10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Chair

Establishment of an in vitro Test Battery as a New Approach Methodology for Non-Genotoxic Carcinogens in Human Colon Cell Models

3R Practice / Alternative Methods

TOX07

Carcinogenic chemicals can be divided into two categories, namely genotoxic and non-genotoxic carcinogens, depending on whether their mode of action (MoA) involves the direct interaction with DNA or not. While genotoxic carcinogens damage DNA, which can be detected by a battery of in vitro assays, non-genotoxic carcinogens (NGTxCs) show a diverse spectrum of different MoAs. So far, their carcinogenic potential can only be identified in long-term rodent bioassays, indicating the need for the establishment of a test battery for NGTxCs, consisting of human-based in vitro assays reflecting important mechanisms of carcinogenesis.In this project belonging to the European PARC consortium, non-malignant human colonic epithelial cells (HCEC) as well as the colorectal cancer cell line HCT116 were used. To establish the test battery a broad range of different chemicals including known NGTxCs as positive controls (PC), known non-carcinogens as negative controls (NC), as well as endpoint-specific positive controls were applied. These substances were tested using the endpoints cytotoxicity, proliferation, formation of ROS and genotoxicity.For all test substances concentration-response data were obtained, which allowed the determination of EC50 values and cytotoxic potency. Both cell models displayed overall comparable cytotoxicity of the test compounds. These data were complemented by EdU-based assessment of cell proliferation, revealing only one PC with promoting effects. Furthermore, a 96-well plate based assay was developed to detect ROS formation. The results of this high-throughput assay were compared with those from a flow cytometry-based approach revealing overall comparability. Finally, γ-H2AX induction as marker of genomic instability was measured in both cell models, confirming the non-genotoxic MoA of the tested PCs and NCs.Current efforts to increase the throughput of the test battery are focused on multiplexing the assays for the endpoints genotoxicity, proliferation and formation of ROS. To further aid the development, the addition of cellular transformation as an endpoint utilising the soft agar assay will be implemented.

Toxicology

Short talk

Prof. , Dr

Inhibition of Keratinocytes" Tolerance by Chemicals and Impact on the Activation of Dendritic CellsAruna, O. A.; Lobes, N, Blömeke, B.Department of Environmental Toxicology, Trier University, Germany

Epithelial cells, specifically keratinocytes, the main cells in the epidermis, maintain homeostasis. However, upon reaching a critical degree of dysregulation, they are involved in the recruitment of innate immune cells and the activation of dendritic cells (DCs). Upon encounter with high molecular weight allergens, keratinocytes are known to release critical cytokines such as Interleukin-1 alpha (IL-1α), IL-1β, Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), IL-33, and Thymic Stromal Lymphopoietin (TSLP), which instruct dendritic cells to promote Th2 immune responses. However, the exact conditions under which keratinocytes recognize low molecular weight chemicals and mediate activation of dendritic cells and initiation of adaptive T cell responses are not well understood.Earlier we found that higher concentrations of small molecular weight chemicals were needed to promote activation and maturation of DC in the presence of keratinocytes compared to DC alone (Hennen et al., 2017). Others reported that DC activation and maturation is only triggered when keratinocytes are significantly damaged by high concentrations of substances (Matejka et al. 2012).In this project, we characterized how HaCaT keratinocytes respond to low molecular weight chemicals and activate DC in more detail. HaCaT cells were exposed to chemicals for 2 hours, then incubated in fresh medium for 24 hours to generate Damage Associated Molecular Patterns (DAMPs). The medium was collected, centrifuged to remove debris, and the resulting keratinocyte-conditioned medium (KCM) was incubated with THP-1 cells (surrogate of DCs). We then analysed the expression of costimulatory and adhesion molecules, such as CD86 and CD54, using flow cytometry.Contrary to Matjeka et al. (2012), which reported that HaCaT keratinocytes only release DAMPs during chemical-induced cell death, leading to dendritic cell (DC) maturation, our findings, studying a set of chemicals with varying properties indicate that partially damaged keratinocytes (30% reduction of cell respiration) can promote DC activation. In conclusion, like high molecular weight substances, certain low molecular weight chemicals can activate keratinocytes followed by DC activation.ReferencesHennen et al. 2017, doi: 10.14573/altex.1606171.Matjeka et al. 2012, doi: 10.1016/j.jaci.2011.09.021

Prof.

