EUROTOX 2018 ControlCenter

Online Program Overview Session: SOC 3

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Short oral communications 3

Session chair: Philippe Vanparys Belgium
 
Shortcut: SOC 3
Date: Wednesday, 5 September, 2018, 10:00
Room: Hall 300
Session type: Short Oral Communication

Contents

Click on an contribution to preview the abstract content.

OP03-01

Use of reconstructed human skin and IL-18 to determine the skin sensitization potency of red and black tattoo inks (#117)

W. Bil1, S. V. D. Bent3, S. Spiekstra1, T. Rustemeyer3, S. Gibbs1, 2

1 VU university medical center, Dermatology Research Lab, Amsterdam, Noth Holland, Netherlands
2 Academic Center for Dentistry, Oral Cell Biology, Amsterdam, North Holland, Netherlands
3 VU University Medical Center, Dermatology, Amsterdam, North Holland, Netherlands

During the last decade, the number of people with one or more tattoos has increased considerably within the European population. Despit this, safety assessment is still very limited for tattoo inks. The aim of this study was to test the skin sensitization potential of tattoo inks in vitro using reconstructed human skin (RhS) and the contact sensitizer biomarker IL-18. Two red and three black tattoo inks, one additive (Hamamelis Virginiana extract) and one irritant control (lactic acid) were tested. In order to mimic dermal exposure, the culture medium of RhS (reconstructed epidermis on a fibroblast populated collagen hydrogel) was supplemented with test substances in a dose dependent manner for 24 hours. Cytotoxicity (histology, MTT assay) and sensitizing potential (IL-18 secretion, ELISA) was then assessed. Our results showed that all inks demonstrated cytotoxicity. Notably, one red ink and one black ink were able to cause an inflammatory response, indicated by substantial IL-18 secretion compared to control irritant, strongly suggesting that these inks are sensitizers. Further HPLC analysis of the black inks, identified considerable amounts of polycyclic aromatic hydrocarbons (PAHs) in one of the inks. PAHs are known for their sensitizing and carcinogenic properties. In conclusion, it is a worrying finding that all tattoo inks were cytotoxic and two were able to cause an inflammatory IL-18 response, indicating that an individual will be prone to develop allergic contact dermatitis when exposed to these tattoo inks. This research therefore stipulates the importance of stricter tattoo ink safety assessment in the future.

Keywords: allergic contact dermatitis, human reconstructed skin, IL-18, in vitro, safety assessment, skin sensitization, tattoo ink
OP03-02

Inflammatory conditions affect the acetaminophen-induced liver injury in a novel 3D model of human multicellular microtissues (#169)

T. D. Kok1, J. Jiang1, J. Kelm2, M. van Herwijnen1, J. Kleinjans1

1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
2 Insphero AG, Schlieren, Switzerland

The individual sensitivity to drug induced liver damage is known to vary strongly between subjects and the presence of inflammatory conditions is one of the factors known to modulate such interindividual differences. In this study, we used a 3D multicellular liver model consisting of primary human hepatocytes and Kupffer cells, to evaluate the inflammation-associated increased toxicity of acetaminophen (APAP) among humans. Compared to conventional monolayer cultures, the 3D InSight™ liver microtissues we used here provide cells with a microenvironment that more closely resembles normal liver physiology in humans. To investigate the mechanisms involved, we exposed these microtissues, to 0, 0.5, 5 and 10 mM APAP for 24h with/without lipopolysaccharide (LPS). Agilent microarrays were used to analyze the transcriptomic changes. In combination with LPS, the median-dose of APAP was already sufficient to inhibit respiratory chain and antioxidant-related gene expressions, suggesting the induction of reactive oxygen species and oxidative stress. Furthermore, the expression of Fcγ receptor (FcγR)-coding genes was significantly down-regulated after the median- and high-dose APAP exposures, regardless the presence of LPS. The expression of TLR4, a receptor known to stimulate the production of various pro-inflammatory cytokines, was however constantly elevated after all LPS/APAP co-exposures. This may result in reduced Kupffer cell phagocytosis and unbalanced cytokine patterns. Exposure to APAP combined with LPS, compared to the LPS only exposures, the levels of IL-8, a pro-inflammatory cytokine, were constantly raised, but the IL-6 concentrations, a cytokine regulating hepatic regeneration, dropped along with the increase in APAP doses. In addition to the disrupted mitochondrial functions, the presence of LPS significantly increased APAP susceptibility after exposure to the median- and high-dose.

