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Molecular Impact of Electronic Cigarette Exposure on Human Bronchial Epithelium In Vitro.
E. Moses1, T. Wang1, G.R. Jackson3, E. Drizik1, C. Perdomo1, D. Brooks2, G. O’Connor1, S. Dubinett4, P.J. Hayden4, M. Lenburg1, and A. Spira1. 1Boston University School of Medicine, Boston, MA; 2Boston University School of Public Health, Boston, MA; 3MatTek Corporation, Ashland, MA; and 4UCLA, Los Angeles, CA.
Thursday Morning, March 17 9:30 AM to 12:15 PM CC Room R06 Platform Session: Electronic Cigarette Research Chairperson(s): Irfan Rahman, University of Rochester, Rochester, NY; and Sonali Das, Strand Life Sciences Pvt. Ltd, Bangalore, India.
Background: Electronic cigarettes (ECIGs) are an emerging alternative tobacco product thought to be safer than traditional tobacco cigarettes (TCIGs). Despite the increasing prevalence of ECIG use, few studies have evaluated the potential physiological effects of ECIG exposure. Objectives: In this study we aimed to determine the global gene expression effects of ECIG exposure on bronchial epithelium in vitro. Methods: Human bronchial epithelial cells (HBECs) grown at Air Liquid Interface (ALI) (EpiAirway) were exposed to direct ECIG vapor or TCIG smoke. ECIG vapor exposures included tobacco and menthol flavor with and without nicotine. Results: We found that while some gene expression changes induced by ECIG exposure are shared with TCIGs, ECIGs have a distinct effect on airway epithelial cells, inducing gene pathways related to cell cycle and mitosis. Of particular interest, ECIG exposure induces the expression of genes involved in oxidative and xenobiotic stress pathways, similar to, but generally lower in magnitude, than the effects of TCIGs. Furthermore, ECIG exposure decreases the expression of genes involved in cilia assembly and movement. Conclusions: These results indicate that ECIG vapor may induce significant cellular stress and molecular alterations within airway epithelium. These results underscore the urgent need for further studies to fully characterize the type and magnitude of cellular and molecular responses to ECIG exposure, in order to determine whether their use may lead to deleterious health outcomes.
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Evaluating the Inflammatory and Genotoxic Effects of Smokeless Tobacco Using a Human Organotypic Model of Oral Epithelium. Abstract: 3066 Poster: P218
Wednesday, March 16 1:15 PM to 4:45 PM CC Exhibit Hall Poster Session: Alternative In Vitro Toxicity Models Recent Advances in Safety Assessment Author Attended: 1:15 PM–2:45 PM
M. Bachelor, J. Oldach, B. Breyfogle, P. Hayden and M. Klausner. MatTek Corporation, Ashland, MA.
The use of tobacco use has been implicated as a major cause of oral cavity disease leading to thousands of deaths per year. Snus, a smokeless tobacco applied to the oral cavity, has been proposed as a less harmful alternative to smoking although its safety has not been adequately evaluated. The objective of this study was to evaluate the cytotoxic, genotoxic and inflammatory effects of snus using an in vitro model of human oral mucosa (EpiOral). EpiOral tissues were exposed topically to 5–25 milligrams of snus or snus (CRP1) extracts for 24-48 hours. Tissues exposed to 5 mg of snus had comparable viability to vehicle treated controls while those exposed to 25 mg displayed approximately a 20% decrease in viability after 24 and 48 hours of exposure as determined by MTT analysis. Histological evaluation revealed hyperchromic staining in tissues exposed to 5 mg of snus at 24 hours post-exposure whereas tissues exposed to 25 mg of snus displayed a significant amount of sloughing of the apical layers. Following exposure, an inflammation-specific cytokine panel was used to analyze markers of inflammation 24 and 48 hours post exposure. Of the cytokines analyzed, a significant increase in IP-10 was observed 24 hours post exposure in tissues exposed to 25 mg of snus or 5–10% snus extract. As a measure of genotoxicity, the presence of γ-H2AX foci was evaluated in exposed tissues. γ-H2AX is a phosphorylated derivative of the H2AX histone and is tightly bound to double strand DNA break sites, therefore serving as a biomarker of genotoxic insult. γ-H2AX foci were readily detected in the apical layer of tissues exposed to 25 mg of snus and 10% snus extract at 24 and 48 hours post treatment. Exposure of snus extracts to EpiOral also revealed significant increases in CYP1A1 (600 fold) and CYP1B1 (40 fold) gene expression. These results demonstrate the utility of this organotypic oral tissue model to evaluate smokeless tobacco product safety.