Enhancing the Physiological Relevance of Human Brain Models by Incorporating Sex Hormones

Differences between female and male nervous systems, in part originating from a short-term testosterone surge from the developing testes of male fetuses, are not well understood, but have widespread effects – from the prevalence of neurological disorders to the sensitivity to neurotoxicants. In rodents however, estrogens, not testosterone, induce masculinization, highlighting a species-specific mechanism that limits the translational value of animal models. Additionally, sex as a biological variable is rarely considered for in vitro systems, leaving a substantial knowledge gap in our understanding of sex differences in human brain development.This project examines testosterone-induced sex differences in a human brain microphysiological system (bMPS). To this end, induced pluripotent stem cells from both male and female donors were differentiated into spheres that consist of different neuron types as well as astrocytes and oligodendrocytes. During a sensitive differentiation window, these bMPS were exposed to testosterone and analyzed at cellular and molecular level.We report expression levels of selected receptors and enzymes that corroborate the role of androgen signaling over estrogen signaling. After testosterone exposure, the size of the spheres was increased, mirroring reports of larger brain sizes in men after accounting for body size. Furthermore, the testosterone-exposed cultures displayed an increase in number and marker expression for oligodendrocytes, consistent with rodent studies on masculinization effects. Utilizing bulk RNAseq, we identified transcriptional shifts related to anterior-posterior patterning of the brain model, which may link to increased susceptibility to neurodevelopmental disorders.Thus, sex hormone-induced masculinization is a promising approach for enhancing human relevance in neuronal models and is highly relevant for studies on neurotoxicity or neurodevelopmental disorders.Funded by the Deutsche Forschungsgemeinschaft (DFG, 507269789), the Wendy Klag Center at the Johns Hopkins University and the Alternatives Research & Development Foundation (ARDF).

Prof. Dr.

Evaluation of precision cut kidney and liver slices as a potential 3R-compliant method to assess organ toxicity

For the investigation of organ toxicity under 3R principles, cell culture, co-cultures or even organoids are not sufficient to seize the high complexity of whole organs. Ex vivo cultivation of precision cut tissue slices (PCTS) could act as a link between the classical in vitro and in vivo systems, reflecting the complex organ anatomy and the interplay of different cell types and allowing kinetic analyses while lowering the animal numbers.PCTS of liver (PCLS) and kidney (PCKS) were incubated with known hepato- and nephrotoxins and analyzed after different time points. Viability assays were performed to determine IC50 values which were compared with IC50 values measured in cultured kidney and liver cells.Treatment with the hepatotoxin acetaminophen resulted in a dose-dependent loss of viability of PCLS and PCKS. While the IC50 values of PCKS and cultured kidney cells were comparable, the values of PCLS and cultured liver cells differed tremendously, with PCLS being much more sensitive. The same was observed after treatment with the nephrotoxin cyclosporin A, which also exhibited acute toxicity to the liver. When comparing different viability assays, the MTT assay proved to be more robust but less sensitive than the quantification of ATP levels.In conclusion, PCLS proved to be much more sensitive than cultured liver cells in the tests used here, while PCKS were less sensitive than cultured kidney cells. Both the MTT and ATP tests yielded robust results, but required too many technical replicates. Therefore, it would be beneficial to identify further reliable and less variable endpoints to minimize the use of animals.

Plenarsaal HS A

Kurzvorträge Toxikologie﻿

Short talks 2

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Short talks 2
3R Practice / Alternative Methods

Termin

Chair

Programm

Short talks 23R Practice / Alternative Methods

Termin

Chair

Programm

Short talks 2
3R Practice / Alternative Methods