Biofabrication of liver constructs and cell seeded hollow fiber membranes
Manon Bouwmeester1, Nynke Kramer2, Kerstin Schneeberger1, Bart Spee1

1Department of Companion Animals, Faculty of Veterinary Medicine, Utrecht University
2Institute of Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University

Human liver organoids represent a novel, patient-specific model, but thus far they do not recapitulate the complex liver physiology. Combining tissue engineering with organoid technology could lead to a suitable model, in which the 3D microenvironment of hepatocytes can be mimicked. We focus on bioprinting perfusable liver constructs containing human liver organoids alone and together with liver mesenchymal stem cells (LMSCs). Constructs were printed in custom designed flow perfusion chambers. Organoids remained viable for at least eight days in static culture conditions in a bioprinted construct and retained hepatic characteristics. Our preliminary data indicates that the perfusable liver constructs are suitable for toxicology studies, as cell damage could be induced by treatment with toxic compounds.

Additionally, for the purpose of transport studies we cultured human organoids on hollow fiber membranes (HFM). Our aim was to devise a system to measure transport from the basolateral side of the hepatocytes to the apical bile canaliculi side. Currently, we are examining the polarity of human liver organoids on HFM after coating with different extracellular matrix (ECM) components, such as collagen IV, I and different laminins.

Keywords: hepatic organoids, 3D model, bioprinting, hollow fiber membrane

Occurrence and risk assessment of aflatoxin B1 in maize in Mexico
Ixchel Gilbert Sandoval1 , Sebas Wesseling and Ivonne M.C.M. Rietjens

Division of Toxicology, Wageningen University, The Netherlands
1Corresponding author. E-mail address: ixchel.gilbertsandoval@wur.nl

Aflatoxin B1 (AFB1) is a well-known genotoxic carcinogen produced by fungi, which can be present in food commodities and products thereof. The chronic dietary exposure to AFB1 is of concern due to the increased risk of developing liver cancer. In Mexico, maize is a high consumed staple food that might contain AFB1. Yet the exposure and risk assessment of AFB1 from maize are limited for the Mexican population. The aim of this study was to analyse the occurrence of AFB1 in 39 maize samples collected in Mexico, and to assess accompanying exposure and risk. AFB1 levels were quantified by LC/MS with a limit of detection (LOD) of 0.2 ng/g. The risk was assessed using the Margin of Exposure (MOE) and a quantitative liver cancer risk estimation approach. Five out of 39 samples were tested positive for AFB1 in the range of 0.36-9.33 ng/g. The average AFB1 exposure levels were calculated as a lower bound or an upper bound (negative samples with AFB1 set at 0 ng/g or equal to the LOD of 0.2 ng/g, respectively). The exposure assessment for the lower and upper bound resulted in 1.6 and 1.9 ng AFB1/kg body weight/day, based on an average maize consumption of 175.9 g/person/day for a 70 kg person. This resulted in MOE values of 109 and 87, and in an estimated increased cancer risk of 21 and 26 cases per million upon a lifetime of 75 years. Altogether, the assessment reveals the need for risk management of AFB1 in Mexico.

Keywords: Aflatoxin B1, maize, Risk Assessment, MOE, liver cancer

Cell-type specific effects of two valproic acid isomers in the neural embryonic stem cell test
De Leeuw Victoria C, Hessel Ellen V, Piersma Aldert H

Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Non-animal testing methods, amongst others in vitro and in silico techniques, are indispensable for improving human risk assessment of chemicals and reducing the number of laboratory animals being used for toxicological assessment. For complex processes such as embryonic development, a series of complementary in vitro and in silico methods is needed to study effects of chemicals. The murine neural embryonic stem cell test (ESTn) could serve as one of these tests, as it mimics a number of processes in early differentiation of the embryonic neuroectoderm and brain. In previous research in our lab a biomarker gene set was derived based on testing a range of compounds in ESTn. The present study aims to further explore its response characteristics by exposure to two isomers, Valproic acid (VPA) and 2-Ethylhexanoic acid (EHA), to investigate whether these can be distinguished based on gene expression. It is hypothesised that by using a limited number of markers for various cell types differences in potency of the compounds can be observed. Based on morphological scoring a concentration of 0.1 mM was chosen to analyse gene expression effects, which was in between the ID50 of VPA and EHA. At this concentration, gene markers for stem cells (Fut4, Cdh1) showed increased expression, while neuronal markers (Nes, Tubb) were virtually unaffected. Markers for neural crest cells (Msx2, Snai2) and astrocytes (Gfap) showed decreased expression. Findings were confirmed with immunostainings. These effects on cell type markers point to a similar mode of action with different potencies between VPA and EHA.
Keywords: Developmental neurotoxicity, reproductive toxicity, in vitro, valproic acid, embryonic stem cells

