MatTek scientists will be attending and presenting posters at the Society of Toxicology Annual Meeting in Anaheim, California. Read more to see what they’ve been working on and request copies of their posters. We can’t wait to see you at SOT 2020!
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In vitro – in vivo assay validation for vaginal irritation of chemicals (Abstract: 1541, Poster P659)
A. Armento, T. Landry, M. Klausner, Y. Kaluzhny, and S. Ayehunie. MatTek Corporation, Ashland, MA.
Session: Reproductive Toxicology I
Monday, March 16 | 10:45AM – 12:30PM
The goal of the study is to validate the utility of the 3D human in vitro vaginal tissue model as an alternate for rabbit vaginal irritation using a set of coded test articles (TAs). A double-blinded study was conducted in which the investigator and the animal testing facility were provided with N=14 coded test articles and assayed in vitro and in vivo following topical application at 2% dose of each TA and 5 repeat exposures over 6 days. Dose volumes were proportionally adjusted based on the surface area and N=5 rabbits and N=3 EpiVaginal tissues were used per TA. While the RVI score was used to monitor in vivo irritation; MTT, TEER, and histological analysis were used as endpoints for the in vitro assays. The results showed that four TA including two known irritants, benzalkonium chloride (BZK) and nonoxynol 9 (N9) were predicted as irritants by MTT viability and TEER (<50% reduction). While BZK was identified as a mild/severe irritant in the RVI assay, the effect of N9 in rabbits was highly variable – in three independent animal RVI studies, irritation was detected only in 25-30% of the animals. Furthermore, the other two TAs, copper sulfate and sodium dodecyl sulfate (SDS), that were determined as vaginal irritants in vitro were not identified as irritants in the RVI test. Additionally, cytokine analysis from culture supernatants showed that N9, SDS and BZK induces a significant increase (>2 fold) in IL-1α/IL-1β release by Epivaginal tissues. In contrast, only BZK showed a significant increase in IL-1α/IL-1β from rabbit vaginal lavages. Based on the in vitro in vivo data, we expanded the study to include N=55 test articles and the in vitro reproducibility of the results for the 55 test articles were monitored using reconstructed tissues from cells obtained from N=4 donors. In short, a combination of MTT, TEER, and IL-1α/IL-1β were found to be valuable makers of in vitro vaginal irritation. In conclusion, the in vitro assay method performs equally well or better compared to the standard RVI assay and could be a useful tool to assess vaginal toxicity of formulations, drugs, and medical devices.
Inter- and Intra-Laboratory Reproducibility of a Three-Dimensional Model of Human Buccal Tissue (Abstract: 1422, Poster: P502)
Session: Alternatives to Mammalian Models II
Monday, March 16 | 2:45PM – 4:30PM
Human 3D tissue models of human oral epithelia have been developed as alternatives to animal testing. These models are powerful tools for investigating basic biological function in response to various challenges including oral care products and environmental insults. To evaluate the inter-laboratory reproducibility of the commercially available 3D buccal epithelial model, EpiOral, MTT tissue viability was monitored. Tissues were shipped to Lion Corp (Idawara, Japan) and Kurabo Industries (Osaka, Japan) and the MTT values for negative control (NC) tissues (exposed to ultrapure water, 60 min) and the time to reduce tissue viability to 50% (ET-50), following exposure to 1% Triton X-100, were determined. NC tissues gave an average MTT value of 1.633 (n=15 lots) and 1.829 (n=11) at Lion and Kurabo, respectively, in independent tissue lots; tissues packaged and stored overnight at MatTek gave NC values of 1.545 (n=22 lots). ET-50 of tissues tested at Lion and Kurabo averaged 64.3 ± 12.9 (n=15) and 54.7 ± 10.3 (n=11) min, respectively; tissues tested at MatTek gave 79.6 ± 13.7 min (n=22). All lots tested in Japan and the US fell within the acceptance QC range for the NC (MTT > 1.0) and ET-50 (34.8-105.8 min). In addition, histology of non-cornified buccal tissue produced in 2019 was highly reproducible (n=22 lots). For intra-laboratory reproducibility, functional analysis was performed by exposing the tissues to two inducers in independently produced tissue lots (n=3). Tissues were exposed to TNF-α/IL-1β to induce an inflammatory response or to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) to evaluate xenobiotic metabolism. Adenylate kinase release, MTT activity, pro-inflammatory cytokine release and xenobiotic phase I enzyme expression were analyzed. Exposure of the oral tissues to the inflammatory inducers, TNF-α/IL-1β, showed statistically significant increases in secreted IL-8, IP-10, and MMP-1. TCDD induction also showed increased gene expression of the Phase I metabolism enzymes, CYP1A1 and CYP1B1. Overall, excellent reproducibility was observed for MTT and adenylate kinase endpoints over n=3 independent lots. In addition, reproducible trends in cytokine release and gene expression versus the NC tissues were observed in the n=3 tissue lots. In summary, the EpiOral model exhibited good inter-lab and intra-lab reproducibility and can serve as a tool to reduce animal experimentation for oral toxicity studies.
