Reading School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom.
School of Pharmacy, The Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, United Kingdom.
Toxicol In Vitro. 2020 Dec;69:105004. doi: 10.1016/j.tiv.2020.105004. Epub 2020 Oct 1.
Here we report a new planarian (Dugesia lugubris) fluorescent assay as a rapid and cheap pre-screening tool to predict strong skin irritants. Our aim was to provide a simple and cost-effective in vivo method that avoided use of higher vertebrates. Adapting previously reported methods for planaria mobility alongside an acute toxicity assay, different irritants at five concentrations (0.1%, 0.05%, 0.025%, 0.01% and 0.005% w/v) were tested but both methods failed to discriminate the irritation potential of the test compounds. Therefore, a new alternative fluorescence assay was developed, hypothesising that increasing damage from the irritant to the planarian outer protective membrane will increase accumulation of sodium fluorescein in the flatworm. Fourteen test chemicals were selected representing strong, moderate, mild and non-irritants. In general, results showed increasing sodium fluorescein accumulation within planaria following acute exposure to increasingly strong skin irritants; on exposure to the strong irritants, benzalkonium chloride, citronellal, methyl palmitate, 1-bromohexane and carvacrol, fluorescence within the planaria was significantly greater (P < 0.05) than the negative controls and the common non-irritants PEG-400, dipropylene glycol and isopropyl alcohol; fluorescence values of planaria tested with negative controls and non-irritants were not significantly different. For all test compounds, Fluorescence Intensity of the planaria was compared with literature Primary Irritation Index data and generated a statistically significant (P < 0.005) Pearson correlation (r) of 0.87. Thus, the planarian fluorescent assay is a promising tool for rapid early testing of potential strong skin irritants, and non-irritants, and avoids use of higher vertebrate models.
在这里，我们报告了一种新的涡虫（Dugesia lugubris）荧光测定法，作为一种快速且廉价的预筛选工具，用于预测强皮肤刺激性物质。我们的目的是提供一种简单且具有成本效益的体内方法，避免使用高等脊椎动物。本研究通过改编先前报道的涡虫运动性方法和急性毒性测定法，测试了五种浓度（0.1%、0.05%、0.025%、0.01%和 0.005%w/v）的不同刺激性物质，但这两种方法均未能区分测试化合物的刺激性潜力。因此，我们开发了一种新的荧光替代测定法，假设刺激性物质对涡虫外保护膜的损伤越大，荧光素钠在扁形动物体内的积累就会越多。选择了 14 种测试化学品，代表强、中、弱和非刺激性物质。一般来说，结果表明，随着扁形动物急性暴露于越来越强的皮肤刺激性物质，其体内的荧光素钠积累量增加；暴露于强刺激性物质苯扎氯铵、香茅醛、肉豆蔻酸甲酯、1-溴己烷和香芹酚时，扁形动物体内的荧光强度明显高于阴性对照和常见的非刺激性物质聚乙二醇 400、二丙二醇和异丙醇（P<0.05）；用阴性对照和非刺激性物质测试的扁形动物的荧光强度无显著差异。对于所有测试化合物，与文献中的初级刺激性指数数据相比，扁形动物的荧光强度都生成了具有统计学意义（P<0.005）的皮尔逊相关系数（r）为 0.87。因此，这种涡虫荧光测定法是一种很有前途的工具，可用于快速早期测试潜在的强皮肤刺激性物质和非刺激性物质，同时避免使用高等脊椎动物模型。
