Nichols John W, Ladd Melanie A, Fitzsimmons Patrick N
US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, Duluth, MN, USA.
Appl In Vitro Toxicol. 2018;4(4):365-378. doi: 10.1089/aivt.2017.0005.
substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured activity declined with incubation time and was reduced by acetone (used as a spiking solvent) at concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a concentration-dependent manner. The substrate concentration dependence of activity was evaluated to estimate and values for each compound. Derived kinetic constants suggested that all three PAHs are transformed by the same reaction pathway and indicated an inverse correlation between and chemical log . Binding effects on activity were evaluated by measuring unbound chemical concentrations across a range of S9 protein levels. Reaction rates were proportional to the unbound concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings suggest that previous work with high log compounds was conducted at inappropriately high substrate concentrations resulting in underestimation of true activity. Preliminary calculations also indicate that PAH metabolism in fish may approach saturation during standardized testing efforts, potentially resulting in concentration-dependent accumulation and/or steady-state levels of accumulation greater than those which occur in a natural setting.
制药行业开发的底物消耗方法正越来越频繁地用于支持鱼类的化学生物累积评估。然而，将这些方法应用于高log 化学品带来了特殊挑战。使用鳟鱼肝S9组分测量了三种多环芳烃（PAH）的生物转化。测量的活性随孵育时间下降，并且在丙酮（用作加标溶剂）浓度大于0.5%时降低。添加阿拉霉素（一种用于支持UDP-葡萄糖醛酸基转移酶活性的成孔肽）也以浓度依赖性方式降低活性。评估活性的底物浓度依赖性以估计每种化合物的 和 值。推导的动力学常数表明，所有三种PAH都通过相同的反应途径转化，并表明 与化学log 之间呈负相关。通过测量一系列S9蛋白水平下的未结合化学物浓度来评估对活性的结合效应。反应速率与未结合浓度成正比，除非这些浓度接近饱和水平，这直接证明了游离化学物假说。这些发现表明，以前对高log 化合物的研究是在不适当的高底物浓度下进行的，导致对真实活性的低估。初步计算还表明，在标准化测试过程中，鱼类体内的PAH代谢可能接近饱和，这可能导致浓度依赖性积累和/或稳态积累水平高于自然环境中的水平。
