Fujita Masaharu, Yamamoto Yusuke, Tahara Haruna, Kasahara Toshihiko, Jimbo Yoshihiro, Hioki Takanori
FUJIFILM Corporation, Safety Evaluation Centre, Kanagawa, Japan.
FUJIFILM Corporation, Safety Evaluation Centre, Kanagawa, Japan.
J Pharmacol Toxicol Methods. 2014 Jul-Aug;70(1):94-105. doi: 10.1016/j.vascn.2014.06.001. Epub 2014 Jun 10.
The Direct Peptide Reactivity Assay (DPRA) was developed as an alternative simple and versatile method for predicting skin sensitization. Here, we describe a novel Amino acid Derivative Reactivity Assay (ADRA) involving 2 amino acid derivatives: N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), in which each amino-terminal residue is introduced into a naphthalene ring. ADRA measurements were conducted at 281nm, which improved baseline stability, and were less influenced by other substances in the reaction solutions than DPRA measurements that are conducted at 220nm.
Chemically synthesized NAC and NAL were dissolved in phosphate buffers of pH9.5 and 12.0, respectively. Each solution, test chemical solution, and phosphate buffer, were mixed in 96-well microplates and incubated in the dark for 24h at 25°C. Following incubation, samples were diluted 10 times with a mixed solution of 25% acetonitrile/0.5% trifluoroacetic acid (TFA) in water, and NAC and NAL levels were quantified in each sample and control using a high-performance liquid chromatography (HPLC) system. The reactivity of NAC/NAL was calculated as the percent depletion based on the decrease in the non-reacted NAC/NAL concentration in the samples relative to the average concentration in the control; the average NAC/NAL reduction score was calculated. A 2-class classification model was developed using ADMEWORKS/ModelBuilder, and an optimal average score that could discriminate between sensitizers and non-sensitizers was determined.
A total of 82 test chemicals were applied to ADRA for comparison against DPRA. The prediction accuracy of ADRA was 88%, which was similar to that of DPRA.
ADRA was used to quantify NAC/NAL at 281nm, which showed high accuracy for the prediction of skin sensitization, similar to that of DPRA. Therefore, ADRA could be used to expand the range of chemicals tested in skin sensitization analyses.
直接肽反应性测定法（DPRA）是作为一种预测皮肤致敏性的简单通用替代方法而开发的。在此，我们描述了一种新型氨基酸衍生物反应性测定法（ADRA），它涉及两种氨基酸衍生物：N-（2-（1-萘基）乙酰基）-L-半胱氨酸（NAC）和α-N-（2-（1-萘基）乙酰基）-L-赖氨酸（NAL），其中每个氨基末端残基都引入到一个萘环中。ADRA测量在281nm处进行，这提高了基线稳定性，并且与在220nm处进行的DPRA测量相比，受反应溶液中其他物质的影响较小。
化学合成的NAC和NAL分别溶解在pH9.5和12.0的磷酸盐缓冲液中。将每种溶液、测试化学品溶液和磷酸盐缓冲液在96孔微孔板中混合，并在25°C黑暗中孵育24小时。孵育后，用25%乙腈/0.5%三氟乙酸（TFA）的水溶液将样品稀释10倍，并使用高效液相色谱（HPLC）系统对每个样品和对照中的NAC和NAL水平进行定量。NAC/NAL的反应性根据样品中未反应的NAC/NAL浓度相对于对照中平均浓度的降低计算为消耗百分比；计算平均NAC/NAL降低分数。使用ADMEWORKS/ModelBuilder开发了一个二类分类模型，并确定了能够区分致敏剂和非致敏剂的最佳平均分数。
总共82种测试化学品应用于ADRA以与DPRA进行比较。ADRA的预测准确率为88%，与DPRA相似。
ADRA用于在281nm处对NAC/NAL进行定量，其对皮肤致敏性的预测显示出与DPRA相似的高精度。因此，ADRA可用于扩大皮肤致敏性分析中测试化学品的范围。