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Use of a New Human Small Intestinal Tissue Model to Screen Drug Induced Gastrointestinal Toxicity Abstract: 3069 Poster: P221
Wednesday, March 16 1:15 PM to 4:45 PM CC Exhibit Hall Poster Session: Alternative In Vitro Toxicity Models Recent Advances in Safety Assessment Author Attended: 1:15 PM–2:45 PM
S. Ayehunie2, M. Wagoner1, H. Barthlow1, C. Scott1, Z. Stevens2, T. Landry2, A. Armento2, M. Klausner2 and P. Hayden2. 1AstraZeneca, Waltham, MA and 2R&D, MatTek Corporation, Ashland, MA.
The mechanisms of drug-induced gastrointestinal (GI) toxicity are often poorly understood, in large part due to the lack of physiologically relevant in vitro models. The inherent physiology differences between humans and preclinical animal models add another layer of complexity in interpreting GI toxicity studies. The objective of this work was to evaluate the utility of a new in vitro primary human cell-based small intestinal (SMI) tissue model to predict drug-induced GI toxicity. A blinded study was performed using 8 therapeutic compounds, including 5 for which dog and rat GLP toxicity studies were not predictive of human GI toxicity. These 5 compounds were selected from discontinued AstraZeneca clinical development programs and represent a diverse set of target classes and chemistries. As negative controls, the 3 other drugs included in this study were well tolerated in humans. To model GI toxicity, we examined cytotoxicity by MTT viability and LDH release assays and tissue barrier integrity using transepithelial electrical resistance (TEER) measurements, following two repeat exposures over 96 hours. The SMI model detected drug-induced disruption of intestinal barrier function (TEER) in 5/5 problematic drugs at concentrations up to 30 fold below clinical exposure levels. Importantly, the SMI tissue showed no effect at concentrations up to 1,000 fold higher than the clinical exposure levels for the three negative controls. Overall, TEER measurements in the SMI tissue were a sensitive, predictive tool to assess clinically relevant exposures of drugs that induced human GI toxicity that were incorrectly predicted in preclinical GLP toxicology studies. These results suggest that the in vitro system will serve as a useful tool for both investigational and traditional GI drug safety studies.
Pre-Validation of In Vitro – In Vivo Assays for Vaginal Irritation Abstract: 3070 Poster: P222
Wednesday, March 16 1:15 PM to 4:45 PM CC Exhibit Hall Poster Session: Alternative In Vitro Toxicity Models Recent Advances in Safety Assessment Author Attended: 1:15 PM–2:45 PM
Ayehunie, T. Landry, P. Hayden and M. Klausner. R&D, MatTek Corporation, Ashland, MA.
A validated and predictive in vitro test system for assessing the vaginal irritation of test articles (TA) is needed to screen women’s care products. The goal of the study was to validate the utility of the in vitro tissue model as an alternate vaginal irritation test by comparing head-to-head testing with the rabbit vaginal irritation (RVI) assay (ISO10993-10) for a 485SOT 2016 Annual Meeting coded set of test articles (TA). A double-blinded study was conducted in which the investigator and the animal testing facility were provided with N=14 coded TA 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. The RVI score was used to monitor in vivo irritation; MTT, TEER, and histological analysis were used as endpoints for the in vitro model. 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). BZK was identified as a mild/severe irritant in the RVI assay, but the effect of N9 in rabbits was highly variable – in three independent 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. Cytokine analysis from culture supernatants showed that N9, SDS, and BZK induced 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. In short, a combination of MTT, TEER, and IL-1α/ IL-1β were found to be valuable markers of in vitro vaginal irritation. In vitro assay method performed equally well or better compared to the standard RVI assay and is anticipated to be a useful tool to assess vaginal toxicity of formulations, drugs, and medical devices.
