Grantee Research Project Results

2024 Progress Report: Assessment of Underlying Molecular Mechanisms Promoting Adipogenic Outcomes in Complex Mixtures

EPA Grant Number: R840459
Title: Assessment of Underlying Molecular Mechanisms Promoting Adipogenic Outcomes in Complex Mixtures
Investigators: Kassotis, Christopher Dennis , Korzeniewski, Steven
Institution: Wayne State University
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2022 through May 12, 2025
Project Period Covered by this Report: September 1, 2023 through August 31,2024
Project Amount: $598,487
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 , Endocrine Disruptors , New Approach Methods (NAMs) , Early Career Awards , Human Health , Safer Chemicals , Mixtures , Chemical Safety for Sustainability , Non-Vertebrate Animal Testing , CSS

Objective:

(1) We will evaluate mixtures of increasing complexity for ability to promote adipogenesis. Specifically, we will examine mixtures of increasing complexity for their deviations from expected adipogenic effects using concentration addition and independent action models. We hypothesize that mixtures of increasing complexity will shift towards independent action, but that the most complex mixtures will not be well predicted by either model. We will also develop an effect-based model to predict adipogenic activity based on component bioactivities, which we hypothesize will provide a chemical agnostic approach to risk assessments of realistic environmental mixtures. (2) This proposed research program will rigorously assess available mixture models through controlled assessments of contaminants acting through shared, distinct, and mixed mechanisms. We will utilize in silico approaches to select chemicals predicted to promote adipogenesis through distinct mechanisms. We will use in vitro and in vivo models of metabolic health disruption to assess both individual chemicals and their mixtures and compare with predicted outcomes based on concentration addition and independent action. (3) Our results will promote a greater functional understanding of complex mixture effects that can be utilized to bolster risk assessments of diverse contaminant exposures. By using this stepwise approach of increasing mixture complexity, we expect a primary output of clearly describing the weaknesses and strengths of these standard mixture models. Through our testing of dust extracts, we expect that we can support a new method of mixture risk assessments through using component mechanistic effect levels to determine cooperative effects on complex health endpoints and/or outcomes.

Progress Summary:

The first mixture (Aim 1A) was tested both in vitro and in vivo for all measured outcomes. Each of the chemicals and the mixture were able to activate the peroxisome proliferator activated receptor gamma and each of the chemicals and the mixture were pro-adipogenic in the human mesenchymal stem cell model. This mixture represents a simple mixture acting through a shared (single) mode of action on metabolic disruption endpoints and the concentration addition model performed best at predicting observed responses, as expected. The second mixture (Aim 1B) was tested both in vitro and in vivo for all measured outcomes. While these chemicals were selected from the ToxCast database to hit one of six pro-adipogenic pathways only, we found that these predictions did not match their actual bioactivities as measured in reporter gene assays. Unfortunately, this complicates the tiered approach to our mixture model assessment, but it still reflects a demonstrably more complex mixture than 1A and a demonstrably less complex mixture than 2A. Our preliminary assessment of the 2A chemicals supports a much more complex chemical mixture from the standpoint of underlying pro-adipogenic mechanisms and should provide a considerable increase in mechanistic complexity for evaluation in the mixture models.

Our results describe that 1) metabolism disrupting chemicals are considerably more widespread in the environment, with a number of these contaminants being reported as active on metabolic health for the first time; 2) that the mixtures generally always produce greater effects in combination than would be predicted based on individual component chemical testing. While determinations of synergy are rare and not observed here, additivity is a common phenomenon and are entirely ignored by current federal chemical regulations. Moreover, we provide additional data supporting that single mechanism mixtures are more accurately predicted via concentration addition than via independent action for mixtures of chemicals acting through more simple mechanisms of action. We are now proceeding to test more complex chemical mixtures and already see more equality in responsiveness for concentration addition and independent action, with the most complex mixtures still pending. We anticipate that our next aims will help to identify which models work best when multiple mechanisms are involved in the outcomes of interest.

Future Activities:

Manuscript writing was delayed in this funding period due to keeping on track with experimental objectives in bench work. The next year of the funding period will include writing and submission of manuscript on Aim 1A by end of 2024. We will also present this project at one or more conferences during the year three period. We will complete analysis of all in vitro and in vivo testing for Aim 1B and prepare this manuscript for submission by December 2024 / January 2025. In vitro testing is ongoing for Aim 2A, with reporter gene assays and hMSC assays complete by March 2025 and zebrafish experiments complete by the end of December 2024. Analysis and writing will commence in earnest in early 2025 with the expectation of submission by Summer 2025. Results will be presented at one or more meetings during year two. We expect to acquire our dust samples for Aim 2B by the end of 2024 and will begin in vitro testing in early 2025.

Supplemental Keywords:

Adipogenesis; Metabolic Health; Obesogen; Mixture; Dust; Chemicals; Organics; Cumulative Effects; Organism; Cellular; Animal

Relevant Websites:

Kassotis Lab Exit

Progress and Final Reports:

Original Abstract

2023 Progress Report

Final