Novel Approach for Characterizing Exposure and Response to Engineered Nanomaterials in the Gut (Abstract: 2142, Poster P492)
J. Oldach, P. Llanos, M. Klausner, A. Armento, Y. Kaluzhny, and S. Ayehunie. MatTek Corporation, Ashland, MA.
Session: Nanotoxicology: In Vitro
Tuesday, March 17 | 9:00AM – 10:45AM
Despite the expanding number of applications for engineered nanoparticles (ENPs), human health concerns associated with ingested nanoparticles are poorly understood. In this study, we utilized a 3D human small intestinal tissue model to develop a physiologically relevant test system to assess toxicological profiles of ingestible nanomaterials. We examined barrier integrity and cytotoxicity of the human 3D intestinal tissue model following exposure (4 doses of each nanoparticle to Cupric (II) oxide (CuO) (50 nm in size), zinc oxide (ZnO, 35-45 nm in size), and titanium oxide (TiO2, 40 nm in size). To monitor the reproducibility of the test method the nanoparticles were tested using 3-4 intestinal tissue lots. To determine the effect of the nanoparticles on the small intestinal tissues, we examined 1) barrier integrity (TEER), 2) tissue viability (MTT), and inflammatory response (ELISA assay). In the various experiments performed, the tissues were exposed to 40 ul of different doses of sonicated nanoparticles under the rocking condition for 4 hr. After 4 hrs, dosed tissues were further cultured for overnight under static conditions. Using IC15 (concentration that reduces barrier function or tissue viability by 15%) as a cut-off, we observed a dose-response reduction of barrier integrity and tissue viability for CuO and ZnO. However, Titanium oxide did not induce toxicity for the concentrations tested. Furthermore, culture supernatants collected from at 24 hr of the culture period were also analyzed for inflammatory response and the result showed a dose-dependent release of IL-8 for CuO and ZnO. Overall, the TEER measurement was a sensitive endpoint compared to the MTT tissue viability assay. In summary, the use SMI tissue model to examine the toxicity profile of ingested nanotoxicity will also enhance our understanding of nanoparticle-host cell interaction, improve dose/design, generate a physiologically relevant data set, and provide greater insight into in vivo responses.
A Novel Macrophage-Containing Organotypic Small Intestinal Tissue for Modeling Gut Inflammation (Abstract: 1857, Poster P147)
S. Ayehunie1, H. Scott1, Z. Stevens1, K. Marengo1, T. Landry1, M. Klausner1, A. Langerveld2, E. Lehigh2, S. Wheeler2, A. Armento1, and Y. Kaluzhny1.