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Development of an In Vitro Test to Assess the Inhalation Toxicity of Nanomaterials Abstract: 3506 Poster: P196
Thursday, March 17 9:30 AM to 12:45 PM CC Great Hall A Poster Session: Nanotoxicology: Carbon Based Author Attended: 9:30 AM–11:00 AM
Monita Sharma1, Barbara Rothen-Rutishauser2, Hana Barosova2, Savvina Chortarea2, Fikad Zerimariam2, Martin Clift2, Vicki Stone3, Patrick Hayden4, Anna Maione4, Amy Clippinger1. 1PETA International Science Consortium Ltd., UK, 2Adolphe Merkle Institute, University of Fribourg, CH, 3Heriot-Watt University, UK, 4MatTek Corporation, USA.
The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this assay led an international expert group to convene in February 2015 to discuss the approaches to evaluate inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. Subsequently, work began to develop an in vitro co-culture system using relevant lung cells exposed to MWCNTs at the air-liquid interface (ALI) while considering human-relevant a MatTek Corp) exposed to NMs at ALI using the VITROCELL™ Cloud system. The long term goal of this work is to develop an in vitro testing strategy using human-relevant methods to predict pulmonary toxicity and to enable effective risk assessment of substances including MWCNTs.
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A Novel Assay for Evaluating Wound Healing in a Full-Thickness In Vitro Human Skin Model Abstract: 3516 Poster # P208
Thursday, March 17 9:30 AM to 12:45 PM CC Great Hall A Poster Session: Skin Responses and Toxicology Author Attended: 11:15 AM–12:45 PM
Bachelor, A. Armento, J. Oldach, G. Stolper, M. Li, M. Klausner and P. Hayden. MatTek Corporation, Ashland, MA.
Cutaneous wound healing involves interactions between dermal fibroblasts and epidermal keratinocytes as well as cell-extracellular matrix interactions. The current study describes wound healing experiments conducted in a full thickness in vitro human skin model (EpiDermFT). This model exhibits stratified epidermal components and a fully developed basement membrane and resembles in vivo skin in regard to both morphology and barrier function. Small epidermal only wounds were induced in the model using a 3mm punch biopsy and subsequently evaluated at various recovery time points by two methods. Historically, EpiDermFT has been used to evaluate re-epithelization of the wound by: a) manually bisecting the tissues through the center of the wound, b) staining with hematoxylin and eosin, and c) quantifying migration from the wound origin. Accurate bisection of the wound in difficult and often leads to variability in assay results. Here we describe a novel method of visualizing wound re-epithelization in situ simplifying analysis and reducing introduction of variables inherent in tissue processing that could potentially confound data. Following wounding, tissues were fixed and immunostained with markers of epidermal differentiation as well as a marker of fibroblasts allowing simultaneous visualization of migrating keratinocytes (keratin 14), differentiated suprabasal cells (involucrin), and dermal fibroblasts (vimentin) within the wound. Histological and immunohistochemical analysis showed keratinocyte migration at 2 days following wounding. In both methods, wounded tissues cultured without growth factors (2% human serum) had a reduced healing rate in which keratinocytes did not cover the entire wound within a 6 day timeframe. In contrast, wounded tissues cultured with growth factors demonstrated a dramatic increase in healing rate as keratinocyte migration completely covered the wounded area by day 6. In conclusion, this novel method of evaluating re-epithelization by utilizing immunohistochemical markers of differentiation is a quicker and more reproducible method of analyzing wound healing.
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Development of an In Vitro Inhalation Toxicity Test for Improved Protection of Human Health Abstract: 3589 Poster # P281
Thursday, March 17 9:30 AM to 12:45 PM Great Hall A Poster Session: Late Breaking Poster Session I: Respiratory Toxicology
G.R. Jackson, A. Hunter, J. DeBay, P. Hayden. MatTek Corporation, Ashland, MA.
