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Research

Research

September 2015: An In Vitro Evaluation of Nicotine Kinetics Utilizing a Human Airway Model (Mucilair™) Combined With an Integrated Multi-Organ Culture Plate

Poster presented at the Congress of the European Societies of Toxicology

Abstract: The framework set out in the report entitled “Toxicity Testing in the 21st Century” (2007) focused on the need for the development of alternative methods to animal testing. Emphasis was placed on the use of human cells combined with adverse outcome pathways for determining adverse effects. Predicting systemic toxicity from in vitro data requires a platform that incorporates multiple organs interconnected via a fluidics network, in such a way that pharmacokinetic data can be integrated into the responses observed. The aim of this study was to evaluate a new Dynamic Multi-Organ Plate (DMOP) that provides the ability to use human tissues or cells, which are in communication via fluidics and dialysis. A simple transwell-based, two organ compartment model containing a human airway model (MucilAirTM) was used to measure nicotine kinetics and biochemical effects. A standard 6-well plate was fitted with a fluidics/dialysis network. The dialysis membrane allows for exchange of test article and metabolites while maintaining each cell type under optimized media conditions. There is no net gain or loss of fluid. Nicotine (1000 µM) was added to the apical side of the MucilAir™ model. Nicotine in the basolateral chamber diffused into the fluidics system via dialysis and was carried to the delivery well. A static sample from the basolateral and delivery chambers was collected every hour for 6 hrs. Perfusate from the fluidics system was collected at integrated one hour time intervals for 6 hr at a flow rate of 1 µL/min. Nicotine was first detected in the basolateral chamber at 1 hr and reached a maximum concentration (3-4 µg/mL) by 5 hr. Nicotine was first detected in the delivery chamber at approximately 3 hr and increased linearly up to 0.3 µg/mL. Nicotine in the run through perfusate was also detected at 3 hr. When kinetic data is combined with biochemical changes in key biomarkers extrapolating from in vitro to in vivo becomes more accurate. These data demonstrate that a meso-scale multi-organ culture platform linked by fluidics/dialysis can provide a means of evaluating pharmacokinetic and pharmacodynamic parameters to study the movement of chemicals and their metabolites between organs in order to extrapolate in vitro toxicity to in vivo systemic effects.

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June 2015: Applied In Vitro Toxicology — Changing the Paradigm of Toxicity Testing From Observational to Predictive: An Update on Two Global In Vitro Screening Initiatives

Moderator: James McKim

Participants: Tina Bahadori, Warren M. Casey, Richard S. Paules, Russell S. Thomas

In this roundtable discussion, Dr. McKim brings together a few leaders in both Tox21 and ToxCast to discuss the strategies behind each initiative, progress made to date, examples of how the data is being used, and what we can expect in the future.

Moderator: James McKim, Editor-in-Chief, Applied In Vitro Toxicology

Participants:

Tina Bahadori, Program Director, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC

Warren M. Casey, Director, NTP Center for the Evaluation of Alternative Toxicological Methods (NICEATM)

Richard S. Paules, Acting Chief, Biomolecular Screening Branch, Division of the NTP, National Institute of Environmental Health Sciences

Russell S. Thomas, Director, National Center for Computational Technology; U.S. Environmental Protection Agency, Washington, DC.

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April 2015: Food & Chemical Toxicology — The common food additive carrageenan is not a ligand for Toll-Like-Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model

Abstract: Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter mol- ecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/ mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins.

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March 2015: Evaluation of an Integrated Human Multi-Organ Culture Plate For Predicting Systemic Toxicity

