Developmental toxicity of arecoline, the major alkaloid in betel nuts, in zebrafish embryos

BACKGROUND: The major alkaloid in the betel nut, arecoline, has been reported to be potent in inducing developmentally toxic effects by generally lowering the embryo weight and retarding development of the embryo. This study examined the adverse effects of arecoline and tried to unravel the mechanism through the tools of molecular biology. METHODS: Arecoline was administered to zebrafish embryos by incubation at concentrations ranging from 0.01-0.04% (wt/vol) and lethality and morphological changes were recorded. The expression of genes was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. In addition, the protective effects of several antioxidants were tested. RESULTS: The survival rate of treated embryos during a three-day incubation significantly declined as the arecoline concentration increased. Treated embryos showed general growth retardation and lower rate of heartbeat. When examined at the 24-hr stage, the relative amounts of transcripts of p53, p21, and cyclin D1, and the spatial expression patterns of these genes in treated groups, were comparable to those of the untreated early stages of embryos. Finally, the addition of glutathione (GSH) or its precursor, N-acetyl-L-cysteine (NAC), ameliorated the developmental retardation of embryos by arecoline. CONCLUSIONS: Arecoline-treated embryos exhibited general developmental retardation in a dose-dependent manner. Our results from RT-PCR, in situ hybridization, and antioxidant-protection experiments indicate that the mechanism underlying growth retardation by arecoline in embryos is predominately due to a general cytotoxic effect induced by depletion of intracellular thiols. Birth Defects Research (Part A) 70:28-36, 2004. (C) 2004 Wiley-Liss, Inc.