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

Prof. Dr.

Chair

A next generation risk assessment (NGRA) case study using pendimethalin based on in vitro transcriptomics and physiology based toxicokinetic modelling

3R Practice / Alternative Methods

TOX07

The combination of new approach methods and physiology based toxicokinetic (PBTK) models in a workflow referred to as next generation risk assessment (NGRA) aims to allow for an animal-free assessment of toxicological risk associated with chemical exposure. As an integral part, PBTK-based quantitative in vitro to in vivo extrapolation (QIVIVE) is utilized to convert points of departure (POD) derived by high-content in vitro methods (e.g. transcriptomics) to external doses. Here, we applied NGRA to the herbicidal agrochemical pendimethalin.The scope of this study was the implementation and evaluation of transcriptome data generated for in vitro toxicological assessments. Therefore, we extrapolated the obtained concentration-response relationships to external doses based on QIVIVE-modeling following the NGRA approach.The TempO-SeqTM method by BioSpyderTM was utilized for high-content transcriptome analysis to measure substance-induced alterations in the expression of 22538 genes using the human whole transcriptome panel. Following cytotoxicity testing, HepG2 cells were exposed to pendimethalin (CAS no. 40487-42-1) in concentrations from 0.76 μM to 56 μM for 48 hours. Transcriptomic PODs were derived by three different data analysis approaches and subsequently extrapolated to external doses by QIVIVE using a rat 9-compartment PBTK model. The PBTK model input parameters for hepatic clearance were determined experimentally, while QSAR calculations or literature data were used to obtain the remaining parameters.The expression of 769 genes was altered dose-dependently in response to pendimethalin treatment which allowed for the calculation of corresponding benchmark doses (BMDs). The 25th potency ranked gene and the 1st mode were utilized to obtain a gene level POD of 2.0 μM and 2.3 μM, respectively, while the most sensitive pathway median BMD of 3.0 μM was derived as a pathway level POD. Following QIVIVE, the related external exposures for the rat were 26.8, 29.1 and 39.8 mg/kg body weight (BW). These results revealed to be in good accordance with a NOAEL of 41.3 mg/kg BW per day based on hepatotoxicity observed in a 90-days rat study.In summary, our case study successfully applied the NGRA concept for the agrochemical pendimethalin in a quantitative manner. The combination of transcriptome analysis in HepG2 cells and PBTK modeling for QIVIVE derived a POD related external dose level that is in good accordance with the results of rat in vivo studies.

Toxicology

Short talk

Assessing the impact of prenatal Paracetamol exposure on early human forebrain development using cerebral organoids

An increasing number of epidemiological studies suggest that prenatal Paracetamol (APAP) exposure impairs fetal neurodevelopment in a time- and dose-dependent manner (Alemany 2021, Bauer 2018, Brandlistuen 2013, Ricci 2023). The underlying molecular mechanisms remain largely unexplored.Here, we present a novel 3D cell culture workflow based on human iPSC-derived forebrain organoids to assess the impact of prenatal APAP exposure on human neurodevelopment. Immediately following neural induction, we initiate an 8-week treatment at 2 concentrations, based on the maximum plasma concentration reported in pregnant females after oral administration (Graham 2013, Kumpulainen 2007, Rayburn 1986). As APAP readily crosses the placental- and blood-brain-barrier, our setup is well suited to simulate the in vivo fetal exposure (Anderson 1998, Conings 2019, Nitsche 2017, Raffa 2018). Further, it perfectly aligns with the 3R principles, replacing animal models with human iPSC-derived organoids, omitting animal use, and refining the experimental setup to minimize potential harm and adhere to ethical standards.For phenotypic assessment of forebrain organoids, we use whole mount light-sheet fluorescence expansion microscopy (LSFEM) (Rodriguez-Gatica 2022). LSFEM allows to portray the organoids' overall architecture and to focus on specific sub-compartments. The latter enables characterization of cellular phenomena at nanoscale with reasonable normalization and in respect to the 3D tissue-architecture sourrounding individual cells.We show that APAP reduces the organoids' cross-sectional area by 20% and disrupts their overall structure, with a 33% reduction of their vital volume, a 10% increase in neural rosette density and a 33% reduction of the rosettes' individual volumes. Moreover, APAP affects cell proliferation and causes a shift in the organoids' gene expression profiles. Upcoming analyses on apoptosis, mRNA and miRNA sequencing are expected to further elucidate the underlying molecular mechanisms.Our observations suggest an accumulative effect of chronic APAP exposure on early human neurodevelopment. Further, our experimental setup provides a controllable in vitro platform to study prenatal neurotoxic effects of various pharmaceutical compounds, compliant to the 3R principles.Fig. 1 Effect of APAP on overall volume and neural rosette architectureFig. 2a Effect of APAP on cell proliferationFig. 2b Effect of APAP on organoid gene expression profiles

Nephrotoxin- and oxidant-induced cell death and DNA damage in hiPSCs, differentiating hiPSCs and differentiated renal proximal tubular epithelial cells

Proximal tubular epithelial cells (PTEC) are consistently exposed to potentially toxic metabolites and oxidants. The kidney's regenerative capabilities enable the replacement of damaged cells through either stem cell differentiation or the restoration of PTEC's proliferative functions. However, kidney function tends to decline, indicating that the replacement cells may not attain full functionality. Investigating the impact of toxins on the regeneration process is essential to better understand the possible causes of this decline in renal cell function. Consequently, we examined how nephrotoxins and oxidants affect human induced pluripotent stem cells (hiPSC) at various stages of differentiation. hiPSCs were differentiated into proximal tubular epithelial-like kidney cells (PTELC). We analyzed proliferation, apoptosis, senescence, the expression of DNA repair and oxidative defence genes in hiPSCs, differentiating cells on day 3 of the differentiation process, and differentiated cells on day 9 that were treated with cisplatin, cyclosporin A and oxidants.Differentiated PTELC exhibited typical morphological characteristics of human PTEC after 9 days of differentiation. During this process, stem cell markers were downregulated, while PTEC markers were significantly upregulated at both the mRNA and protein levels. Notably, genes associated with DNA repair and antioxidant defence were predominantly downregulated in the differentiating cells compared to hiPSC and PTELC. As a result, treatment with cisplatin and ionizing radiation resulted in the highest levels of γH2AX foci in cells at this intermediate differentiation stage. The differentiating cells also displayed increased sensitivity to cisplatin and oxidative stress, whereas all differentiation stages demonstrated similar sensitivity to cyclosporine A. Both oxidative stress and cisplatin triggered apoptosis across all differentiation stages, while cyclosporine A primarily induced senescence. Epithelial-mesenchymal transition (EMT) was most strongly stimulated by cyclosporine A, followed by cisplatin. By contrast, oxidizing agents did not significantly enhance the expression of EMT markers.Our results point to a significant sensitivity of differentiating cells to DNA damaging agents and oxidants, which could potentially impede regenerative processes. Our model of hiPSC differentiating in vitro into PTELC emerges as a promising 3R-compatible tool for delving deeper into the area of kidney regeneration.

Short talk 9
3R Practice / Alternative Methods (2)

Appointment

Chair

Programme

Short talk 93R Practice / Alternative Methods (2)

Appointment

Chair

Programme

Short talk 9
3R Practice / Alternative Methods (2)