In summary, we demonstrated that the 3D InSight™ liver microtissue model can be used in toxicological research and that it is particularly suitable for mechanistic exploration of inflammation-associated drug toxicity. With this system, we identified potential mechanisms that may explain the increased susceptibility to APAP as a consequence of LPS-induced inflammation.

Keywords: Acetaminophen, microtissues, gene expresion, LPS, Inflammation
OP03-03

Evaluation of 3D skin model-based assays using difficult to test substances: an EPAA multi-sector project (#170)

A. Irizar1, A. Mehling2, E. Adriaens3, S. Casati4, B. Hubesch5, M. Klaric6, I. Manou7, B. Mueller8, E. Roggen9, E. van Vliet6

1 The International Fragrance Association (IFRA), Brussels, Belgium
2 BASF SE, Duesseldorf, Germany
3 Adriaens Consulting BVBA, Aalter, Belgium
4 European Commission, Joint Research Centre (JRC), Ispra, Italy
5 Hubesch Consult BVBA, Brussels, Belgium
6 Cosmetics Europe, Brussels, Belgium
7 European Partnership for Alternative Approaches to Animal Testing (EPAA) Industry Secretariat, Brussels, Belgium
8 Symrise, Holzminden, Germany
9 3Rs Management and Consulting ApS, Kongens Lyngby, Denmark

The European Partnership for Alternative Approaches to Animal Testing (EPAA) is a public-private voluntary initiative that promotes the replacement, reduction and refinement (3Rs) of animal use for meeting regulatory requirements through better and more predictive science. Its membership includes 8 industry sectors, e.g. chemicals, cosmetics, fragrances, and pharmaceuticals, as well as members from European Commission services, including EURL ECVAM. The EPAA works to enhance the development of alternative approaches and more importantly their regulatory acceptance and harmonization at national, European and international levels.

Whilst there are validated OECD accepted in vitro methods available for assessing the skin sensitisation potential of substances, these methods have limitations regarding their applicability. For example, they may not always be suitable for testing substances or mixtures with low water solubility (highly hydrophobic substances) and for predicting sensitisation potency. To overcome these limitations, assays based on 3D skin tissue models are being developed to detect sensitisers and predict their potency. These 3D skin models better mimic the skin structure and function and offer advantages as test substances can be applied directly onto the model skin. Moreover they cover multiple mechanistic key events of the adverse outcome pathway (AOP) for skin sensitisation.

The EPAA project on “difficult to test substances” was designed in order to evaluate the most advanced, commercially available, 3D skin model-based assays, namely the SenCeeTox, SENS-IS and RHE IL-18 assays. The project was run in collaboration with Cosmetics Europe and the RHE IL-18 consortium. Evaluation of the assays was conducted using a blinded set of 12 substances selected based on observed difficulties when using the currently available OECD test guidelines and with relevance to industry. Firm animal and/or human evidence of their skin sensitisation potential and potency was available to assess the reliability of the assays. Results showed that the 3D skin model-based assays were able to assess all substances. Assay predictions were encouraging for hazard identification, but more work is needed for the reliable assessment of sensitisation potency.

Keywords: Skin sensitisation, non-animal alternative methods, 3D skin model-based assays
OP03-04

Liver-Chips and Liver Spheroids as Models to Determine Hepatic Safety and Metabolism (#252)

A. J. Foster1

1 AstraZeneca, Drug Safety & Metabolism, IMED Biotech Unit, Cambridge, United Kingdom

A. Foster1, B. Chouhan2, S. Regan1, H. Rollison1, S. Amberntsson2, L. Andersson2, A. Srivastava1, K-J. Jang3, D. Petropolis3, K. Kodella3, J. Rubins3, M. Darnell2, D. Williams1, G. A. Hamilton2, L. Ewart1, P. Morgan1

1Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca,Cambridge, UK & 2Sweden; 3Emulate, Inc., Boston, US.

