Grantee Research Project Results

2023 Progress Report: System toxicological approaches to define and predict the toxicity of Per and Polyfluoroalkyl Substances

EPA Grant Number: R839481
Title: System toxicological approaches to define and predict the toxicity of Per and Polyfluoroalkyl Substances
Investigators: Tanguay, Robyn L. , Field, Jennifer , Reif, David , Truong, Lisa , Ng, Carla , DeWitt, Jamie
Institution: Oregon State University , North Carolina State University
EPA Project Officer: Packard, Benjamin H
Project Period: May 1, 2019 through May 7, 2025
Project Period Covered by this Report: May 1, 2023 through April 30,2024
Project Amount: $1,981,500
RFA: National Priorities: Per- and Polyfluoroalkyl Substances (2018) RFA Text | Recipients Lists
Research Category: Water Quality

Objective:

1) Study the toxicity of a large collection of volatile and non-volatile PFASs and PFAS mixtures with the zebrafish assay. Hypothesis: PFAS compounds with similar structures will bind to the same biomolecular targets, induce expression of the same or highly overlapping gene sets, and induce similar toxic responses.

2) Conduct developmental immunotoxicity (DIT) studies in mice. Hypotheses: Developmental exposure to PFASs will compromise antigen-specific antibody responses (a measure of adaptive immunity) and natural killer cell cytotoxicity (a measure of innate immunity). Developmental findings in the mouse will accord with developmental findings in the zebrafish.

3) Create pharmacokinetic models that can explain and predict the concentrations of PFASs in the organs of mice and adult zebrafish as a function of exposure dose and chemical structure. Hypotheses: The bioaccumulation and internal distribution of PFASs depend on passive diffusion, transporter-mediated membrane uptake and efflux, and protein binding. The interaction of PFASs with proteins and membranes will depend on i) the presence of polar or charged functional groups and on ii) the length of the linear fluorinated alkyl chain.

Progress Summary:

We developed a practical method for estimating concentrations of suspect PFAS compounds. This method involves comparing the area counts of anionic and zwitterionic/cationic target PFAS to the average area of their stable-isotope labeled surrogates. By doing so, we create "average PFAS calibration curves" for substances detected using both negative- and positive-ionization modes in liquid chromatography quadrupole time-of-flight mass spectrometry. These calibration curves are fitted using log-log and weighted linear regression models. We then assessed these models for accuracy and prediction intervals in determining the target PFAS concentrations. The key benefit of using average PFAS calibration curves is that it allows for concentration estimation without requiring specific knowledge of the suspect’s nanomolar response or exact structure.

We have secured, validate and tested the toxicity of an ever-growing library of PFAS compounds. These include 58 Wellington Laboratories PFAS analytical standards, a 139-member US EPA PFAS Library, an additional 25 commercial reference standards and a series of PFAS mixtures. We also completed three adult zebrafish dietary studies by contaminating a commercial diet with PFAS. These were 43-day dietary exposure studies at several exposure levels starting at 17-dpf. We investigated the toxicity and transcriptional responses following exposure to a series of short-chain compounds and determined that the sulfonamide head group was the most biologically active. We recently we prioritized 15 bioactive PFAS that induced significant morphological effects and performed RNA sequencing to characterize early transcriptional responses and determined that a broad range of differentially expressed gene counts were identified across the PFAS exposures. Most PFAS that elicited robust transcriptomic changes affected biological processes of the brain and nervous system development. While PFAS disrupted unique processes, we also found that similarities in some functional head groups of PFAS were associated with the disruption in expression of similar gene sets. We completed a quantitative assessment of laboratory animal diets for PFAS contamination and found that some commercial sources have background levels that could interfere with toxicological data interpretation.

We have developed, validated, and tested the toxicity of an expanding library of PFAS compounds. This includes 58 analytical standards from Wellington Laboratories, a 139-compound library from the US EPA, an additional 25 commercial reference standards, and various PFAS mixtures. We also conducted three dietary studies on adult zebrafish, contaminating commercial feed with PFAS over 43 days, starting at 17 days post-fertilization. These studies explored the toxicity and transcriptional responses to various short-chain PFAS compounds, identifying the sulfonamide head group as the most biologically active.

