Grantee Research Project Results
2023 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 , Leszczynski, Jerry
Current Investigators: Sepulveda, Maria , Lee, Linda S. , Gantz, JD , Hoskins, Tyler , Hoverman, Jason , Kar, Supratik
Institution: Purdue University , Jackson State University , Hendrix College
Current 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, 2022 through August 31,2023
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:
To date, we have made most progress under Objective 1 where we have quantified the binding energy 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) employing extra precision docking. The goal is to rank PFAS based on their binding energy to the target proteins and to determine if binding affinity is driven by chain length and functional group. Since no X-ray crystalized protein structure exists for Chironomus dilutus hemoglobins, we collected hemolymph from our midges and conducted proteomics analyses and predicted the protein structure. Docking analyses have been performed for the 11 single PFAS for all 6 proteins twice, first in the presence of heme and once without heme in the active site. From these analyses, we couldn’t identify a strong pattern of binding energy with respect to chain length (C4 < C6 < C7/C9/C10 -10 kcal/mol for all proteins) compared to PFAS. Therefore, our original hypothesis that PFAS could displace heme was incorrect which led us to a new set of analyses using flexible docking instead of induced fit docking (IFD), followed by binding pose metadynamics (BPMD), and finally molecular dynamics simulation for 200 nanoseconds. This approach further validated 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. The goals for this first objective have not changed. Once we finish the current computational analyses and identify the most and least toxic PFAS, we will experimentally check the toxicity of single PFAS using in vivo toxicity testing (Objective 3). We will next move to modeling PFAS binary mixtures. For Objective 3, we have established a colony of Chironomus dilutus at Purdue University. We have also developed protocols for quantifying hemoglobin gene expression in midges and have created primers targeting three different types of hemoglobins. We have also developed protocols for quantifying PFAS in midge larvae.
Future Activities:
For Objective 1, For Objective 2, we will be conducting the equilibrium dialyses experiments between December 2023 and April 2024. For Objective 3, we are in the process of finalizing an experimental protocol for a short-term PFAS midge in vivo exposure targeting changes in hemoglobin expression. We will be testing 11 PFAS in short tests with midges during 2024 and compare these results to those under Objective 1.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 4 publications | 1 publications in selected types | All 1 journal articles |
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Gallagher A, Kar S, Sepúlveda MS. Computational Modeling of Human Serum Albumin Binding of Per-and Polyfluoroalkyl Substances Employing QSAR, Read-Across, and Docking . Molecules. July 2023;28(14):5375. |
R840456 (2023) |
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Supplemental Keywords:
Computational toxicology, invertebrates, plasma, serum, circulating proteinsRelevant Websites:
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.