Poster presented at the Society of Toxicology

Abstract: The framework put forth in the report entitled “Toxicity Testing in the 21st Century” (2007) focused on the need for the development of alternative methods to animal testing. Emphasis was placed on the use of human cells combined with adverse outcome pathways for determining adverse effects. Predicting systemic toxicity in vitro requires a platform that incorporates multiple organs interconnected via a fluidics network. The aim of this study was to evaluate a new Dynamic Multi-Organ Plate (DMOP) that provides the ability to use human tissues or cells, which are in communication via fluidics and dialysis. A simple transwell-based, two organ compartment model containing Caco-2 cells and rat primary hepatocytes was used initially. A standard 6-well plate was fitted with a fluidics/dialysis network. The dialysis membrane allows for exchange of test article and metabolites while maintaining each cell type under optimized media conditions. Caco-2 cells were grown to confluency on a transwell insert and then transferred to the DMOP. To mimic oral dosing, Acetaminophen (APAP) (200 µM) was added to the upper chamber of transwells containing confluent monolayers of Caco-2 cells. Dialysis samples from the lower chamber were collected at a flow rate of 1 µL/min. Integrated time samples were collected at 1, 2, 3, and 6 hr. APAP was first detected in the 1-2 hr dialysate with a relative recovery of approximately 70%. APAP was added to the second compartment containing a monolayer of primary rat hepatocytes and dialysis samples collected over 2 hr. APAP, as well as its glucuronide and sulfate conjugates, were detected in the perfusate. Relative recovery of APAP in the hepatocyte dialysate was >95%. These data demonstrate that a meso-scale multi-organ culture platform linked by fluidics/dialysis can provide a means of evaluating the movement of chemicals and their metabolites between organs in order to predict systemic effects in vitro.

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>March 2015: Evaluation of the Food Additive Carrageenan in a Caco-2 Intestinal Absorption Model

Poster presented at the Society of Toxicology

Abstract: Carrageenan (CGN) belongs to a group of viscosifying polysaccharides that are extracted from certain species of red seaweeds in the family Rhodophyceae. CGN is composed of a linear backbone of galactose sugars linked together by alternating alpha 1-3 and beta 1-4 glycosidic linkages. CGN sugar units are both sulfated and non-sulfated. There are three major commercial forms of CGN (lambda-, kappa-, and iota-) which differ by the number and position of the sulfate moieties. CGN is used by the food industry as a thickening and stabilizing agent. Several recent studies using a human colonic cell line (NCM460) have reported that CGN is a ligand for TLR4 and that binding of CGN to TLR4 leads to inflammation. To date, direct binding to TLR4 by CGN has not been shown. Therefore, the aim of this study was to characterize the possible binding of CGN to TLR4 and subsequent activation of downstream signaling events. To accomplish this, a stably co-transfected HEK293 cell line that expresses full-length TLR4 and the secreted alkaline phosphatase (SEAP) under the control of NF-kB response element (Imgenix) was used. Cells were treated with the positive control, lipopolysaccharide (LPS), and to food grade CGN, commercially available lambda-, kappa- and iota-CGN, and to clarified locust bean gum (CLBG) a negative control. All test agents were administered to the cells at concentrations ranging from 0.1-5000 ng/mL and incubated for 6 hr at 37oC in the presence of 10% fetal bovine serum. Each treatment group was prepared in triplicate wells and the experiment repeated on two separate days. Following the exposure period activation of TLR4 signaling was assessed by measuring SEAP in the culture medium. LPS increased SEAP in a concentration dependent manner with a maximum response at 50 ng/mL. In contrast, there was no detectable increase in SEAP following exposure to CLBG, or to any form of CGN. These data indicate that under standard cell culture conditions CGN is not a ligand/agonist for TLR4 in this HEK cell model.

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>March 2015: Applied In Vitro Toxicology> — Roundtable Discussion — A Vision of Toxicity Testing in the 21st Century

Moderator: Jim McKim

Participants: Alan Goldberg, Nicole Kleinstreuer, Francois Busquet, Melvin Andersen

In this roundtable discussion, Dr. McKim brings together some of the great minds in the field of toxicity testing to discuss a very important issue: The National Academy of Science report on toxicity testing in the 21st century. The central question will be: How do we as scientists begin to implement the concepts put forth in this report, with a focus on adverse outcome pathways (AOPs) and the methods available and still required?

Moderator: Jim McKim, Editor-in-Chief, Applied In Vitro Toxicology. Founder and CEO of IONTOX, LLC.

Panelists include:
Alan Goldberg, Consulting Editor, Applied In Vitro Toxicology.

Nicole Kleinstreuer, ILS/National Toxicology Program Interagency Center for Evaluation of Alternative Toxicological Methods (NICEATM).

Francois Busquet, Center for Alternatives to Animal Testing (CAAT Europe).

Melvin Andersen, Institute for Chemical Safety Sciences and ScitoVation (Formerly The Hamner Institute), Research Triangle Park, North Carolina.

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