Recently, we prioritized 15 bioactive PFAS that caused significant morphological changes, performing RNA sequencing to examine early transcriptional responses. Our findings revealed a broad range of differentially expressed genes across PFAS exposures, with most notable changes affecting brain and nervous system development. We observed that while PFAS compounds disrupted distinct biological processes, some shared head groups were associated with similar gene expression disruptions. Additionally, we assessed PFAS contamination in laboratory animal diets, discovering that some commercial sources have background levels that could impact toxicological data interpretation.

We also completed developmental immunotoxicity (DIT) studies in mice using “PFMOAA” (perfluoro-2-methoxyacetic acid), a three-carbon perfluoroether carboxylic acid, alongside evaluations of PFMOAA, Nafion Byproduct 2, and FHxSA.

Furthermore, we advanced two physiologically based toxicokinetic (PBTK) models for PFAS: one for PFOA in adult zebrafish and a new model for various PFAS in adult mice. The zebrafish model has been enhanced with improved physiological parameters specific to zebrafish and a refined description of protein binding. The mouse model, based on Cheng & Ng’s 2017 rat model, has been re-parameterized to reflect mouse physiology. This new model includes a mechanistic description of PFAS interactions with phospholipids in cell membranes, impacting PFAS kinetics and tissue distribution. It effectively captures the distinct distribution kinetics and tissue accumulation patterns of PFOA and PFBS, demonstrating the model's robustness and adaptability.

Future Activities:

We will gestationally expose second cohort of offspring to hexafluoropropylene dimer acid (HFPO-DA).
Will gestationally expose offspring to perfluoro-3,5,7,9-tetraoxadecanoic acid (PFO4DA) at postnatal day (PND) 21, blood and organs will be collected from one male and one female offspring per litter and archived for later analyses.
At PND28, spleens and thymuses will be collected from one male and one female offspring per litter for immunophenotype. At PND56, blood and organs will be collected from one male and one female offspring per litter and archived for later analyses and spleens and thymuses will be collected for immunophenotype.

References:

Publictations (This reporting period)

Rericha Y, St. Mary L, Truong L, McClure RS, Martin JK, Leonard S, Thunga P, Simonich MT, Waters KM, Field JA, Tanguay RL. Distinct transcriptomic responses to structurally diverse per-and polyfluoroalkyl substances (PFAS) precede developmental toxicity in zebrafish. Frontiers in Toxicology. 2024;6. doi: 10.3389/ftox.2024.1425537

Cao D, Schwichtenberg T, Duan C, Xue L, Muensterman D, Field J. Practical Semiquantification Strategy for Estimating Suspect Per- and Polyfluoroalkyl Substance (PFAS) Concentrations. Journal of the American Society for Mass Spectrometry. 2023;34(5):939-47. doi: 10.1021/jasms.3c00019.

Presentations (This reporting period)

Invited Speaker “Thinking outside the tank to unravel chemical activity mechanisms” Seoul National University of Science & Technology, Seoul Korea April 11th 2024.

Invited Speaker “Use of High Throughput Zebrafish Model for Screening of Potential Developmental Toxicants: Benefits Over Cellular Model and Remaining Challenges in Translation” Society of Toxicology Annual Meeting, Salt Lake City, UT, March 13, 2024.

Invited Speaker “Thinking outside the tank to unravel chemical activity mechanisms” University of Florida, Gainesville FL, February 21st, 2024.

Invited Speaker “Predicting Developmental and Neurotoxicity of Chemicals and Mixtures Using Multidimensional Zebrafish Data” Amity University, Noida India January 10th, 2024.

