The effect of maternal betel quid exposure during pregnancy on adverse birth outcomes among aborigines in Taiwan.

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) 67:000–000, 2003. © 2003 Wiley-Liss, Inc.

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

The toxicological assessment of plant products and pharmaceutical chemicals is a necessary requirement to ensure that all compounds are safe to be exposed to humans. Many countries are trying to reduce the use of animals; thus, alternative techniques, such as ex vivo tests, in vitro assays, and ex uteri embryos, are used. Toxicological assays using zebrafish embryos are an advantageous technique because they are transparent, have rapid embryonic development, and do not require invasive techniques. This paper comprehensively reviews how toxicity testing with plant products is conducted in zebrafish embryos. The search terms zebra fish, Danio rerio, zebrafish, zebra danio, Brachydanio rerio, zebrafish, and embryos were used to search for English-language articles in PUBMED, SCOPUS, and WEB OF SCIENCE. Twelve articles on plant product toxicity studies using zebrafish were selected for reading and analysis. After analyzing the articles and comparing with results in mammals, it was possible to prove the similarity among the results and thus corroborate the further development of zebrafish as a valid tool in toxicity tests.

Zebrafish as an alternative method for determining the embryo toxicity of plant products: a systematic review

The areca nut-chewing habit is common in Southeast Asia, India, and Taiwan, and arecoline is the most abundant and potent component in the areca nut. The effects of arecoline on birth defects have been explored in many species, including chicken, mice, and zebrafish. The effects of arecoline on embryos after long-term exposure are well established; however, the effects of short-term embryo exposure to arecoline are not understood. Using zebrafish, we study the effects of short-term exposure of arecoline on embryos to model the human habit of areca nut-chewing during early pregnancy. Arecoline, at concentrations from 0.001% to 0.04%, was administered to zebrafish embryos from 4 to 24 hours post fertilization. The morphological changes, survival rates, body length, and skeletal muscle fiber structure were then investigated by immunohistochemistry, confocal microscopy, and conventional electron microscopy. With exposure of embryos to increasing arecoline concentrations, we observed a significant decline in the hatching and survival rates, general growth retardation, lower locomotor activity, and swimming ability impairment. Immunofluorescent staining demonstrated a loose arrangement of myosin heavy chains, and ultrastructural observations revealed altered myofibril arrangement and swelling of the mitochondria. In addition, the results of flow-cytometry and JC-1 staining to assay mitochondria activity, as well as reverse transcription-polymerase chain reaction analyses of functional gene expression, revealed mitochondrial dysfunctions after exposure to arecoline. We confirmed that short-term arecoline exposure resulted in retarded embryonic development and decreased locomotor activity due to defective somitic skeletal muscle development and mitochondrial dysfunction.

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Short-term exposure of zebrafish embryos to arecoline leads to retarded growth, motor impairment, and somite muscle fiber changes.

Betel nut is a widely masticated natural product, which is consumed by over 600 million people across the globe. The ancient habit of betel nut chewing, either as dry or raw/wet nut, in association with betel leaf and a host of region specific additives, including chewing tobacco, is believed to be an important etiological factor for human cancer. Alkaloids and their betel nut specific nitrosamine derivatives produced upon metabolic activation interact with DNA and other cellular targets to produce highly variable mutagenic, genotoxic, cytostatic, immunostatic and teratogenic effects. At molecular level the betel nut or its constituents strongly influence gene expression patterns, especially that of tumor suppressor genes. Structural damage to nucleus and mitochondria, etc. are also induced. The review dwells upon these aspects of betel nut induced carcinogenesis to show that genetic susceptibility to cancer through generations progressively increased due to exposure to betel nut.

K. Paul

Papers

Teratogenicity of arecoline hydrobromide on developing chick embryos: a preliminary report.