Grantee Research Project Results

2024 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: R840457
Title: 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 May 1, 2025
Project Period Covered by this Report: September 1, 2023 through August 31,2024
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: Health Effects , Computational Toxicology , Endocrine Disruptors , New Approach Methods (NAMs) , Human Health , Safer Chemicals , Mixtures , Chemical Safety for Sustainability , CSS

Objective:

The 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.

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 allow us to understand the toxicology of PAH mixtures in humans and other mammals and serve as a potential system for future studies with other toxic compounds affecting the intestinal tract.

Progress Summary:

In this reporting period, we performed MTT assays to determine the lethal concentration 50 (LC₅₀) for individual PAHs and PAH mixtures in mouse intestinal cell line (C57BL/6) and intestinal porcine enterocyte cell line (IPEC-J2). The PAHs 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. We determined that 0.025 % (v/v) Dimethyl sulfoxide (DMSO) was the optimum solvent concentration to dissolve PAHs without causing toxic effects to the cells. We optimized the single-channel and multi-channel flow cell bioreactor (3D-FCB). The single-channel 3D-FCB can accommodate one cell line and treatment under microfluidic conditions. Whereas the multichannel can accommodate up to 8 different cell lines or treatments. Both setups were optimized for the flow rate of culture media, temperature, and the length of tubes connecting the different components, and bacterial contamination. The single and multichannel 3D-FCB setups were tested with mammalian cells seeded on scaffolds - Clonal Embryonic stem cell line (C57BL/6),  Intestinal Porcine Enterocyte cell line (IPEG-2). After 7 days, the cells were immunostained with muc2 and lgr5, and the expression was observed using confocal imaging (Leica DM2500 SPE). The results indicated that the cells were living (nucleus expression), differentiated (muc2 and lgr5 expression), and growing around the villi structures of the scaffolds, similar to what is seen in the gut tissue.  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.  We also performed in vivo pilot experiments using Neratinib (EGFR/HER2 inhibitor). BALB/c mice were randomly separated into two groups and were treated with saline (control) or neratinib (30 mg/kg, oral gavage, five days per week for 3 weeks). Hematoxylin and Eosin staining was performed on 4-μm tissue sections analyzed under a phase-contrast microscope. Neratinib treatment caused severity of colonic epithelial injury, especially degeneration of surface lining colonocytes and infiltration of immune cells distal colon at day 24.

Future Activities:

This grant project's upcoming activities aim to advance existing research by focusing on the effects of PAH mixtures on human, pig, and mouse IEC (intestinal epithelial cells) using 3D-FCB. The key objectives for these future activities are:

1. Application of 3D FCB system: 1. Perform qPCR to quantify the gene expression of muc2 and lgr5 in C57BL/6 and IPEC-J2 cells in single channel 3D-FCB; 2. Optimize the design for high throughput 3D-FCB to accommodate 8 cell lines/treatments in a single setup; 2. Evaluate effect of PAH mixtures on IPEC-J2, C57BL/6, and primary Human Small Intestine Epithelial (FHs 74 int) cells; 3. Test the 3D FCB system to simulate chronic exposure of PAH mixtures for 21 days. 
2. 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.
3. 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.
4. 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 expression

Progress and Final Reports:

Original Abstract

2023 Progress Report

Final