Drug-induced liver injury continues to be a major focus in candidate selection and clinical development in consideration of therapeutic index. Accordingly more predictive and translational model systems are required to assess human hepatotoxicity risk. Organs-on-Chip technology recreate dynamic microenvironments (flow) and represent promising models to assess mechanistic hepatotoxicity, due to enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Increased sensitivity of these models to drug-induced hepatotoxicity has been demonstrated vs 2D static cultures. However, a comprehensive evaluation of liver spheroid co-cultures and microengineered Organ-Chip models has not been undertaken. Here we report on evaluation of primary human hepatocyte spheroids and the Liver-Chip using two hepatotoxins with different mechanisms of hepatotoxicity, acetaminophen (APAP) and fialuridine (FIAU).

We assessed the sensitivity of the models across a range of cytotoxicity biomarkers (ATP, albumin (ALB), αGST, miR-122) and their metabolic functionality by incorporating quantitation of APAP and FIAU metabolites. Comparisons were made at equivalent time points and dose and were expressed as EC50, absolute or % control. Dose dependent increases in αGST and miR-122, as well as decreases in ALB, were observed in both the Liver-Chip and the hepatic spheroid model. APAP metabolite formation was maintained until day 10 and dose dependent increases were observed in both models at times/doses where no cytotoxicity was detected. For FIAU, the most sensitive endpoints were ALB in the Liver-Chip and miR-122 in the spheroids respectively, both on day 7.

Overall, both models demonstrated integrated toxicity and metabolism with released toxicity biomarkers observed earlier in the Liver-Chip.

Keywords: Liver-Chip, spheroids, microphysiological systems, organ-on-a-chip
OP03-05

Successful proof of concept of a micronucleus genotoxicity assay performed on reconstructed epidermis exhibiting intrinsic metabolic activity. (#576)

R. Barcham1, N. Orsini2, E. Andres1, A. Hundt1, A. - P. Luzy2, C. Dini1

1 Oroxcell, Life Sciences, Romainville, France
2 Nestlé Skin Health, Preclinical group− Early Development department, Sophia-Antipolis, France

We investigated the commercially available Episkin LM™ reconstructed epidermis test system as a potential 3D model for human genotoxicity assessment by cytokinesis-block micronucleus assay to mitigate limitations of the currently accepted micronucleus test.

We established appropriate culture conditions for cytokinesis-block micronucleus assay in maximizing the frequency of binucleated cells by choice of culture medium and calibration of the system exposure to the cytokinesis inhibitor Cytochalasin B, without affecting the basal frequency of micronuclei in the model. We confirmed that the application of the classic solvents had no significant effect on this basal level of micronuclei.

We determined the performance of cytokinesis-block micronucleus assay in Episkin LM™ reconstructed epidermis to predict in vivo genotoxins by testing the genotoxicity potential of 17 well known in vivo genotoxic, progenotoxic and non-genotoxic reference chemicals over a 48 hours and 72 hours exposure period.

We found that cytokinesis-block micronucleus assays in Episkin™ reconstructed epidermis following the 48 h-topical regimen had a specificity of 60 - 75% and a sensitivity of 83 - 85%, resulting in an overall accuracy of 76 - 82% for genotoxicity assessment in tissues depending on the assessment of the reference chemicals with equivocal genotoxic profiles in the literature.

The positive micronucleus test results obtained without addition of any exogenous metabolic activation system confirmed the ability of Episkin LM™ reconstructed epidermis to intrinsically bioactivate progenotoxic chemicals. The evidence showed that the 72-hour exposure protocol significantly improved the detection of progenotoxins.

Taken together, our data demonstrated that the Episkin LMTM reconstructed epidermis system is a relevant in vitro tool in the study of genetic toxicology.

