Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress

Abstract

High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15μM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5μM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.

Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure

“…6 closely resembles the experimental data for the first and last exposure day. A similar model for repeated exposure in kidney cells was described by Wilmes et al (2012). To have a better fit, additional experiments with sampling points on intermediate days are necessary.…”

BK/TD models for analyzing in vitro impedance data on cytotoxicity

“…Among these few attempts, the EU 7th Framework project, Predict-iv has been conducted to improve the predictivity of in vitro systems by developing mechanistic strategies. Thus, within this project, datasets have been generated in order to investigate long term repeated dose toxicity in three targeted organs (i.e., the liver, the kidney and the central nervous system), and a mechanism-based model of cellular toxicity has been developed for renal epithelial cells (RPTEC/TERT1) exposed repeatedly for 14 days to the nephrotoxin cyclosporine A (Wilmes et al, 2013). In the present study, we proposed an approach providing long-term, real-time toxicological data using impedance metrics and also a methodology for analyzing these data.…”

EURL ECVAM strategy for achieving 3Rs impact in the assessment of toxicokinetics and systemic toxicity

“…This also concerns in vitro toxicity testing where the in vitro kinetics should be measured, i.e. the concentration-time profile of the chemical and/or metabolites intracellular as well as in the incubation medium (Blaauboer, 2010;Wilmes et al, 2013). And it pertains as well to animal toxicity testing as long as it is still performed in order to improve study design and value for risk assessment (Creton et al, 2012).…”