Grantee Research Project Results
2022 Progress Report: System toxicological approaches to define and predict the toxicity of Per and Polyfluoroalkyl Substances
EPA Grant Number: R839481Title: 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 April 30, 2022 (Extended to April 30, 2025)
Project Period Covered by this Report: May 1, 2022 through April 30,2023
Project Amount: $1,981,500
RFA: National Priorities: Per- and Polyfluoroalkyl Substances (2018) RFA Text | Recipients Lists
Research Category: Water Quality
Objective:
- 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.
- 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.
- 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:
Progress Objective 1:
In vivo Screening Results Progress to date.
The following sources were used to secure PFAS compounds for testing:
- Wellington Laboratories PFAS Analytical Standards (58)
- US EPA PFAS Library (139)
- Commercial Reference Standard Library (25)
US EPA PFAS Library Evaluations. Per- and polyfluoroalkyl substances (PFAS) are a class of widely used chemicals with limited human health effects data relative to the diversity of structures manufactured. To help fill this data gap, an extensive in vivo developmental toxicity screen was performed on 139 PFAS provided by the US EPA. Dechorionated embryonic zebrafish were exposed to 10 nominal water concentrations of PFAS (0.015–100 µM) from 6 to 120 h postfertilization (hpf). The embryos were assayed for embryonic photomotor response (EPR), larval photomotor response (LPR), and 13 morphological endpoints. A total of 49 PFAS (35%) were bioactive in one or more assays (11 altered EPR, 25 altered LPR, and 31 altered morphology). Perfluorooctanesulfonamide (FOSA) was the only structure that was bioactive in all 3 assays, while Perfluorodecanoic acid (PFDA) was the most potent teratogen. Low PFAS volatility was associated with developmental toxicity (p < 0.01), but no association was detected between bioactivity and five other physicochemical parameters. The bioactive PFAS were enriched for 6 supergroup chemotypes. The results illustrate the power of a multi-dimensional in vivo platform to assess the developmental (neuro)toxicity of diverse PFAS and in the acceleration of PFAS safety research. This Manuscript is published PMID: 32827657.
Phenotypically Anchored Transcriptomics. While the number of -omics studies addressing PFAS toxicity is increasing, definitive modes of action of PFAS toxicity have not yet been established. This may in part be due to the challenge of translating data between different model organisms, exposure paradigms, and life stages. Therefore, transcriptomic analysis of numerous PFAS in a single, highly standardized system will enable direct comparison between compounds and greater insight into modes of action. The present study characterized transcriptomic responses to structurally diverse bioactive PFAS in developmental zebrafish under standardized conditions previously employed to evaluate other chemical classes. Our aim was to phenotypically anchor transcriptional changes to whole-animal phenotypes. When possible, exposures were conducted at concentrations that induced approximately 80% incidence morphological effects at 120 hpf, but zebrafish were sampled at 48 hpf before those effects were observable. In doing so, this study identified unique and commonly affected gene expression profiles among PFAS and began to elucidate mechanisms of toxicity. This Manuscript is in late stages of development for publication in PLOSONE..
Dietary Perfluorohexanoic Acid (PFHxA) Study. Diet is a predominate route of human exposure, and PFAS are frequently measured in food. Manufacturing trends have shifted from legacy PFAS to shorter-chain alternatives that are suggested to be safer, such as perfluorohexanoic acid (PFHxA). However, the current amount of data to support safety assessments of these alternatives is not yet sufficient. In this study we investigated the effects of a 42-day dietary exposure to 1, 10, or 100 ng/g PFHxA in juvenile zebrafish. The zebrafish model was leveraged to interrogate morphometrics, fecundity, and numerous behavior endpoints across multiple generations. Dietary PFHxA exposure did not result in measurable body burden and did not affect growth, fecundity, adult social perception behavior, or associative learning. PFHxA exposure did induce abnormal adult anxiety behaviors in the F0 generation that persisted transgenerationally in the F1 and F2. Abnormal larval and juvenile behavior was observed in the F1 generation, but not in the F2. PFHxA juvenile dietary exposure induced subtle and multigenerational behavior effects that warrant further investigation of this and other alternative short-chain PFAS. This Manuscript is published PubMed PMID: 35878277.