Keywords: Skin micronucleus assay, Genotoxicity, Reconstructed epidermis, 3D human skin model, in vitro, Skin metabolism
OP03-06

A novel in-vitro human skin explant test to predict adverse immune reactions to biologics and aggregated monoclonal antibodies (#579)

L. Bibby1, A. Ribeiro1, S. Ahmed2, A. Dickinson1, 2

1 Newcastle University, Medical School, Academic Haematology, Newcastle upon Tyne, United Kingdom
2 Alcyomics Limited, Bulman House, Newcastle upon Tyne, United Kingdom

To aid preclinical prediction of adverse effects of a hypersensitivity (HS) nature to biological drugs, we have developed a human in-vitro skin explant test, which uses human blood and non-artificial autologous skin that assesses histopathological damage indicative of adverse immune activation. This can be used as a valuable preclinical tool for safety assessment of biologics and aggregated monoclonal antibodies.

We tested 17 commercial biologics in the skin explant assay (n=10) and correlated the results with clinical occurrence of adverse reactions. Results showed a statistically significant positive correlation between adverse clinical occurrences and a positive response in the skin explant test (r=0.815, p<0.0001). Drugs known to cause adverse HS reactions e.g. OKT3® and Campath® gave a positive Grade II or above response in 90% and 70% of individuals, respectively. An analogue of the TGN drug, TGN1412, gave an extreme positive response in 90% of individuals. Drugs which have been defined as clinically negative for HS reactions, such as Enbrel® and Cimzia®, showed a negative response (<40% positive skin explants), with 10% and 30% of individuals reacting with the drug, respectively. We also assessed adverse reactions to biosimilars (Remsima® and Inflectra®) against the reference product (Remicade®). Overall, a negative response was observed for all three biologics in the skin explant test. Interestingly, drug responses varied donor to donor, indicating that that test may be able to predict the best tolerated therapy for a patient. Aggregation of monoclonal antibodies (mAbs) was induced by temperature stress and characterised by analytical ultra-centrifugation and transmission electron microscopy, revealing 5% aggregation of total protein content. Results showed aggregated Herceptin® and Rituximab® caused mild atopic damage, determined from observed histopathological damage and positive for HSP70 in the skin. Our results showed exposure to temperature can cause conformational changes in the mAb structure that, ultimately, can induce adverse immune reactions detected by the skin explant assays.

Our novel human in-vitro assay showed that it was highly sensitive for determining adverse immune reactions to biologics and aggregated mAbs and is a promising tool for the prediction of immunotoxicity.

Keywords: hypersensitivity, immunotoxicity, immunogencicty, human in vitro testing, aggregation, biologics, monoclonal antibodies
OP03-07

IN VITRO “skin patch tests” for the detection of sensitization, allergy and relative potency assessment. (#580)

S. Ahmed1, A. Tulah1, L. Bibby1, 2, A. M. Dickinson1, 2

1 Alcyomics Ltd, Newcastle upon Tyne, United Kingdom
2 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom

Sensitization to chemicals and cosmetics resulting in allergy is an important health issue. Until recently, the most favorable method to test compounds for sensitization was the mouse local lymph node assay (LLNA). Changes in EU legislation (2013) have resulted in banning the use of animal testing for cosmetics. A number of alternative predictive test methods for the identification of compounds with the potential to cause skin sensitization have been developed, however, one single alternative test cannot replicate an animal test in its entirety, so multiple test methods are needed to determine the effects. Here we describe a human in vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method uses a human autologous system to test for sensitivity and adverse reactions to compounds, in which activity is measured as histopathological grading of skin damage, caused by induced immune sensitization response, which correlates with T cell proliferation and IFN-γ production. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously LLNA misclassified compounds (e.g. Nickel Sulphate). Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of r=0.9 (p<0.0001). Additionally, it has proven to be a sensitive method for predicting allergy responses to cosmetics and can be used to determine adverse effects of repeat dosing. Human skin equivalent models are useful in vitro testing platforms and a reliable alternative to animal testing. Here we also describe a unique full-thickness 3D autologous skin equivalent model made from primary human tissue as a valuable platform for high throughput testing. The 3D equivalent model showed similar structure to normal healthy skin, observed by H&E staining and positive immunofluorescence staining for epidermal differentiation markers (loricrin, involucrin, cytokeratin 14 and collagen 3). Collectively, the data suggests these human in vitro skin tests could provide the basis for a novel approach for characterization of the sensitizing activity of compounds as a first step in the risk assessment process.