1MatTek Corporation, Ashland, MA; and 2Genemarkers, Kalamazoo, MO
Tuesday, March 17 | 10:45 AM -12:30PM
As gatekeepers of intestinal immune homeostasis, macrophages play a critical role in inflammation in the gut. In this study, we reconstructed a new macrophage-containing primary cell-based full-thickness small intestinal (SMI+M) tissue model and characterized it for 1) macrophage incorporation (immunohistochemistry, IHC), 2) barrier properties (TEER), and 3) functionality by measuring inflammatory responses following exposure to ligands for TLR4 (lipopolysaccharide; LPS), NOD-1 (C12-iE-DAP) and NOD-2 (L18-MDP) either individually or synergistically. For the identification of inflammatory responses, we utilized Affymetrix GeneChip arrays. Results showed that SMI+M tissues have 3D polarity and their morphology and physiological barrier property mimics that of native in vivo tissues. IHC of SMI+M tissues showed CD14+ (macrophage marker) cells in the underlying fibroblast layer. Using gene upregulation level of >1.9 fold as a cut-off following ligand stimulation, tissues without macrophages (SMI-M) showed fewer upregulated genes (1400 genes) compared to SMI+M (4400 genes). Furthermore, when gene upregulation levels by ligand-induced SMI+M were compared against stimulated SMI-M even higher differences in upregulated genes (> 5200 genes) were noted. In SMI+M, upregulated genes include chemokines, chemokine receptors, FC receptors, co-stimulatory molecules, interferons, and HLA’s which are characteristic of immune cells. When we looked at 13 upregulated genes (>6-fold) in SMI+M, the synergistic effect of ligands in inducing inflammatory gene upregulation was much more pronounced compared to stimulated SMI-M tissues. Furthermore, increased cytokine release of IL-6, IL-8, and TNF-α into the culture medium from ligand exposed SMI+M was also confirmed by BioPlex ELISA. In summary, our results demonstrate that the 3D SMI+M tissue model can serve as an in vitro tool to study the complex cellular interactions manifested during inflammation in the gut microenvironment.
Ocular Side Effects of Systemic Medications: Utilization of the 3D Human Corneal Epithelial Tissue Model (Abstract: 2910, Poster P536)
Session: Ocular Toxicology
Wednesday, March 18 | 9:00 AM -10:45 AM
Many systemic medications at high doses have the potential to induce unwanted ocular effects, including light sensitivity, pain, or corneal edema that are due to inflammation and/or cytotoxicity. Current studies utilize animal models which are not suitable for rapid drug screening, have poor species extrapolation and standardization. There is a need for physiologically relevant, human primary corneal epithelial tissue models to address ocular safety for the evaluation of new drug formulations. We have utilized the 3D human corneal epithelial tissue model, EpiCorneal, to analyze the effect of common drugs with known adverse ocular side effects. EpiCorneal tissues are comprised of normal human corneal epithelial cells, express site-specific mucins and tight junctions and attain morphology, barrier properties (TEER>900±200 Ω*cm2), and gene expression similar to the in vivo human cornea. The effect of physiologically relevant concentrations of Chlorpromazine hydrochloride (CPZ), a common psychotropic agent; Hydroxychloroquine sulfate (HCQ), an anti-inflammatory and anti-malaria drug (HCQ); and Fosamax (Alendronate Sodium, FOS), a common anti-osteoporosis agent, were investigated. Effects on tissue viability (MTT assay), barrier function (Transepithelial electrical resistance, TEER), histology, LDH and cytokine release were studied. EpiCorneal tissues were incubated in the medium containing CPZ at 6.25 – 100 μM, HCQ at 6.17 – 500 μg/ml, or FOS at 0.1 – 100 μg/ml for 24h and 48h. For CPZ-treated tissues, the lowest dose to cause a significant decline in barrier function (67.4%) was 12.5 μM at 24h, and 25 μM decreased tissue viability (60.5%) at 48h. For HCQ-treated tissues, a decline in TEER (67.4%) was detected for 18.52 μg/ml at 24h, and in tissue viability (53.6%) for 55.56 μg/ml at 48h. For FOS-treated tissues, a significant TEER decrease (57.8%) was detected at 0.1 μg/ml and in tissue viability (85.7%) for 10 μg/ml, both at 48h. Treatment-specific changes in tissue morphology and dose-response of LDH and cytokine release were also observed. EpiCorneal tissue model has been very useful for evaluating formulations with negligible irritation potential. It will model systemic drug exposure for an extended time, will generate a rapid response, avoid species extrapolation, be more cost-effective and more reproducible than animal methods, and will facilitate drug discovery by allowing screening and optimization of pharmaceuticals prior to clinical studies.