In the original publication [...].

The use of sensitive animals in toxicological studies tends to be limited. Even though cell culture is an attractive alternative, it has some limitations. Therefore, we investigated the potential of the metabolomic profiling of the allantoic fluid (AF) from ex ovo chick embryos to predict the hepatotoxicity of valproate (VPA). To this end, the metabolic changes occurring during embryo development and following exposure to VPA were assessed using 1H-NMR spectroscopy. During embryonic development, our findings indicated a metabolism progressively moving from anaerobic to aerobic, mainly based on lipids as the energy source. Next, liver histopathology of VPA-exposed embryos revealed abundant microvesicles indicative of steatosis and was metabolically confirmed via the determination of lipid accumulation in AF. VPA-induced hepatotoxicity was further demonstrated by (i) lower glutamine levels, precursors of glutathione, and decreased β-hydroxybutyrate, an endogenous antioxidant; (ii) changes in lysine levels, a precursor of carnitine, which is essential in the transport of fatty acids to the mitochondria and whose synthesis is known to be reduced by VPA; and (iii) choline accumulation that promotes the export of hepatic triglycerides. In conclusion, our results support the use of the ex ovo chick embryo model combined with the metabolomic assessment of AF to rapidly predict drug-induced hepatotoxicity.

Aristolochic acids (AAs) are powerful nephrotoxins that cause severe tubulointerstitial fibrosis. The biopsy-proven peritubular capillary rarefaction may worsen the progression of renal lesions via tissue hypoxia. As we previously observed the overproduction of reactive oxygen species (ROS) by cultured endothelial cells exposed to AA, we here investigated in vitro AA-induced metabolic changes by 1H-NMR spectroscopy on intracellular medium and cell extracts. We also tested the effects of nebivolol (NEB), a β-blocker agent exhibiting antioxidant properties. After 24 h of AA exposure, significantly reduced cell viability and intracellular ROS overproduction were observed in EAhy926 cells; both effects were counteracted by NEB pretreatment. After 48 h of exposure to AA, the most prominent metabolite changes were significant decreases in arginine, glutamate, glutamine and glutathione levels, along with a significant increase in the aspartate, glycerophosphocholine and UDP-N-acetylglucosamine contents. NEB pretreatment slightly inhibited the changes in glutathione and glycerophosphocholine. In the supernatants from exposed cells, a decrease in lactate and glutamate levels, together with an increase in glucose concentration, was found. The AA-induced reduction in glutamate was significantly inhibited by NEB. These findings confirm the involvement of oxidative stress in AA toxicity for endothelial cells and the potential benefit of NEB in preventing endothelial injury.

IntroductionLa néphropathie aux acides aristolochiques (AA) se caractérise par une atrophie tubulaire et une fibrose interstitielle sévères mais aussi par une raréfaction des capillaires péritubulaires. L’hypoxie tissulaire induite pourrait contribuer à la progression rapide des lésions tubulo-interstitielles.DescriptionVu la production d’espèces réactives de l’oxygène rapportée dans une lignée de cellules endothéliales humaines (EAhy926) exposées aux AA [Toxicol In Vitro 2020 ;65 :104811], le but de ce travail est d’investiguer in vitro les changements métabolomiques AA-induits et les effets cytoprotecteurs éventuels du nébivolol (NEB), un β-bloquant présentant des propriétés antioxydantes [J Hum Hypertens 2017 ; 31 :376].MéthodesLes cellules EAhy926 sont incubées en présence ou non de AA (50μM ou 100μM) et les effets d’une préincubation au NEB (10nM) sont évalués par le test de viabilité à la résazurine. La production d’H2O2 est mesurée par cytomètre en flux. Pour chaque condition expérimentale, les profils métabolomiques des extraits intracellulaires et des surnageants sont analysés par la spectroscopie 1H-RMN.RésultatsAprès 24h, les AA produisent des effets cytotoxiques marqués dose-dépendants et une production d’H2O2 nette, effets atténués par le NEB. Après 48h d’exposition aux AA, les principaux changements métabolomiques sont une diminution significative de l’arginine, du glutamate, de la glutamine et du glutathion ainsi qu’une augmentation significative de l’aspartate, de la glycérolphosphocholine et de l’UDP-N-acétylglucosamine. Le NEB inhibe significativement les changements en glutathion et glycérophosphocholine. Dans les surnageants des cellules intoxiquées, on observe une augmentation en glucose ainsi qu’une réduction en lactate et en glutamate, cette dernière étant significativement inhibée par le NEB.ConclusionCette étude confirme la toxicité des AA vis-à-vis de la cellule endothéliale et la cytoprotection partielle du NEB. L’analyse métabolomique corrobore l’implication du stress oxydatif dans les lésions endothéliales. Le NEB semble être une molécule intéressante à explorer dans la prévention de l’hypoxie rénale d’origine toxique.

