ENTRIX, Inc., Sasaktoon, SK S7N 5B3, Saskatchewan, Canada.
Environ Sci Pollut Res Int. 2011 Mar;18(3):503-15. doi: 10.1007/s11356-010-0396-x. Epub 2010 Oct 3.
BACKGROUND, GOALS, AND SCOPE: In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.
A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and "negative" chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.
With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.
With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).
Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.
背景、目的和范围：为了应对人们对化学物质可能与人类和野生动物内分泌系统相互作用的日益关注，各国和国际上都启动了各种项目，旨在制定新的指导方针，用于筛选和测试脊椎动物中的这些化学物质。在这里，我们报告了一种体外检测方法 H295R 类固醇生成检测的验证情况，该方法旨在检测可能抑制或诱导雄激素（T）和 17β-雌二醇（E2）等性激素产生的化学物质，这是为了制定经济合作与发展组织（OECD）检测指南做准备。
使用先前经过优化和验证的方案，评估了 28 种具有不同结构和特性的化学物质的潜在作用，以验证 H295R 类固醇生成检测的有效性。这些化学物质包括已知的内分泌活性化学物质和“阴性”化学物质，这些化学物质预计不会对目标终点产生影响，以及一些作用机制未知的测试化学物质。来自七个国家的七个实验室参与了这项工作。除了对激素产生的影响外，还评估了细胞活力和测试物质可能对基于抗体的激素检测分析的直接干扰等混杂因素。在进行暴露实验之前和期间，每个实验室都必须证明它们能够在预先定义的性能标准范围内进行检测。
除了少数例外，所有实验室都满足了关键的质量性能参数，只有 2%和 7%的 T 和 E2 实验分别因超过这些参数而被排除。在分析的 28 种化学物质中，有 13 种和 14 种分别影响 T 和 E2 的产生，而 11 种和 8 种分别对 T 和 E2 的产生没有显著影响。4 种和 6 种化学物质对 T 和 E2 的产生产生了不确定的结果。然而，T 和 E2 中的这两种情况中的四种分别与人员变动后发生的一个实验室有关。有四种化学物质对某些基于抗体的激素检测系统产生了显著干扰。然而，这些化学物质中只有一种显著影响了检测系统将化学物质分类为影响 E2 或 T 产生的能力。
除了一个例外，H295R 类固醇生成检测方案成功地识别了大多数具有已知和未知作用机制的化学物质，这些化学物质作为 T 和 E2 的诱导剂或抑制剂。因此，它可以被认为是一种可靠的筛选方法，用于筛选能够改变性激素产生的化学物质。H295R 类固醇生成检测方案的一个剩余限制是 E2 的基础产量相对较小，并且在确定弱抑制剂对这种激素产生的减少的影响时，这会影响到对其的定量。将本研究中产生的数据与文献中的体内研究数据进行的初步比较表明，H295R 类固醇生成检测有潜力识别影响整个生物体激素平衡的化学物质。特别有希望的是，在验证过程中没有出现任何假阴性，并且假阳性的数量非常低（T 和 E2 各有 1 种化学物质）。
基于本验证研究中获得的结果以及相应修订的测试方案，制定了 OECD 测试指南草案，并于 2009 年 12 月提交给经济合作与发展组织国家协调员测试指南方案工作组征求意见。
