Use of in vitro metabolomics in NRK cells to help predicting nephrotoxicity and differentiating the MoA of nephrotoxicants

Birk, Barbara; Haake, Volker; Sperber, Saskia; Herold, Michael; Wallisch, Svenja K.; Huener, Hans-Albrecht; Verlohner, Andreas; Amma, Meike M.; Walk, T.; Hernandez, Tzutzuy Ramirez; Hewitt, Nicola J.; Kamp, Hennicke; Ravenzwaay, Bennard van

doi:10.1016/j.toxlet.2021.09.011

Cited by 5 publications

(4 citation statements)

References 72 publications

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“…The high content imaging data showed that for two lineages of proximal tubule cells, disturbance of lysosome function preceded the onset of cytotoxic effects for polymyxin B as an exemplary stressor for this AOP. Moreover, using high content screening of additional nephrotoxins, including presumed additional stressors for this AOP (i.e., colistin, polymyxin B nonapeptide, vancomycin, gentamicin, CdCl 2 ) as well as compounds that are thought to cause kidney toxicity via inhibition of mitochondrial DNA polymerase γ (cidofovir, tenofovir, tenofovir disoproxil fumarate) or covalent protein binding (i.e., S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), acetaminophen and 4-aminophenol) ( Birk et al, 2021 ; Mally and Jarzina, 2022 ), the most prominent effects on lysosomal endpoints were induced by stressors of this AOP, even though minor changes were also evident in response to compounds that act primarily via mechanisms unrelated to lysosomes, particularly at concentrations at which cytotoxicity occurred ( Supplementary Figure S1 ).…”

Section: Resultsmentioning

confidence: 99%

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study

et al. 2022

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Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.

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Section: Resultsmentioning

confidence: 99%

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study

et al. 2022

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“…Elevated concentrations of both LPC and LPE represent an increased turnover of phospholipids species and are as well a common finding in investigations of liver pathologies and in the nephrotoxic-related MoA—mitochondrial DNA interaction—in kidney cells (García-Canaveras et al 2011 ; Beyoglu et al 2013 ; Birk et al 2021 ).…”

Section: Discussionmentioning

confidence: 92%

“…Metabolomics has been successfully implemented for more than a decade to identify toxicological mechanisms in rodent studies (Kamp et al 2012 ; Van Ravenzwaay et al 2014 ; Van Ravenzwaay et al 2015 ). More recently, it has been used in combination with in vitro models to expand the investigation of organ toxicity (García-Cañaveras et al 2016 ; Birk et al 2021 ; Huang et al 2021 ; Jeon et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity

et al. 2023

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Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here, we present the development of a 96-well plate LC-MS/MS-based targeted metabolomics screening platform for the classification of liver toxicity modes of action (MoAs) in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform. The applicability of the system was tested with seven substances known to be representative of three different liver toxicity MoAs (peroxisome proliferation, liver enzyme induction, and liver enzyme inhibition). Five concentrations per substance, aimed at covering the complete dose-response curve, were analyzed and 221 uniquely identified metabolites were measured, annotated, and allocated in 12 different metabolite classes such as amino acids, carbohydrates, energy metabolism, nucleobases, vitamins and cofactors, and diverse lipid classes. Multivariate and univariate analyses showed a dose response of the metabolic effects, a clear differentiation between liver toxicity MoAs and resulted in the identification of metabolite patterns specific for each MoA. Key metabolites indicative of both general and mechanistic specific hepatotoxicity were identified. The method presented here offers a multiparametric, mechanistic-based, and cost-effective hepatotoxicity screening that provides MoA classification and sheds light into the pathways involved in the toxicological mechanism. This assay can be implemented as a reliable compound screening platform for improved safety assessment in early compound development pipelines. Graphical abstract

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“…Thus, metabolomics is the “omics” technology that closest represents the phenotype and for this reason has been considered to be closer to classical toxicology than other Omics techniques (Ramirez et al 2013 ). Metabolomics approaches have been successfully employed in toxicity assessment for identifying mechanisms of toxicity and characterizing key molecular events (Birk et al 2021 ; Cuykx et al 2018 ; Kamp et al 2012 ; Mattes et al 2014 ; Van Ravenzwaay et al 2007 , 2015 ). For cell-based metabolomics, however, requirements for large biomass quantities have previously restricted the throughput scalability, limiting the testing to few concentrations and single (static) time points (Cuykx et al 2018 ; García-Cañaveras et al 2016 ; Ramirez et al 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin

Ramirez-Hincapie,

Birk,

Ternes

et al. 2023

Arch Toxicol

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Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC–MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones.

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Use of in vitro metabolomics in NRK cells to help predicting nephrotoxicity and differentiating the MoA of nephrotoxicants

Cited by 5 publications

References 72 publications

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin

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