Boyd Windy A, McBride Sandra J, Freedman Jonathan H
Laboratory of Molecular Toxicology, National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
PLoS One. 2007 Dec 5;2(12):e1259. doi: 10.1371/journal.pone.0001259.
Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.
METHODOLOGY/PRINCIPAL FINDINGS: Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50) of 2 microM.
CONCLUSIONS/SIGNIFICANCE: The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.
政府机构已明确需要用使用替代物种的测试方法来减少、优化或取代当前基于哺乳动物的生物测定法。无脊椎动物物种，如秀丽隐杆线虫，因其生命周期短、维护成本低以及与高等真核生物的高度进化保守性而提供了一个有吸引力的选择。秀丽隐杆线虫的咽部是研究神经肌肉功能以及化学物质对神经肌肉活动（即进食）影响的有利模型。然而，目前的进食方法劳动强度大且只是半定量的。
方法/主要发现：本文描述了一种高通量测定法，该方法使用流式细胞术通过测定数百只线虫在接触荧光标记微球后的大小和肠道荧光来测量秀丽隐杆线虫的进食情况。通过对进食缺陷型秀丽隐杆线虫（eat突变体）的荧光进行定量，以及将野生型线虫暴露于神经活性化合物5-羟色胺和槟榔碱，对该测定法进行了验证。先前确定会导致泵送速率缓慢的eat突变在我们的测定中表现出最低的进食水平。5-羟色胺暴露后进食水平的浓度依赖性增加取决于食物供应情况，而无论是否有食物，槟榔碱暴露的线虫的进食水平都会下降。然后利用环境污染物氯化镉和毒死蜱对野生型秀丽隐杆线虫进食的影响来证明进食测定法的应用。高于200微摩尔的镉暴露导致进食水平急剧下降。暴露于毒死蜱的线虫的进食以浓度依赖性方式下降，半数有效浓度（EC50）为2微摩尔。
结论/意义：秀丽隐杆线虫荧光微球进食测定法是一种快速、可靠的方法，用于评估药物、工业化学品或环境因子的神经毒性作用。该测定法也可能适用于大规模的遗传或RNA干扰筛选，以鉴定咽部或其他神经肌肉系统发育或功能所必需的基因。