Knowledge of acute inhalation toxicity and irritation potential is important for establishing safe handling, packaging, labeling, transport and emergency response procedures for chemicals. The US EPA High Production Volume Chemical Challenge, and the EU REACH programs have further increased the need for inhalation toxicity information. A UN treaty endorsed by the US, EU and others outlines a “Globally Harmonized System” (GHS) of Classification and Labeling of Chemicals. The GHS specifies 5 inhalation toxicity categories. The EPA has established a separate system that uses 4 toxicity categories. Acute inhalation toxicity tests currently accepted within the GHS and EPA systems involve in vivo 4 hr rat inhalation LC50 tests (OECD TG 403/436). In the current work, a newly developed in vitro toxicity test was evaluated in comparison to the established in vivo tests. The in vitro test exposes an organotypic human airway tissue model to test chemicals for 3 hrs, followed by measurement of tissue viability (IC75). 64 chemicals covering a broad range of toxicity classes, chemical structures and physical properties were evaluated. Results show that the in vivo and in vitro tests had 100% concordance for identifying highly toxic chemicals (GHS Cat 1‐2 and EPA CAT I‐II). However, the in vivo tests had only 29.0% (EPA system) or 61.4% (GHS system) sensitivity for identifying less toxic respiratory irritants. Numerous human respiratory irritants including acids, bases, aldehydes, amines and others were not classified as respiratory toxins/irritants by the in vivo tests. The in vitro airway model was very good (sensitivity of 81.1 ‐ 82.4%) for distinguishing respiratory toxins and irritants (corresponding to GHS 1‐3, EPA, I‐III) from non‐ toxins, non‐irritants (corresponding to GHS 4‐5, EPA IV). Overall accuracy of the in vitro test was 81.2 ‐ 84.1%. There were no false negative GHS Cat 1‐2 or EPA Cat I‐II predictions using the in vitro test. These data suggest that tests based on lethality in animals, while good for predicting highly toxic chemicals, produce a high percentage of false negative predictions for moderately/slightly toxic or irritating chemicals. The in vitro test using an organotypic human airway model was equal to current animal tests for predicting highly toxic inhaled chemicals, and better than animal tests for predicting moderately/slightly toxic respiratory irritants. The new in vitro testing approach should provide improved protection of human health compared to the current animal tests.
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Development, Optimization and Validation of an In Vitro Skin Irritation Test for Medical Devices Using the Reconstructed Human Tissue Model EpiDerm Abstract: 3895 Poster # P587
Thursday, March 17 9:30 AM to 12:45 PM Great Hall A Late Breaking Poster Session 6: Medical Devices
H. Kandarova1,2, J.A. Willoughby3, W.H.de Jong4, M.A. Bachelor2, S. Letasiova1, T. Milasova1, B. Breyfogle2, L. de la Fonteyne4, Y. Haishima5, K.P. Coleman6. 1MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; 2MatTek Corporation, Ashland, MA; 3Cyprotex US LLC, Kalamazoo, MI; 4RIVM, Bilthoven, Netherlands; 5NIHS, Tokyo, Japan; 6Medtronic, PLC, Minneapolis, MN.
Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals (OECD TG 439). However, medical device extracts are dilute solutions with low irritation potential, therefore validated RhE‐methods needed to be modified to reflect the needs of ISO 10993. A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers extracts (Casas et al., TIV, 2013). In 2014 a second laboratory assessed the transferability of the assay. Two additional exposure times were tested along with other medical device materials. After the successful transfer and standardization of the protocol, nine EU and USA laboratories were trained in the use of the protocol in preparation for a validation study. All laboratories produced data that was nearly 100% in agreement with predictions for the selected references. Two of the laboratories performed additional tests with heat‐pressed PVC sheets spiked with Genapol X‐080 (Y‐4 polymer), Vicryl suture, and polymers spiked with heptanoic acid and sodium dodecyl sulfate. All materials were extracted for 24 or 72 hours in both saline and sesame oil at 37°C. Significant irritation responses were detected for Y‐4 under all conditions. These results were consistent with those reported by other research groups involved in the upcoming validation study. Vicryl suture was negative and spiked polymers were either positive or negative depending on the extraction solvent. Therefore we conclude that a modified RhE skin irritation assay has the ability to address the skin irritation potential of medical devices, however, standardization and focus on technical issues is essential for accurate prediction. A round robin validation study of in vitro skin irritation testing for the assessment of medical devices extracts is beginning in March 2016.
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in vitro Permeation of Latanoprost Eye Drop Formulations in the 3‐Dimensional Normal Human Corneal Tissue Model Abstract: 3897 Poster # P589
Thursday, March 17 9:30 AM to 12:45 PM Great Hall A Late Breaking Poster Session 6: Alternatives to Mammalian Models
A.M. Gremilogianni1, Y. Kaluzhny2, C.G. Tsoli1, S.A. Pantazopoulou1, M.W. Kinuthia2, P. Hayden2, M. Klausner2, M.A. Koupparis1, N.C. Megkoulas1. 1Laboratory of Analytical Chemistry, QualiMetrixSA, University of Athens, Athens, Greece; 2MatTek Corporation, Ashland, MA.
