The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 39 Kessels Road, Brisbane, Qld 4108, Australia.
Chem Res Toxicol. 2011 Jul 18;24(7):1134-43. doi: 10.1021/tx200114y. Epub 2011 Jun 8.
Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity.
由于疏水性化学物质在水中的浓度较低、易被透析膜和测试容器吸附以及达到平衡的动力学较慢，因此使用传统的微透析方法很难测量其与胶体（如蛋白质或脂质）的结合。在此，我们采用三相分配系统，其中硅橡胶（聚二甲基硅氧烷，PDMS）作为第三相，以确定水相与胶体之间的分配，并同时作为疏水性化学物质的给药装置。该方法的适用性通过牛血清白蛋白（BSA）进行了验证。氯蜱、甲氧氯、壬基酚和芘的测定结合常数（K(BSAw)）与已建立的定量构效关系（QSAR）吻合良好。由于明显的非生物降解，第五种化合物氟氧嘧啶-甲基-庚酯被排除在分析之外。然后，使用 PDMS 耗竭法测定虹鳟鱼（Oncorhynchus mykiss）肝 S9 级分（K(S9w)）和血浆（K(bloodw)）中测试化学品的分配系数。测定的 K(S9w)和 K(bloodw)值与使用质量平衡模型预测的结果一致，该模型使用辛醇-水分配系数（K(ow)）作为脂质分配的替代物，K(BSAw)代表蛋白质结合。对于每种化合物，K(bloodw)均显著大于 K(S9w)，主要是因为血液中的脂质含量高于肝 S9 级分（湿重的 1.84%比 0.051%）。随后，将测定的肝 S9 和血浆结合参数应用于体内外推算模型，将体外肝 S9 代谢降解试验与鱼类体内代谢联系起来。从实验数据计算出的表观分布容积（V(d)）与文献估计值相似。然而，用于将体外代谢清除率与完整肝脏的清除率相关联的计算结合比（f(u））比以前的建模工作中使用的值低 10 至 100 倍。使用实验结合数据预测的生物浓缩因子（BCF）远高于早期研究中获得的预测值，与鱼类中测定的 BCF 值相关性较差。造成这种结果的一个可能原因是与蛋白质结合的化学物质可以迅速解吸，从而促进化学物质的代谢转化。这一假设有待进一步研究，理想情况下，使用疏水性更高的化学物质。
