Proteomics approach for evaluating metabolites toxicity of furosemide, a widely-used diuretics.

Abstract

Introduction. Pharmaceutical by-products (PPs) including metabolites and transformation products of pharmaceuticals are a lot of concerns for both human and ecosystems health. Several studies pointed out PPs as emerging pollutants with a risk of toxicity exceeding the one of their parent molecules. Herein, we used toxicoproteomics to unveil toxicity effects of PPs of furosemide, a widely used diuretics. PPs of interest are pyridinium of furosemide (PYR), saluamine (SAL) and furfural (FRF) that were recently revealed as potential emerging pollutants in water. Methods. A label-free proteomic approach and cell viability assays were performed on hepatocarcinoma Hep-G2 cell line exposed for 96h to PPs (PYR, SAL FRF), furosemide and their mixture (MIX) at a CL10 letal dose. Protein extracts were submitted to a trypsin/Lys-C enzymatic digestion before performing LC-MSE analysis by using a NanoAcquity-C18/SYNAPT-G2Si massspectrometer system. Results. Toxic effects were confirmed on Hep-G2 cell line with a higher toxicity after an exposure to PYR, SAL and FRF while more substantial toxic effects were observed after an exposure to MIX and FUR. Based on the quantification of 1379 proteins, wedeciphered 71, 101, 138, 205 and 34 deregulated proteins after exposure to FUR, PYR, SAL, FRF, and MIX, respectively. A gene ontology enrichment analysis revealed that these proteins were involved in metabolism, immune response, biosynthesis or oxidative stress. For PPs exposure (PYR/SAL/FRF), these proteins were related to several diseases (neurodegenerativediseases/endocrine disruption/cancer) while, for MIX, these diseases were not retrieved, suggesting possible antagonistic effects. Conclusions. This toxicoproteomic work contributes to improve the current understanding of furosemide by-products and their mixture impacts on human cells. Beyond the case of furosemide, our study underlined the need to better take into account drug by-products during toxicological risk assessment