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

International audience; 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 14 days. 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 15 µM 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 5 µM 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

Assessment of the effectiveness of reported Water Framework Directive Programmes of Measures - Part I – Pan-European scale screening of the pressures addressed by member states

In this report we propose a classification of river basin districts (RBDs) in terms of the consistency between reported significant pressures and pressures expected to be relevant in each RBD on the basis of pan-European indicators. We discuss the results and limitations of the classification obtained at European scale, and we suggest a perspective for its use for improving Water Framework Directive (WFD) implementation and the subsequent in-depth analysis of programmes of measures (PoMs) and their effectiveness. Overall, the analysis highlights that some river basin districts report pressures in a way that is in line with expectations from European indicators, while some others report either higher or lower pressure intensity than expected. Reasons for these discrepancies may include lack of relevant local information in the assessment of European indicators, and under/overestimation of pressures by the RBD authorities or reporting member states. The analysis presented here is based on data aggregated at RBD level, as allowed by the available information, and should be regarded mainly as a proof of concept. In the future, the same analysis should be updated with reference to individual water bodies, and taking into consideration the updates of river basin management plans and PoMs under development at the time of writing this report.JRC.H.1-Water Resource

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

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 characterized nephrotoxin Cyclosporine A (CsA) at therapeutic and supra therapeutic concentrations for 14 days. 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 15 µM 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 5 µM 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.JRC.I.1-Chemical Assessment and Testin

p73 poses a barrier to malignant transformation by limiting anchorage-independent growth

p53 is known to prevent tumour formation by restricting the proliferation of damaged or oncogene-expressing cells. In contrast, how the p53 family member p73 suppresses tumour formation remains elusive. Using a step-wise transformation protocol for human cells, we show that, in premalignant stages, expression of the transactivation-competent p73 isoform TAp73 is triggered in response to pRB pathway alterations. TAp73 expression at this stage of transformation results in increased sensitivity to chemotherapeutic drugs and oxidative stress and inhibits proliferation and survival at high cell density. Importantly, TAp73 triggers a transcriptional programme to prevent anchorage-independent growth, which is considered a crucial hallmark of fully transformed cells. An essential suppressor of anchorage-independent growth is KCNK1, which is directly transactivated by TAp73 and commonly downregulated in glioma, melanoma and ovarian cancer. Oncogenic Ras switches p73 expression from TAp73 to the oncogenic ΔNp73 isoform in a phosphatidyl-inositol 3-kinase-dependent manner. Our results implicate TAp73 as a barrier to anchorage-independent growth and indicate that downregulation of TAp73 is a key transforming activity of oncogenic Ras mutants