Grantee Research Project Results
2024 Progress Report: Protein Binding Affinity as the Driver for Studying PFAS Mixture Toxicity
EPA Grant Number: R840456Title: Protein Binding Affinity as the Driver for Studying PFAS Mixture Toxicity
Investigators: Sepulveda, Maria , Lee, Linda S. , Gantz, JD , Hoskins, Tyler , Hoverman, Jason , Kar, Supratik
Institution: Purdue University , Jackson State University , Hendrix College , Kean University
EPA Project Officer: Chung, Serena
Project Period: September 1, 2022 through August 31, 2025 (Extended to August 31, 2026)
Project Period Covered by this Report: September 1, 2023 through August 31,2024
Project Amount: $725,481
RFA: Development of Innovative Approaches to Assess the Toxicity of Chemical Mixtures Request for Applications (RFA) (2022) RFA Text | Recipients Lists
Research Category: Health Effects , Computational Toxicology , Urban Air Toxics , Endocrine Disruptors , Heavy Metal Contamination of Soil/Water , New Approach Methods (NAMs) , Human Health , Safer Chemicals , Mixtures , PFAS Treatment , Chemical Safety for Sustainability , Predictive Toxicology , CSS
Objective:
Environmental exposure to per and polyfluoroalkyl substances (PFAS) occurs as mixtures. Importantly, the bioaccumulation potential and toxicity of a particular mixture is driven by the number of halogenated carbons and functional groups present. PFAS are also highly proteinophilic and known to bind to proteins. Hemoglobins (Hbs) are key proteins responsible for transporting oxygen to tissues and studies show PFAS can bind with Hb interfering with oxygen transport. Chironomids (midges) are highly sensitive to PFAS and >95% of all their hemolymph proteins are Hbs. Using a combination of in silico, in vitro, and in vivo tools, we will test the overarching hypothesis that binding of PFAS to Hbs is a tractable and sensitive physiological signal for predicting the toxicity of PFAS mixtures. Our objectives are to: 1) Develop and parameterize mechanistic toxicity in silico models to determine whether the toxicity of PFAS mixtures deviates from additivity; 2) Inform and validate in silico models using PFAS and Hb binding affinity data; and 3) Inform and validate in silico models using in vivo toxicity tests.
Progress Summary:
Under Objective 1 we have quantified the binding energy and interaction dynamics of our 11 target PFAS to the hemoglobin of Chironomus spp. (thummi, dilutus, and plumosus) as well as three human hemoglobins (oxyhemoglobin, deoxyhemoglobin, and carbomonoxyhemoglobin). Flexible docking analyses, followed by binding pose metadynamics and molecular dynamics simulation, have shown that heme is not being knocked out of the protein by PFAS, but rather PFAS can bind to other sites inducing changes in the shape of the protein and symmetry of the heme group. This binding, we hypothesize, could disrupt the ability of hemoglobin of carrying oxygen, resulting in toxicity. Once we finish the single PFAS computational analyses, we plan to test whether a selected number of binary PFAS mixtures result in protein changes that deviate from additivity. Under Objective 2, we will finalize our experiments examining hemoglobin-PFAS interactions through a combination of equilibrium dialysis and fluorescence spectroscopy approaches. We will also examine changes in fluorescence intensity after mixing human hemoglobin and midge hemolymph to a selected number of binary PFAS mixtures. Under Objective 3, we will continue to test the in vivo toxicity of the target PFAS on midges and compare results with those obtained through in silico and in vitro approaches under Objectives 1 and 2. We will finish by testing the in vivo toxicity PFAS binary mixtures.
Future Activities:
For Objective 1, we will finish analyzing the interactions of PFAS with human oxyhemoglobin and test a selected number of binary PFAS mixtures to determine if the interactions deviate from additivity. For Objective 2, we will calculate binding affinity using equilibrium dialysis data and evaluate changes in structure using fluorescence spectroscopy for the 11 target PFAS. Results will be compared against those obtained from computational modeling under Objective 1. For Objective 3, we will continue to test the in vivo toxicity of the target PFAS. If gene expression results continue to show no effects, we might focus on examining the interactions of PFAS with midge hemolymph using approaches developed under Objective 2. Exposures to binary mixtures of PFAS to test whether mixtures deviate from additivity are also planned.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this projectSupplemental Keywords:
Computational toxicology, invertebrates, plasma, serum, circulating proteins.
Relevant Websites:
Modeling of PFAS Mixture Toxicity Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.