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

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.

Keywords: Biotransformation; Carbamazepine; Developmental toxicity; In vitro; LC-MS; Levetiracetam; NAM; Zebrafish larvae.

Graphical abstract

Fig. 1

Proposed pathway of carbamazepine in humans by myeloperoxidases in activated leukocytes (Furst and Uetrecht, 1993).

Fig. 2

Proposed pathway of carbamazepine in humans. Metabolism in the liver is indicated with broken arrows. Metabolism in the peripheral blood is indicated with continuous arrows (Mathieu et al., 2011).

Fig. 3

Larvae exposed to CBZ or AI at 120 hpf. A) Control larva (1 % DMSO) showing no signs of abnormalities. B) Larva treated with 250 µM CBZ. C) Larva treated with 500 µM CBZ. D) Larva treated with 30 µM AI. The larvae depicted in B, C and D show several malformations. Abbreviations: acridine (AI), carbamazepine (CBZ), edema eyes (EE), edema head (EH), edema pericard (EP), increased pigmentation (IP), malformation yolk (MY), no hatching (NH).

Fig. 4

Larvae exposed to AO or 9ACA at 120 hpf. A) Control larva (1 % DMSO) showing no signs of abnormalities. B) Larva treated with 30 µM AO. C) Larva treated with 60 µM AO. D) Larva treated with 30 µM 9ACA. E) Larva treated with 60 µM 9ACA. The larvae depicted in B, C, D and E show several malformations. Abbreviations: 9-acridine carboxaldehyde (9ACA), acridone (AO), curved tail (CT), deviation eyes (shape) (DES), edema eyes (EE), edema head (EH), edema pericard (EP), increased pigmentation (IP), malformation yolk (bigger) (MYB), no hatching (NH).