(1) Background: White adipose tissue (WAT) is a dynamic and plastic tissue showing high sensitivity to carbohydrate supply. In such a context, the WAT may accordingly modulate its mitochondrial metabolic activity. We previously demonstrated that a partial replacement of glucose by galactose in a culture medium of 3T3-L1 cells leads to a poorer adipogenic yield and improved global mitochondrial health. In the present study, we investigate key mitochondrial metabolic actors reflecting mitochondrial adaptation in response to different carbohydrate supplies. (2) Methods: The metabolome of 3T3-L1 cells was investigated during the differentiation process using different glucose/galactose ratios and by a targeted approach using 1H-NMR (Proton nuclear magnetic resonance) spectroscopy; (3) Results: Our findings indicate a reduction of adipogenic and metabolic overload markers under the low glucose/galactose condition. In addition, a remodeling of the mitochondrial function triggers the secretion of metabolites with signaling and systemic energetical homeostasis functions. Finally, this study also sheds light on a new way to consider the mitochondrial metabolic function by considering noncarbohydrates related pathways reflecting both healthier cellular and mitochondrial adaptation mechanisms; (4) Conclusions: Different carbohydrates supplies induce deep mitochondrial metabolic and function adaptations leading to overall adipocytes function and profile remodeling during the adipogenesis.

Characteristic metabolic adaptations are recognized as a cancer hallmark. Breast cancer, like other cancer types, displays cellular respiratory switches-in particular, the Warburg effect-and important fluctuations in the glutamine and choline metabolisms. This cancer remains a world health issue mainly due to the side effects associated with chemotherapy, which force a reduction in the administered dose or even a complete discontinuation of the treatment. For example, Doxorubicin is efficient to treat breast cancer but unfortunately induces severe cardiotoxicity. In the present in vitro study, selected metabolic inhibitors were evaluated alone or in combination as potential treatments against breast cancer. In addition, the same inhibitors were used to possibly potentiate the effects of Doxorubicin. As a result, the combination of CB-839 (glutaminase inhibitor) and Oxamate (lactate dehydrogenase inhibitor) and the combination of CB-839/Oxamate/D609 (a phosphatidylcholine-specific phospholipase C inhibitor) caused significant cell mortality in both MDA-MB-231 and MCF-7, two breast cancer cell lines. Furthermore, all inhibitors were able to improve the efficacy of Doxorubicin on the same cell lines. Those findings are quite encouraging with respect to the clinical goal of reducing the exposure of patients to Doxorubicin and, subsequently, the severity of the associated cardiotoxicity, while keeping the same treatment efficacy.

