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

Chair

Prof.

Methodological approaches for toxicological in vitro screening of mineral wools using normal rat alveolar macrophages and mesothelial cells

Inhalation Toxicology

TOX02

Man-made vitreous fibers (MMVF), including stone wools, are a group of fibers commonly used for thermal and acoustic insulation. Due to fiber morphology, MMVF might mediate adverse lung effects, with durability, dimension, and dose representing critical determinants. To ensure safety at workplaces and for consumers, and to base risk assessment not only on in vivo biopersistence testing, toxicological in vitro screening is highly desired. Therefore, the aim of the presents study was to define in vivo-relevant in vitro fiber concentrations, appropriate cell models, and critical determinants and pitfalls for in vitro MMVF screening. As lung relevant cell models primary rat alveolar macrophages (AM, non-proliferating) and normal rat mesothelial NRM2 cells (proliferating) were chosen. As exemplary MMVF (fibrous vs. ground to estimate impact of fiber shape) a stone wool fiber (RIF56008) was selected and comprehensively characterized before use. Long amosite asbestos served as insoluble fiber reference. Based on different approaches, 0.5, 5, and 50 µg/cm2 were defined to cover low up to overload concentrations. Depending on the specific readout, submerse cell incubation was done for 4-48 h. To avoid false negative judgement, material uptake was estimated by fluorescence-coupled darkfield microscopy and found positive for all materials tested and both AM and NRM2 cells. In NRM2 cells, RIF56008 mediated material-, morphology- and concentration-dependent membrane damage (lactate dehydrogenase release), decrease in cell count, induction of binucleated cells, and CINC-1 release (rat specific ELISA), with long amosite > RIF56008 > RIF56008 ground (particle-like material reference). In contrast, in AM, asbestos was nearly inactive, with CINC‑1 release only seen for RIF56008. Notably, DNA-strand break induction (alkaline comet assay) was nearly absent in both cell types. In conclusion, clear in vitro fiber effects were mostly seen at the supposed overload concentration (50 µg/cm2), where proliferating NRM2 cells were more sensitive than differentiated AM. To define a predictive, MMVF-adapted in vitro (geno)toxicity screening tool, all frame conditions should be carefully chosen based on in vivo relevance, and it needs further discussion whether proliferating cells (more sensitive), differentiated cells (less sensitive) or both together are most predictive for the in vivo situation. Next, additional readouts should be investigated, ideally with validation by in vivo data.

Toxicology

Short talk

Prof. Dr.

Reproducibility and Robustness of the Alveolar Macrophage Assay: Results of an Interlaboratory Comparison

3R Practice / Alternative Methods

TOX07

Inhaled poorly soluble particles1 may induce toxic effects due to intrinsic toxicity. The Alveolar Macrophage Assay (AMA)2 was developed as a reliable low-tier in vitro approach to identify harmful particles which could pose a risk to human health. Yet, the successful and widespread implementation of in vitro models as alternatives to animal testing requires a high level of reproducibility and standardization. Here, we conducted an interlaboratory comparison of the AMA to demonstrate its robustness. Three European institutes (IBE R&D, BASF and LIST) were involved. Rat alveolar macrophages (NR8383) were exposed to 12 blinded particle dispersions under fetal calf serum (FCS) free conditions. Generation of H2O2 was evaluated after 90 min of exposure using Ampliflu™ Red. Cytotoxicity, macrophage activation and inflammation were assessed by measuring lactate dehydrogenase (LDH), β-glucuronidase (GLU) and tumor necrosis factor alpha (TNFα), respectively, in the cell supernatant after 16 hours of exposure. The transfer of the method from IBE R&D to BASF and LIST showed that the AMA is reproducible and robust if a meticulous standardization is performed and strict quality criteria are followed. Evaluating the cell morphology during the growth phase is essential, since NR8383 can be easily activated by minor perturbations in culture conditions. Moreover, the suitability of each FCS lot for routine cultivation needs to be assessed (i) in the light of historical data, and (ii) by careful controls; inappropriate FCS batch may influence basal levels as well as dynamic ranges of all test parameters. In IBE R&D and BASF, seven of the blinded test particles exhibited a dose-dependent increase in LDH levels, while two particles showed no increase at any tested concentration. For GLU, six substances demonstrated a dose-dependent increase, whereas the other six were negative across all doses. For H2O2, a slight increase at the highest dose was observed for four substances only at BASF, while the others exhibited similar responses between the two institutes. Our interlaboratory comparison study also points out to the critical need for refining methodologies and standard operation procedures. Finally, it paves the way for a formal validation preceding the international acceptance of AMA through adoption of guidelines.This study was sponsored by the EU Project MACRAMÉ³.[1] doi.org/10.1080/08958378.2020.1735581 [2] doi.org/10.1186/s12951-016-0164-2 [3] macrame-project.eu/

Comparative Transcriptomic Analysis of Rats and In Vitro Human Models Exposed to Polyamide-6 via Inhalation

Inhalation is one of the routes of exposure to micro- and nanoplastics in humans. Inhalation hazards are usually assessed by in vivo inhalation toxicity testing in rats. However, interspecies differences limit the relevance of the findings of rat studies to human health. To address these limitations, a transcriptomics approach was used to compare pathways affected in rat and air-liquid interface human in vitro models of both bronchial and alveolar cells following the inhalation exposure to polyamide-6 (PA-6), aiming to identify toxicity pathways associated with nanoplastics exposure and assess the human relevance of the rat in vivo model.RNA-Seq was performed on the lungs of female Wistar rats exposed to PA-6 (2, 10, and 50 mg/m3) for 28 days (5 days/week, 6 hours/day), and on in vitro human models exposed to three doses of PA-6 [Bronchial: MucilAir™ (MA; 10.3, 25.4 and 64.9 µg/cm2); Alveolar: AlveolAir™ (AA; 5.2, 10.6 and 34.3 µg/cm2) and A549 cells (2.3, 12.9 and 28.4 µg/cm2)] for 24 hours using VITROCELL® Cloud. Stranded NGS Libraries were generated using the NEBNext Poly(A) mRNA Magnetic Isolation and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina. Sequencing was performed on an Illumina NextSeq2000 platform. The analyses were performed using DESeq2 R package and BMDExpress 3 software.The rat was the most sensitive species when comparing mass-doses based on surface area (µg/cm2). In contrast, the human in vitro models showed lower response to the exposure of PA-6. Besides interspecies differences, the stronger response in the rat could also reflect the longer exposure (repeated exposure in rats and single exposure in vitro). Focusing on genes that displayed a dose-response relationship revealed further changes. Ontology terms enriched in the rat and linked with adverse effects observed in those animals were also enriched in AA and A549 cells (e.g., GO:0030593 - neutrophil chemotaxis). For MA, a high variability between donors (n=4) and limited gene expression changes were observed.In this study, we used transcriptomics to compare pathways affected by PA nanoplastics in rat lungs and three human in vitro respiratory tract models, and we identified common and differing responses. Further research is needed to determine whether these differences reflect interspecies variations or the limited complexity of in vitro models. This will pave the way for experimental models with human relevance as true alternatives to animal testing.

Plenarsaal HS A

Kurzvorträge Toxikologie

Short talk 4

Short talks

Short talk 4
Inhalation Toxicology

Termin

Chair

Programm

Short talk 4Inhalation Toxicology

Termin

Chair

Programm

Short talk 4
Inhalation Toxicology