Araki Naohiro, Ohno Ken, Takeyoshi Masahiro, Iida Mitsuru
EcoScreen R&D Section, Endocrine Disrupting Chemical Analysis Center, Otsuka Life Science Initiative, Otsuka Pharmaceutical Co. Ltd., 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima 771-0195, Japan.
Toxicol In Vitro. 2005 Apr;19(3):335-52. doi: 10.1016/j.tiv.2004.10.008. Epub 2005 Jan 21.
An accurate and reliable in vitro assay system has been needed for first tier screening of endocrine disrupting chemicals. For the purpose, we previously developed stable AR-EcoScreen cell lines to assess androgen receptor (AR)-mediated transcriptional activation. In this report, we evaluated AR-EcoScreen cell lines as the phase I of prevalidation study by determining the intra-laboratory reproducibility, assay stability, and overall protocol performance of AR-EcoScreen assays. Forty compounds recommended by the ICCVAM were tested for AR agonist and antagonist activity. The mean coefficient of variation (CV) for intra-assay reproducibility in the AR agonist assay was 4.35% for 5alpha-dihydrotestosterone (DHT), and that for the antagonist assay was 5.51% for hydroxyflutamide. The detection limit of the agonist assay was 2.3x10(-11) M for 5alpha-dihydrotestosterone. Furthermore, we examined the overall performance of the method by comparing the predicted result with the ICCVAM classification. Thus, the overall sensitivity, specificity, and accuracy of the agonist assay were 89%, 94%, and 91%, respectively. For the antagonist assay, these values were 94%, 100%, and 96%, respectively. In summary, we concluded that AR-EcoScreen method was ready to proceed to the phase II prevalidation study to asses the inter-laboratory variability and transfer of the protocol.
对于内分泌干扰化学物质的一级筛选，一直需要一种准确可靠的体外检测系统。为此，我们之前开发了稳定的AR-EcoScreen细胞系来评估雄激素受体（AR）介导的转录激活。在本报告中，我们通过确定AR-EcoScreen检测的实验室内重现性、检测稳定性和整体方案性能，将AR-EcoScreen细胞系评估为预验证研究的第一阶段。对ICCVAM推荐的40种化合物进行了AR激动剂和拮抗剂活性测试。在AR激动剂检测中，5α-二氢睾酮（DHT）的实验室内重现性平均变异系数（CV）为4.35%，在拮抗剂检测中，羟基氟他胺的CV为5.51%。激动剂检测对5α-二氢睾酮的检测限为2.3×10⁻¹¹ M。此外，我们通过将预测结果与ICCVAM分类进行比较，检验了该方法的整体性能。因此，激动剂检测的总体敏感性、特异性和准确性分别为89%、94%和91%。对于拮抗剂检测，这些值分别为94%、100%和96%。总之，我们得出结论，AR-EcoScreen方法已准备好进入第二阶段预验证研究，以评估实验室间的变异性和方案的转移性。
