FI-TRACE Group, Department of Chemistry, University of the Balearic Islands, Carretera de Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain.
REACMOL Group, Department of Chemistry, University of the Balearic Islands, Carretera de Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain.
Sci Total Environ. 2020 Oct 10;738:140096. doi: 10.1016/j.scitotenv.2020.140096. Epub 2020 Jun 10.
According to ISO 17402:2008 more knowledge is needed on processes controlling bioavailability of organic species so as to close the still existing gap between chemical measurements and biological effects. The bioavailability concept encompasses the investigation of the degree of penetration of target species across biological membranes. In addition, REACH (Registration, Evaluation, Authorisation and restriction of Chemicals) guidelines promote the use of in-vitro methods against conventional ecotoxicological tests because of the ethical controversy of in-vivo tests. This work is aimed at filling the gap by proposing a multidisciplinary approach based on high-resolution and low-resolution empirical techniques, and theoretical quantum mechanics for the in-vitro investigation of the bioavailability and membranotropic effects of organic emerging contaminants, including bioaccumulation, via passive diffusion across lipid bilayers. Phosphatidylcholine (PC) liposomes are selected as biomembrane surrogates, and contaminant effects are explored by (i) fluorescence anisotropy and generalized polarization assays using membrane fluorescence probes (laurdan and prodan) and UV-Vis spectroscopy, (ii) H NMR measurements to ascertain supramolecular interactions with PC and (iii) molecular dynamics simulations. In particular, un-regulated model compounds with distinct physico-chemical properties that are representative of three different classes of emerging contaminants in environmental compartments are chosen for validation of the holistic approach: (i) diclofenac as a model of anti-inflammatory drug; (ii) triclosan as an anti-microbial agent; and (iii) bisphenol A as a plastic-borne compound, and compared with chlorpyrifos as a legacy insecticide. Laurdan anisotropic measurements are in good agreement with H NMR data and both approaches pinpoint that triclosan and chlorpyrifos are highly bioaccumulative in membranes. Molecular dynamic studies indicate that the lateral diffusion of the lipid bilayer is much lower with the incorporation of either triclosan or chlorpyrifos into the bilayer. The theoretical simulations also allowed estimating absolute bioavailability data under passive diffusion (<0.1%, 63%, 73% and 89% for diclofenac, bisphenol A, triclosan and chlorpyrifos, respectively) given as the percentage of time that a given species is located in the region of the fatty acyl chains. Our findings indicate that PC-based liposome assays serve as a fast and cost-effective in-vitro approach, notwithstanding its low resolution features, for environmental bioavailability studies of emerging contaminants for which insufficient or inconsistent ecotoxicological data are identified in the literature.
根据 ISO 17402:2008，需要更多关于控制有机物种生物可利用性的过程知识，以缩小化学测量值与生物效应之间仍然存在的差距。生物可利用性概念涵盖了目标物种穿透生物膜程度的研究。此外，由于体内测试存在伦理争议，REACH（注册、评估、授权和限制化学物质）指南提倡使用体外方法替代传统的生态毒理学测试。这项工作旨在通过提出一种基于高分辨率和低分辨率经验技术以及理论量子力学的多学科方法来填补这一空白，用于研究有机新兴污染物（包括生物积累）的生物可利用性和膜向性效应，这些污染物通过被动扩散穿过脂质双层进行体外研究。选择磷脂酰胆碱（PC）脂质体作为生物膜替代物，并通过以下方法探索污染物的影响：（i）使用膜荧光探针（laurdan 和 prodan）和紫外可见光谱进行荧光各向异性和广义偏振测定，（ii）通过 H NMR 测量确定与 PC 的超分子相互作用，以及（iii）分子动力学模拟。特别是，选择具有不同物理化学性质的未受监管的模型化合物，这些化合物代表环境中三类不同的新兴污染物，用于验证整体方法：（i）双氯芬酸作为抗炎药物模型；（ii）三氯生作为抗菌剂；以及（iii）双酚 A 作为塑料载体化合物，并与作为遗留杀虫剂的毒死蜱进行比较。Laurdan 各向异性测量与 H NMR 数据非常吻合，这两种方法都表明三氯生和毒死蜱在膜中具有高度的生物积累性。分子动力学研究表明，在将三氯生或毒死蜱掺入双层时，脂质双层的侧向扩散要低得多。理论模拟还允许根据被动扩散下的绝对生物利用度数据进行估计（双氯芬酸、双酚 A、三氯生和毒死蜱分别为<0.1%、63%、73%和 89%），这是给定物种位于脂肪酸链区域的时间百分比。我们的研究结果表明，基于 PC 的脂质体测定法是一种快速且具有成本效益的体外方法，尽管其分辨率较低，但对于文献中确定的环境生物可利用性研究中新兴污染物的研究仍然具有重要意义。