Importance of Head Group in PFAS Toxicity PFAS Studies have predominately focused on long-chain PFAS, with far fewer addressing short-chain alternatives. This study leveraged embryonic zebrafish to investigate developmental toxicity of a short-chain series: perfluorobutane sulfonate (PFBS), perfluoropentanoic acid (PFPeA), perfluorobutane sulfonamide (FBSA), and 4:2 fluorotelomer sulfonic acid (4:2 FTS). Following static exposures at 8 h postfertilization (hpf) to each chemical (1–100 mM), morphological and behavioral endpoints were assessed at 24 and 120 hpf. Only FBSA induced abnormal morphology, while exposure to all chemicals caused aberrant larval behavior. RNA sequencing at 48 hpf following 47 mM exposures revealed only FBSA significantly disrupted normal gene expression. Measured tissue concentrations were FBSA > PFBS > 4:2 FTS > PFPeA. This study demonstrates functional head groups impact bioactivity and bioconcentration. This manuscript is published PMID: 35146398.
Quantitative Assessment of Laboratory Animal Diet PFAS Contamination. Zebrafish are a popular biological model because they share early development pathways with humans. A dietary exposure paradigm is growing in popularity in the zebrafish model because the outcomes often translate to humans. To create a diet of known composition, it is crucial to understand background PFAS levels present in zebrafish diet. Background PFAS, if present, potentially confounds interpretation of toxicological data. To date, no studies document the PFAS background levels in laboratory fish diet and there is only limited information on some pet foods. The objective of this study was to develop and validate an analytical method for up to 50 target PFAS in high lipid and protein content laboratory fish diets and pet foods. Long-chain perfluoroalkyl carboxylic acids (C9-C13) and perfluorooctane sulfonate (PFOS) were quantified in 11 out of 16 laboratory fish diets and in three out of five pet fish foods. Foods for pet birds, lizards, and dogs were below the limit of detection for all PFAS. In two of the laboratory fish diets, PFOS concentrations were >1.3 ng/g and the total PFAS for the three laboratory fish diets exceeded 1.0 ng/g. Hundreds of biomedical laboratories across the world utilize these commercial laboratory fish diets, and these results indicate that numerous zebrafish colonies may be inadvertently receiving significant dietary PFAS exposures. In light of this new information, it is critical to design PFAS studies with appropriate controls with measured background PFAS concentrations in the diet and to urge caution when interpreting the results. This manuscript is published PMID: 35750184.
Progress Objective 2:
Our goal was to evaluate six different PFAS in a mouse model for markers of developmental immunotoxicity (DIT). To date, we have evaluated three different PFAS (PFMOAA, Nafion Byproduct 2, and FHxSA). However, we completed six full experiments as we ran three replicates for PFMOAA, one for Nafion Byproduct 2, and two for FHxSA). These replicates are full DIT experiments that take approximately three months just for the animal work and additional time after to complete assays on collected biological tissues and fluids. These replicates were necessary to attain biologically meaningful sample sizes.
Our experiments with these PFAS indicate that in our mouse model, at the doses administered and for the duration of exposure (gestational day 1-17), we were not able to detect changes in the DIT markers we evaluated. During our NCE year, we plan to evaluate one final PFAS for a total of four individual PFAS evaluated.
Progress Objective 3:
The Pitt team has focused on two physiologically based toxicokinetic (PBTK) models for PFAS: a zebrafish model (for PFOA in adult zebrafish) and a mouse model (for several different PFAS in adult mice). The zebrafish model is an update to the model originally published by Khazaee & Ng in 2018. The mouse model is new and intended to capitalize on the new mouse data generated by ECU and OSU.
The major improvements of the zebrafish model include a better physiological parameterization that includes more fish-specific and zebrafish-specific data, and a refinement of the description of protein binding in the different compartments. New data on adult zebrafish PFOA accumulation and tissue distribution, published since 2020, aided in the improved parameterization and calibration of model performance.
The mouse model was developed from the framework of the rat model originally published by Cheng & Ng in 2017, re-parameterized to reflect the physiology of the mouse. In addition to this new species-specific parameterization, the major contribution of this model is the inclusion of a new mechanistic description of PFAS associating with phospholipids within the cell membrane and the impact of this interaction on both the kinetics of PFAS inter-compartment transport and the tissue distribution (due to different phospholipid levels in different tissues). The parameterization of PFAS-phospholipid interactions is based on new membrane-water partition coefficient (Kmw) data generated by Droge (2019) and Droge et al. (2021). The new model captures the different distribution kinetics and tissue accumulation patterns of PFOA and PFBS (with chemical-specific parameterization), illustrating the model’s robustness and flexibility.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 22 publications | 10 publications in selected types | All 10 journal articles |
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Truong L, Reircha Y, Thunga P, Marvel S, Wallis D, Simonich M, Field J, Cao D, Reif D, Tanguay R. Systematic developmental toxicity assessment of a structurally diverse library of PFAS in zebrafish. JOURNAL OF HAZARDOUS MATERIALS 2022;431. |
R839481 (2020) R839481 (2022) |
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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.