Keywords: Sensitisation, Potency, T cell prolifferation, Cytokines, Skin test, In vitro, Non-animal, 3D Skin model
OP03-08

Lung-on-a-Chip: The interplay of primary human epithelial and endothelial cells improves the alveolar barrier function (#607)

A. O. Stucki1, G. Raggi1, S. Sigrist1, P. Zamprogno1, 2, N. Schneider-Daum3, C. - M. Lehr3, H. Huwer4, J. D. Stucki5, N. Hobi1, 5, O. T. Guenat1, 5

1 University of Bern, ARTORG Organ on Chip Technologies, Bern, Switzerland
2 University of Bern, Graduate School for Cellular and Biomedical Sciences, Bern, Switzerland
3 Saarland University, Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany
4 SHG Clinics, Department of Cardiothoracic Surgery, Völklingen, Germany
5 AlveoliX AG, Bern, Switzerland

Introduction: The pulmonary alveolar barrier is one of the largest entry ports for xenobiotics into the body and consists of a thin barrier of epithelial and endothelial cells. Yet, establishing a biologically relevant model of the pulmonary alveolar-capillary barrier is challenging, due to poor availability of human primary alveolar epithelial cells. In addition, the effect of mechanical stretch on the cells due to breathing is thought to be important in regulating lung functions but has so far not thoroughly been considered due to the lack of appropriate tools.

Aims: The goal of our work is to establish a stable pulmonary alveolar-capillary barrier and understand the interplay between lung alveolar epithelial and endothelial cells in an in vivo-like environment.

Methods: Primary human pulmonary alveolar epithelial cells (pHPAEC) from patients undergoing lung resections were cultured on inserts (0.4µm pore size) or lung-on-a-chips (3.5 µm pore size) with and without four different endothelial cell types. Medium was exchanged every other day, following which trans-epithelial electrical resistance (TEER) was measured for up to 14 days.

Results: The TEER of pHPAEC monoculture rises above 1000 Ωcm2 until day 4-6 and then begins to drop reaching values below 500 Ωcm2 at days 8 to 14. Co-culturing pHPAEC with human VeraVec endothelial cells increased TEER values significantly and lead to a more stable culture (values above 4000 Ωcm2 at day 12). Culturing pHPAEC together with primary human pulmonary microvascular (HPMEC) or umbilical vein (HUVEC) endothelial cells, or with immortalized HPMEC ST1.6R resulted in slightly lower TEER values compared to VeraVec cultures but significantly higher compared to monoculture of pHPAEC.

Conclusion: The integrity of the alveolar-capillary barrier is a key parameter that prevents any unwanted leakage from either side of the barrier. TEER is often used to assess the quality of the barrier integrity. Here, we show that a co-culture of primary human alveolar epithelial cells together with endothelial cells does not only significantly increase the barrier integrity but is also a factor of a long-term stability.

*NH and OTG contributed equally to this work

Keywords: Lung-on-Chip, Lung alveolus-on-chip, alveolar barrier, air-blood barrier
OP03-09

3D NephroScreen: high throughput drug-induced nephrotoxicity screening on a proximal tubule-on-a-chip model (#614)

L. Gijzen1, M. K. Vormann1, S. Hutter1, J. Vriend2, S. J. Trietsch1, J. Joore1, M. Wilmer2, L. Suter-Dick3, R. Masereeuw4, H. L. Lanz1

1 Mimetas BV, Leiden, Netherlands, Netherlands
2 Radboud UMC, Department of Pharmacology and Toxicology, Nijmegen, Netherlands
3 University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland
4 Utrecht Institute for Pharmaceutical Sciences, Div. Pharmacology, Utrecht, Netherlands

Renal toxicity remains a major issue in clinical trials, and stresses the need for more predictive models fit for implementation in early drug development. Here, we describe a perfused, leak-tight renal proximal tubule cell (RPTEC) model cultured within a high throughput microfluidic platform (Mimetas’ OrganoPlate®)2, along with recent results from a 12-compound nephrotoxicity screen performed within the “NephroTube” CRACK IT consortium in collaboration with sponsors and the NC3Rs.