Permeation of topically applied ocular drugs occurs predominantly through the cornea and therefore absorption studies using corneal tissues play a critical role in ocular drug formulation. The purpose of the study was to evaluate the permeation of Latanoprost eye drops through in vitro reconstructed normal human corneal tissue model. Seven different formulations of Latanoprost eye drops were tested for ocular permeation using a reconstructed corneal tissue model (EpiCorneal). 50‐100 μl of eye drop formulations were applied topically onto the EpiCorneal tissue surface and incubated at standard cell culture conditions (SCC, 37°C, 5% CO2). At permeation times of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0 and 12.0 hrs, the tissues were moved into new wells filled with receptor medium (Krebs Ringer Buffer, pH 7.4) and returned into SCC. The samples were collected and analyzed with a fully‐validated HPLC‐PDA method for Latanoprost acid determination. Tissue integrity (Lucifer Yellow leakage assay) and tissue viability (MTT assay) were determined after the permeation study. For each formulation, plots of the cumulative % of Latanoprost acid that permeated through the tissue versus time were constructed. From the steady state flux region of the plot (<4hrs for most formulations) the Papp was calculated. Xalatan eye drops (containing 0.02% BAC solution) had the fastest permeation rate (Papp=8.81 cm·s‐1) while Monoprost (preservative‐free) had the lowest permeation rate (Papp=1.15 cm·s‐1). Formulations containing Poloxamer 407 had higher Papp (6.05 and 6.27) when compared to formulations without surfactants (1.69 to 2.57). Tissue integrity and viability were maintained in all experiments as evidenced by Lucifer Yellow and MTT results. The EpiCorneal tissue demonstrated very high reproducibility and presented a permeation profile of different formulations similar to in vivo.
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Molecular Mechanism of Ocular Surface Damage: Applications to Dry Eye and Wound Healing Models on In Vitro Reconstructed Human Corneal Tissues Abstract: 3900 Poster # P592
Thursday, March 17 9:30 AM to 12:45 PM Great Hall A Late Breaking Poster Session 6: Alternatives to Mammalian Models
Y. Kaluzhny, M. Klausner, M. Kinuthia, A. Plotkin, P. Hayden. MatTek Corporation, Ashland, MA.
Current methods used to investigate mechanisms of corneal wound healing (CWH) and pathogenesis of dry eye disease (DED) utilize monolayer cell cultures or animals; hence, there is a need for more physiologically relevant, human‐ based in vitro models for ocular surface research. This study utilized EpiCorneal tissue model comprised of normal human corneal epithelial cells that are cultured at the air‐liquid interface. Corneal wounds were introduced by abrasion or chemicals (1N NaOH). Wounded tissues were cultured in the presence or absence of human corneal keratocytes (HCK) or EGFR inhibitor (erlotinib, 10 uM). A DED model was generated by placing EpiCorneal tissues under desiccating stress conditions (DSC, 40% RH, 40°C, and 5% CO2) that stimulate morphological, cellular, and molecular changes relevant to dry eye. CWH was analyzed by transepithelial electrical resistance (TEER), histology, confocal microscopy, and gene expression. TEER recovered to 933.7/502.4 Ω·cm2 in the presence/or absence of HCK in 4 days post‐wounded cultures. mRNA expression was analyzed using a 96‐gene wound healing microarray. 13 genes (including collagen, integrin, chemokine, and protein kinase families) were up‐regulated in the EpiCorneal tissues 24h post‐abrasion in the absence of HCK and 16 genes (including WNT, FGF, small GTPases, chemokine, and integrin families) were up‐regulated in the presence of HCK, but not in control cultures. DED was analyzed by TEER, histology, tissue viability, mucins and tight junction (TJ) protein expression. Dramatic reduction in tissue thickness was observed after 48h in DSC that coincided with decreased expression of mucins, increased TEER and atypical expression of TJ proteins. Topical application (25 µl/tissue) of lubricant gel drops (GenTeal, Alcon) improved tissue morphology and barrier function. The results demonstrate that the in vitro organotypic human corneal tissue structurally and functionally reproduces CWH and DED. The model will avoid species extrapolation, be more cost effective and more reproducible than animal methods, and will facilitate drug discovery by allowing screening and optimization of active pharmaceuticals prior to clinical studies.
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