Developmental toxicity of dextromethorphan in zebrafish embryos/larvae

Abstract

Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use zebrafish as a model to investigate the potential toxicity of dextromethorphan during embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24, 48 and 72 h post fertilization (hpf), respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the phase I and phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan.

Figure 1

Effects of exposure to dextromethorphan on developing zebrafish larvae. A) Untreated control larva at 168 hpf. B) 2 mM dextromethorphan-exposed larva in the 24 hpf exposure set. At the 96 hpf time point, the larva exhibited edema in the cardiac sac (as indicated by an arrow). C) 25 μM dextromethorphan-exposed larva in the 24 hpf exposure set. At 168 hpf, the larva exhibited lordosis (curving body trunk; indicated by a curved arrow), malformed mouth/jaw (indicated by a black arrowhead) and non-inflated swim bladder (indicated by a white arrowhead). D) 0.1 μM dextromethorphan-exposed larva in the 24 hpf exposure set. At 168 hpf, similar morphological abnormalities as those found in C) were observed. In contrast, these morphological abnormalities were not manifested by 0.1 μM dextromethorphan-exposed larvae in the 48 hpf and 72 hpf exposure sets at the same time point (168 hpf).

Figure 2

Heart rates of zebrafish larvae exposed to different concentrations of dextromethorphan. The data shown were obtained at 96 hpf from the larvae in the 24 hpf exposure set. Heart rate was measured in beats per minute.

Figure 3

Developmental expression of the zebrafish SULT3 ST1 and SULT3 ST3 in untreated and dextromethorphan-treated zebrafish embryos/larvae. RT-PCR was employed to analyze the expression of mRNAs encoding (A) SULT3 ST1, (B) SULT3 ST3, and (C) β-actin, respectively, in untreated and 50 μM dextromethorphan-treated zebrafish embryos/larvae. Final PCR mixtures were subjected to 0.9% agarose electrophoresis. Samples analyzed correspond to 24 hpf untreated zebrafish embryos (lane 1), 48 hpf untreated and dextromethorphan-treated zebrafish embryos (lanes 2 and 3), 72 hpf untreated and dextromethorphan-treated zebrafish larvae (lanes 4 and 5), 120 hpf untreated and dextromethorphan-treated zebrafish larvae (lanes 6 and 7), and 168 hpf untreated and dextromethorphan-treated zebrafish larvae (lanes 8 and 9). The PCR products corresponding to the zebrafish SULT3 ST1, SULT3 ST3, or β-actin, as visualized by ethidium bromide staining, are marked by arrowheads. DNA size markers co-electrophoresed are shown on the right for each of the three panels. The two bright bands are those of the 1,000 bp and 500 bp DNA markers.