Grantee Research Project Results
2023 Progress Report: Oral toxicity assessment of PAH mixtures using an in vitro 3D cell culture bioreactor mimicking the in vivo intestinal tract environment
EPA Grant Number: R840457Title: Oral toxicity assessment of PAH mixtures using an in vitro 3D cell culture bioreactor mimicking the in vivo intestinal tract environment
Investigators: Rodrigues, Debora , Liu, Xinli
Institution: University of Houston
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2022 through August 31, 2025
Project Period Covered by this Report: September 1, 2022 through August 31,2023
Project Amount: $749,926
RFA: Development of Innovative Approaches to Assess the Toxicity of Chemical Mixtures Request for Applications (RFA) (2022) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Health Effects , Computational Toxicology , Human Health , Chemical Safety for Sustainability , New Approach Methods (NAMs) , Mixtures , CSS
Objective:
The overarching objective of this study is to design 3D cell cultures with a flow cell bioreactor to obtain an in vitro system that resembles a mammalian intestinal tract and its microenvironment. This system aims to provide an ideal, affordable, and high throughput toxicological method to replace current in vivo experiments to evaluate polyaromatic hydrocarbon (PAH) mixtures that can be found in food.
In this study we will use the bioreactor with grown human, mouse, and pig cell lines to compare the toxicology of PAH mixtures. We will validate the bioreactor results with in vivo mice models. In addition to the growth of mammalian cells, we also aim to investigate the interaction between the PAH mixtures and the gut microbiome and determine whether they can potentially biodegrade PAHs and produce toxic byproducts. This investigation will have two benefits: first, it will allow us to gain an understanding of PAH mixtures toxicology in humans and other organisms and determine which or if both cell lines are appropriate to understand the pathophysiology of humans related to PAHs and serve as potential systems for future studies with other toxic compounds affecting the intestinal tract
Progress Summary:
In this reporting period we designed the flow cells and the scaffolds with villi structures to grow intestinal epithelial cells (IECs) in 3D. Most of the analyses were done after 3 weeks, however, the human and pig cell lines could be incubated for up to 2 months in the scaffold. The analyses involved determining cell differentiation by using immunofluorescence confocal imaging and quantitative real time PCR with specific markers. We were able to confirm that the initial intestinal cells were differentiated into stem and goblet cells and mucin secretion was happening only in the cells growing in the scaffold as opposed to the cells grown in 2D monolayer cultures. As the next steps of this study, we will use the human, mouse, and pig cells grown in the bioreactors to investigate toxicity of the individual and mixtures of PAHs. The PAHs to be used in this study are benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, chrysene, dibenz[a,h]anthracene, and indeno[1,2,3-cd]pyrene. These PAHs pose significant health concerns due to their high toxicity and association with genotoxic and carcinogenic properties. In parallel with developing the bioreactors, we undertook the essential task of determining the best solvent to well dissolve these PAHs and their optimal concentrations to avoid any confounding toxicity effects to the cells related to the solvent. We investigated the toxicity of various solvents and concentrations of Dimethyl sulfoxide (DMSO), Acetonitrile, and Acetone. The results demonstrated that DMSO was the most appropriate solvent since it did not present cytotoxicity to the cell lines investigated in concentrations below 0.05%. Additionally, during this reporting period, we also have optimized the process to determine accurately the concentration of PAHs using Gas Chromatography-Mass Spectrometry (GC-MS) to ensure that the PAH concentrations prepared in 0.05% DMSO to be used in our experiments aligns accurately with our research objectives. Significant progress has also been made on obtaining the fecal microbiota from pigs, mice, and humans and determining the suitability of the Gut Microbiota Medium (GMM) to support the growth of microbiomes derived from these three species. This data holds substantial importance as it forms the foundation for our comparative study, enabling us to delve into the effects of PAHs on the gut microbiome across various organisms.
Future Activities:
This grant project's upcoming activities aim to advance existing research by focusing on IEC (intestinal epithelial cells) derived from human, pig, and mouse cell lines within a 3D bioreactor and their microbiota. The key objectives for these future activities are:
- Comparative cytotoxicity Analysis: We will determine the PAH mixture toxicity and dose responses in IEC from human, mice and pigs grown in 2D conditions and in the 3D in vitro system that resembles the intestinal tract. The goal is to determine the dose responses of PAHs in these cell lines under different growth conditions.
- Model Organism Evaluation: We will compare the observed genotoxic effects in cell lines from the 3D FCB. This assessment will allow us to determine whether pigs and mice can be good models for human investigations in future studies on the impact of various toxic compounds on the gastrointestinal system.
- Interaction of PAHs and the gut microbiome: we will determine whether the gut microbiome could potentially degrade and generate byproducts of PAHs in the gut, which could increase or reduce the toxicity of hydrocarbons ingested by humans.
- Validation of the 3D FCB system: In vivo experiments on mice will be used to validate the in vitro data for PAH mixtures for the effects of PAH mixtures in the mice gut and microbiota.
In summary, these future activities aim to leverage insights from 3D FCB in vitro experiments and conduct a comparative analysis among different cell lines. This research will provide a framework for evaluating the effects of PAHs and other environmental contaminants on the GI tract, potentially expanding the 3D FCB system's coverage. Additionally, based on research results, we plan to disseminate findings through publications, conferences, and meetings, fostering collaboration within the scientific community.
Supplemental Keywords:
Polyaromatic Hydrocarbon mixture (PAH), Intestinal Epithelial Cell (IEC), Gut Microbiota Medium (GMM), flow-cell-bioreactor (FCB), 3D cells culturing, Intestinal track model, Toxicity, Environmental contaminants, GI tract organs, Gut microbiome, Health risk, 3D villi scaffold, Gene expressionThe 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.