Human RPTEC (SA7K clone, Sigma) were grown against a collagen I ECM in a 3-channel OrganoPlate®, yielding access to both the apical and basal side. Drug-induced toxicity was assessed by exposing kidney tubules to 4 benchmark and 8 blinded compounds with known clinical effects supplied by the sponsors for 24 and 48h. The tightness of the barrier was evaluated by diffusion of a dextran dye from apical to basal compartment. Parallel to this, cell viability with a WST-8 assay and the presence of LDH in the supernatant were assessed. Finally, kidney tubules were lysed, and RNA was extracted for gene expression analysis of acute kidney injury markers.

Upon perfusion flow, RPTEC form leak-tight confluent tubular structures against the collagen I ECM in the OrganoPlate®. The NephroScreen revealed significant decreased barrier tightness and cell viability in 7 out of 12 compounds. Furthermore, the release of LDH was significantly increased in 9 out of 12 compounds. An increased expression in HMOX1, TNFα and NGAL was observed in 9, 5 and 7 out of 12 compounds respectively whereas claudin-2 showed a decrease in 6 out the 12. Overall, more effects were observed after 48h in comparison to 24h exposure.

The kidney-on-a-chip model in the OrganoPlate® provides a promising in vitro renal toxicity tool to answer the desire to provide a better alternative to animal studies in terms of throughput, costs and predictivity and ultimately will be commercialised after further validation.

Keywords: organ-on-a-chip, nephrotoxicity, toxicity screen, 3D renal proximal tubule cell (RPTEC) model
OP03-10

Exploring in vitro the potential effects of repeated drug treatment on the distribution of other xenobiotics at the human blood-brain barrier (BBB) (#627)

M. Heymans1, C. Lontouo1, E. Sevin1, F. Gosselet1, M. Culot1

1 University of Artois, Blood-brain barrier laboratory, LENS, France

Drug-induced activation of transcription factors has been reported to exert either beneficial or detrimental effects at the blood-brain barrier (BBB). Both CAR and PXR activation at the BBB, by respectively phenobarbital and carbamazepine, have been suggested to contribute to the mechanisms of drug resistance in epilepsy. The latter by upregulating the activity of drug-metabolizing enzymes (e.g. CYPs) and the expression of efflux transporters (e.g. ABCB1). On the other hand, the anti-cancer drug, bexarotene, which is an RXR agonist, has been suggested to play a beneficial role in Alzheimer’s disease, however its possible effect at the BBB has been neglected so far.

Recently, our laboratory developed a human in vitro BBB model based on cord blood-derived hematopoietic stem cells, which are co-cultured with bovine brain pericytes to induce the typical phenotype of BBB endothelial cells (i.e. complex tight junctions, low permeability and expression of efflux pumps) {1}.

After adapting this model to study the effects of repeated drug treatment at the BBB, we studied the effects of a selection of drugs, including some known agonists of nuclear receptors (e.g. AhR, CAR, LXR, PPAR-gamma, PXR, RAR, RXR agonists).

Our preliminary data suggest that some drugs affect the expression of drug transporters at the BBB which results in changes in the brain distribution of the drug itself or other drugs.

These experiments could highlight possible relationships between the drug-induced activation of nuclear receptors and disease mechanisms or treatments, as any change in the expression of transporter or metabolizing enzymes at the BBB might affect the distribution of all their substrates to the brain.

References:

1. Cecchelli, R., Aday, S., Sevin, E., Almeida, C., Culot, M., Dehouck, L., Coisne, C., Engelhardt, B., Dehouck, M.P. & L., Ferreira – 2014. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells. PLoS ONE 9(6): e99733. doi:10.1371/journal.pone.0099733.

Keywords: blood-brain barrier, repeated drug treatment, in vitro, nuclear receptors