Invited Speaker “Protecting Human Health Through High Throughput Discovery of Gene and Environment Interactions in Zebrafish” National Institute of Environmental Health Sciences (NIEHS), Centre for Health Analytics Research and Trends (CHART), Trivedi School of Biosciences, Ashoka University & Centre for Chronic Disease Control (CCDC) Sonepat Haryana India, January 8, 2024.

Invited Speaker “Protecting Human Health Through High Throughput Discovery of Gene and Environment Interactions in Zebrafish”, Indo-US International Workshop on Emerging Issues in Environmental Public Health-Dissecting Genetic and Exposome Perspectives, National Environmental Engineering Research Institute Council of Scientific and Industrial Research, Nagpur India January 4th, 2024

Invited Speaker “Using Multidimensional Zebrafish Data to Advance Environmental Health Sciences”, Rutgers University, Piscataway, NJ December 7th, 2023.

Invited Speaker “Using Multidimensional Zebrafish Data to Protect and Improve Human Health “University of Illinois Interdisciplinary Environmental Toxicology Program, November 16, 2023.

Invited Speaker “Scientific Rationale and Opportunities to Use Integrated Models for DNT Assessments”, Food and Drug Administration Workshop State of the Science on Assessing Developmental Neurotoxicity (DNT) Using New Approach Methods (NAMs), Harvey W. Wiley Federal Building Auditorium, College Park, Maryland USA November 14-15, 2023.

Invited Speaker for the 2023 National Institutes of Health EDI Director’s Speaker Series, Wednesday, November 6, 2023, Virtual.

Protecting Human Health through High Throughput Discovery of Gene and Environment Interactions in Zebrafish, University of California Los Angeles, November 1, 2023

Thinking Outside the Tank to Protect Human Health and the Environment, Keynote Presentation for the ToxMSDT Kick-Off Workshop September 28-30, 2023, University of California Davis.

Full organism approaches to support the risk assessment of chemical mixtures, Workshop: In the Mix-Use of New Approach Methods for Chemical Mixtures Risk Assessment, 2023 Eurotox Meeting, Ljubljana Slovenia, September 12th, 2023.

Using Zebrafish for Chemical Mixtures Risk Assessment, 12teh World Congress on Alternatives to Animal Testing, Niagara Falls, Canada, August 29th, 2023.

The Scientific Rationale for Using Complex Integrated Systems for Human Safety Assessments, HESI annual meeting-Not Just Another NAMs Meeting, London, UK June 7th, 2023.

Expanding the zebrafish neurobehavioral screening toolbox to more rapidly detect neurobehavioral toxicants across life stage, 18th International Neurotoxicology Association Meeting, Durham, NC, May 22nd, 2023.

Using Multidimensional Zebrafish Data to Protect and Improve Human Health, Baylor College of Medicine-RICE Superfund Seminar Series, May 16th, 2023 (Virtual)

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Publications Views
Other project views:	All 38 publications	12 publications in selected types	All 12 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Rericha Y, St Mary L, Truong L, McClure RS, Martin JK, Leonard S, Thunga P, Simonich MT, Waters KM, Field JA, Tanguay RL. Distinct transcriptomic responses to structurally diverse per-and polyfluoroalkyl substances (PFAS) precede developmental toxicity in zebrafish. Frontiers in Toxicology.;6:1425537.	R839481 (2023)	Full-text from PubMed Abstract from PubMed Full-text: Frontiers - Full Text HTML Exit
Journal Article	Cao D, Schwichtenberg T, Duan C, Xue L, Muensterman D, Field J. Practical semiquantification strategy for estimating suspect per-and polyfluoroalkyl substance (PFAS) concentrations. Journal of the American Society for Mass Spectrometry. 2023 Apr 5;34(5):939-47	R839481 (2023)	Abstract from PubMed Full-text: ACS Publications - Full Text HTML Exit

Supplemental Keywords:

immunotoxicity, zebrafish, toxicity, development, modeling, bioaccumulation, dietary, pharmacokinetic

Relevant Websites:

OSU/PNNL data portal

Progress and Final Reports:

Original Abstract

The 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.