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Clozapine is an atypical antipsychotic drug used to treat treatment-resistant schizophrenia. Its side effects, including liver enzyme abnormalities, experienced by many patients preclude its more common use as a first-line therapy for schizophrenia. Toxicoproteomic approaches have been demonstrated to effectively guide the identification of toxicological mechanisms. Here, to further our understanding of the molecular effects of clozapine, we performed a data-independent acquisition (DIA)-based quantitative proteomics investigation of clozapine-treated human liver spheroid cultures. In total, we quantified 4,479 proteins across the five treatment groups (vehicle; 15 µM, 30 µM, and 60 µM clozapine; and 10 ng/mL TNFα + IL-1β). Clozapine (60 µM) treatment yielded 36 differentially expressed proteins (FDR0.05). Gene-set enrichment analysis indicated perturbation of several gene sets, including interferon gamma signaling (e.g., interferon gamma receptor 1) and prominent autophagy-related processes (e.g., upregulation of sequestosome-1 (SQSTM1), MAP1LC3B/LC3B2, GABARAPL2, and nuclear receptor coactivator 4). The effects of clozapine on autophagy were confirmed by targeted mass spectrometry and western blotting using conventional SQSTM1 and LC3B markers. Combined with prior literature, our work suggests a broad contribution of autophagy to both the therapeutic and side effects of clozapine. Overall, this study demonstrates how proteomics can contribute to the elucidation of physiological and toxicological mechanisms of drugs.

International audience; Concentration of uranium (U), a naturally encountered radioactive element in earth’s crust, can be enhanced in freshwater ecosystems (µg.L-1 – mg.L-1) due to various anthropogenic activities. The consequent aquatic organism exposure to U leads to its accumulation in all organs, particularly in the gonad, and in subcellular fractions (mainly the cytosol); then it is known to affect fish at several biological levels, and more particularly, at a reproduction endpoint, with a decrease in the total number of eggs, spawn events and larvae survival. The understanding of U reprotoxicity requires the fine knowledge of its speciation at molecular level, i.e., its interaction with cytosolic biomolecules. In this study, we focus on the U-protein interactions in gonads. A non-denaturating extraction protocol combined with size exclusion chromatography (SEC) allowed the separation of metal-protein complexes in ovaries of U-contaminated wild roaches before their elemental detection (ICP MS). This enables unprecedented information to be obtained about U distribution in ovaries of autochthonous fish, Rutilus rutilus, which is different in some points from that obtained in the model species, Danio rerio under controlled laboratory conditions at a similar concentration level. Finally, the ability to transpose results from model to autochthonous fish was briefly discussed.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 μM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 μM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.