究表明,细胞色素 P450(CYP)介导的内在生物转化能力并不成熟,因为大多数 CYP 在器官形成期结束时才开始活跃。有关斑马鱼胚胎中参与异种生物转化的其他 I 期酶的数据十分有限。这些信息对于需要生物活化才能发挥致畸潜力的原致畸剂至关重要。因此,本研究旨在探讨卡马西平(CBZ)和左乙拉西坦(LTC)这两种需要生物活化才能发挥致畸潜力的抗癫痫药物是否会在斑马鱼胚胎中生物转化为非 CYP 介导的代谢物,以及其中一种或多种代谢物是否会对该物种的发育造成毒性。第一步,斑马鱼胚胎在 5.25 至 120 hpf 期间分别暴露于 LTC 和 CBZ 及其非 CYP 介导的人类代谢物依替拉西坦羧酸(ECA)和 9-吖啶甲醛(9ACA)、吖啶(AI)和吖啶酮(AO),并进行形态学评估。然后,使用 LC-MS 方法评估了所有化合物的吸收和代谢物的形成。由于在暴露实验中,斑马鱼幼体对 LTC 和 ECA 的吸收率分别很低或没有吸收,因此我们无法确定 LTC 和 ECA 是否具有致畸性。不过,在 24 hpf 和 120 hpf,我们观察到 LTC 向 ECA 的生物转化,这表明特殊类型的 B 酯酶在 24 hpf 时已经活跃。CBZ 及其三种代谢物都有致畸作用,因为它们都能观察到畸形胚胎的显著增加。所有三种代谢物的致畸作用都比 CBZ 强,其中 AI 的致畸作用最强,其次是 9ACA 和 AO。在 24 hpf 的斑马鱼胚胎中,CBZ 被生物转化为 9ACA 和 AO,而在 120 hpf 的幼鱼中,CBZ 被生物转化为 9ACA,因此髓过氧化物酶(MPO)同源物在 24 hpf 时就已经具有活性。此外,还发现 9ACA 可生物转化为 AI 和 AO,AI 可生物转化为 AO。因此,这些代谢物中的一种或多种可能会导致斑马鱼幼体在接触 CBZ 后出现致畸效应。
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来源期刊
Current Research in Toxicology
Current Research in Toxicology Environmental Science-Health, Toxicology and Mutagenesis
CiteScore
4.70
自引率
3.00%
发文量
33
审稿时长
82 days
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