Introducing WikiPathways to link molecular pathways to Adverse Outcome Pathways to support regulatory risk assessment

Marvin MartensA, Egon WillighagenA, Penny NymarkC,D, Roland GrafströmC,D, Lyle D BurgoonE, Hristo AladjovF, Fernando Torres AndónG, Chris T EveloA,B

A Department of Bioinformatics - BiGCaT, NUTRIM, Maastricht University
B Maastricht Centre for Systems Biology (MaCSBio), Maastricht University
C Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
D Department of Toxicology, Misvik Biology, Turku, Finland
E US Army Engineer Research and Development Center
F Organization for Economic Co-operation and Development (OECD) Environment Directorate: Paris, France
G Istituto Clinico Humanitas: Rozzano, Lombardia, Italy

In the last decade, omics-based approaches such as transcriptomics, proteomics and metabolomics have become valuable tools in toxicological research, and are finding their way into regulatory toxicity. A promising framework to bridge the gap between the molecular-level measurements and risk assessment is the concept of Adverse Outcome Pathways (AOPs). These pathways comprise mechanistic knowledge and connect biological events after exposure to a chemical or nanomaterial at a molecular level with an adverse effect. However, the implementation of omics-based approaches in the AOPs and acceptance by the risk assessment community is still a challenge. Therefore, tools are required for omics-based data analysis and visualization, and to link the data to the traditional AOPs.

Here we show how WikiPathways, an open science pathway database, can serve as a viable tool for this purpose. Therefore, an AOP Portal (aop.wikipathways.org) has been created with a rapidly growing collection of molecular-level AOPs on which omics datasets can be mapped an analyzed. Moreover, we are making WikiPathways more interoperable with aopwiki.org, the main knowledge-base that collects and stores AOPs. By introducing AOPs in WikiPathways, we aimed to make it a useful tool for the regulatory toxicity community and for toxicological research in general. Eventually this could lead to implementation of WikiPathways as a data-source for decision-making in REACH dossiers for risk assessment of chemicals. This project has received funding from the European Union’s Horizon 2020 research and innovation programme project EU-ToxRisk under grant agreement No. 681002 and EINFRA-22-2016 programme project OpenRiskNet under grant agreement No. 731075.

Functional and biokinetic differences in HepaRG’s 2D, sandwich, hollow-fiber and spheroid cultures for intrinsic clearance assays
Susana Proença1, Manon Bouwmeester1, Elena Lopez1, Theo Sinnige1 and Nynke Kramer1

1Institute of Risk Assessment Sciences, University of Utrecht

Obtaining long-term functional hepatocytes suitable for in vitro culture with few ethical problems, has posed a challenge to researchers in the past years. From the available human hepatic cell lines, HepaRG has been shown to be the most promising, although it still falls short compared to primary hepatocytes in terms of expressing biotransformation enzymes and transporters. In this study, we set up and compared the functionality of HepaRG in several cultures configurations: standard monolayers, collagen-sandwich, static hollow-fiber and spheroids cultivated in ultra-low attachment plates. Characterization of these models allows us to assess the extent to which differences in in vitro biokinetics contributes to the variation in the readout of in vitro clearance and hepatotoxicity assays. Quantitative PCR of the four cellular models was performed to analyze the expression of genes relevant for hepatocyte differentiation, proliferation and xenobiotics metabolic activity. To assess the cytochrome P450 enzyme activity, we exposed the cell models to a cocktail of drugs (bupropion, midazolam, dextromethorphan, phenacetin, chlorzoxanone and tolbutamide) and analysed the rate of depletion of parent compounds and the formation of the respective metabolite by LCMS/MS. Immunocytochemistry study of the different models was performed to better understand the state of polarization of cells in the cultures. Despite having different experimental setups and thus, different polarization, sandwich, spheroids and hollow fiber cultures display higher liver specific functions when compared to monolayers.