Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada.
U.S. Environmental Protection Agency, Oak Ridge Institute for Science and Education, Duluth, Minnesota 55804, United States.
Environ Sci Technol. 2021 Jul 6;55(13):9012-9023. doi: 10.1021/acs.est.1c00509. Epub 2021 Jun 16.
Evaluating interspecies toxicity variation is a long-standing challenge for chemical hazard assessment. This study developed a quantitative interspecies thermal shift assay (QITSA) for , quantitative, and modest-throughput investigation of chemical-protein interactions in cell and tissue samples across species. By using liver fatty acid binding protein (L-FABP) as a case study, the QITSA method was benchmarked with six per- and polyfluoroalkyl substances, and thermal shifts (Δ) were inversely related to their dissociation constants ( = 0.98). The QITSA can also distinguish binding modes of chemicals exemplified by palmitic acid. The QITSA was applied to determine the interactions between perfluorooctanesulfonate (PFOS) and L-FABP in liver cells or tissues from humans, mice, rats, and zebrafish. The largest thermal stability enhancement by PFOS was observed for human L-FABP followed by the mouse, rat, and zebrafish. While endogenous ligands were revealed to partially contribute to the large interspecies variation, recombinant proteins were employed to confirm the high binding affinity of PFOS to human L-FABP, compared to the rat and mouse. This study implemented an experimental strategy to characterize chemical-protein interactions across species, and future application of QITSA to other chemical contaminants is of great interest.
评估种间毒性差异是化学危害评估的长期挑战。本研究开发了一种定量种间热移测定法（QITSA），用于定量和适度高通量研究细胞和组织样本中化学-蛋白质相互作用。通过使用肝脂肪酸结合蛋白（L-FABP）作为案例研究，将 QITSA 方法与六种全氟和多氟烷基物质进行了基准测试，热移（Δ）与它们的离解常数（ = 0.98）呈反比。QITSA 还可以区分脂肪酸的结合模式。该 QITSA 用于确定全氟辛烷磺酸（PFOS）与来自人类、小鼠、大鼠和斑马鱼的肝细胞或组织中的 L-FABP 之间的相互作用。PFOS 对人 L-FABP 的热稳定性增强最大，其次是小鼠、大鼠和斑马鱼。虽然内源性配体部分导致了种间的巨大差异，但重组蛋白被用于确认 PFOS 与人 L-FABP 的高结合亲和力，与大鼠和小鼠相比。本研究实施了一种实验策略来描述跨物种的化学-蛋白质相互作用，未来将 QITSA 应用于其他化学污染物将具有很大的意义。