Abstract Objective: Electronic cigarettes (e-cigarettes) are battery-powered devices that aerosolize a liquid (i.e. “e-liquid”) composed of propylene glycol (PG) and/or glycerol (GLY), flavorings and most commonly, nicotine. Suspension of fine particles in a gas forms the aerosol, that is inhaled (i.e. “vaped”). As these small hydrophilic molecules have a short serum/urine half-live, we hypothesized that short-term cessation of vaping in regular users would change serum proteomic signatures. Design and method: Thirty regular e-cigarette users were enrolled in this randomized, investigator-blinded, three-period crossover-study. The periods consisted of nicotine-vaping (nicotine-session), nicotine-free vaping (nicotine-free-session), and complete cessation of vaping (stop-session), all maintained for five days before the session began. Multiparametric metabolomic analyses on urine and serum were used to verify subjects’ protocol compliance, which was 95%. Serum proteomic analysis was performed with Sequential Windowed Acquisition of All theoretical Fragment Ion Mass Spectra (SWATH-MS) Results: Complete cessation of vaping allowed to identify 13 proteins in the serum, which were differentially modulated among sessions. The following proteins of coagulation cascade: anti thrombin III, heparin cofactor II and alpha-2-antiplasmin were higher in the stop-session compared to nicotine- and nicotine-free session. Compared to stop-session, two proteins of complement cascade were lower in the stop-session compared to the nicotine-session. Finally; lower levels of apolipoprotein E were also found in the stop-session compared to the nicotine-free session. (Table 1) Conclusions: In conclusion, our study reveals that short-term e-cigarette cessation in regular users modified several proteins involved in the coagulation pathways, complement cascade and lipid transport. E-cigarette cessation could have a beneficial effect on the cardiovascular health by decreasing the level of proteins involved in the pathogenesis of atherosclerosis, irrespective of the presence of nicotine. This will need further researches in order to confirm our findings in cellular and animal models and to find potential biomarkers of vaping toxicity.

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte’s death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by 1H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis-epoxysuccinate (cis-ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis-ES either alone or in combination with DOX in H9C2 cells. A 1H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis-ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis-ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.

Abstract Objective: We previously demonstrated in young and healthy occasional tobacco smokers that acute exposure to high wattage vaping with and without nicotine decreases transiently transcutaneous oxygen tension and arterial oxyen tension. Propylene glycol and glycerol are e-cigarette constituents that facilitate liquid vaporization and nicotine transport. As these small hydrophilic molecules have a short serum/urine half-live, we hypothesized that short-term cessation of vaping in regular users would change urine metabolic signatures. Design and method: Thirty regular e-cigarette users were enrolled in this randomized, investigator-blinded, three-period crossover-study. The periods consisted of nicotine-vaping (nicotine-session), nicotine-free vaping (nicotine-free-session), and complete cessation of vaping (stop-session), all maintained for five days before the session began. Multiparametric metabolomic analyses on urine and serum were used to verify subjects’ protocol compliance. Metabolomic analysis was performed on urine samples by proton nuclear magnetic resonance. Results: Partial least squares discriminant analysis (PLS-DA) performed on baseline serum metabolome did not allow to split up any of the three experimental session. PLS-DA performed on baseline urine metabolome allowed to split up the nicotine and the stop sessions (Figure 1.C), the nicotine-free and the stop-sessions (Figure 1.D) but not the nicotine from the nicotine-free session (Figure 1.B) as well as all three sessions (Figure 1.A). Heatmap outlines the main metabolites with variables of importance values > 1 of both session as well as those identified by the PLS-DA. Baseline urine propylene glycol was lower in the stop-session compared to the nicotine-and nicotine-free sessions (all-p < 0.0001). Baseline urine 3-hydroxyisovalerate and urine-pyruvate were higher in the nicotine-session vs. the nicotine-free and the stop-sessions (all-p < 0.05). Compared to the stop-session, baseline urine trimethylamine-oxide and hippurate were lower in the nicotine-session (all-p < 0.05). Finally, baseline N-Phenylacetyl-glycine was lower in the nicotine-session when compared to the stop-session (p < 0.05). Conclusions: In conclusion, our study reveals that short-term e-cigarette cessation in regular users modified the urine metabolomic signature in a way signature in a way that suggests several modulations in hypoxia related pathways. This will need further researches focusing on cellular and molecular alterations (i.e., hypoxia-inducible Factors-1, nuclear factor-kappa B), in cell and animal models.

Treatments of metastatic melanoma underwent an impressive development over the past few years, with the emergence of small molecule inhibitors targeting mutated proteins, such as BRAF, NRAS, or cKIT. However, since a significant proportion of patients acquire resistance to these therapies, new strategies are currently being considered to overcome this issue. For this purpose, melanoma cell lines with mutant BRAF, NRAS, or cKIT and with acquired resistances to BRAF, MEK, or cKIT inhibitors, respectively, were investigated using both 1H-NMR-based metabonomic and protein microarrays. The 1H-NMR profiles highlighted a similar go and return pattern in the metabolism of the BRAF, NRAS, and cKIT mutated cell lines. Indeed, melanoma cells exposed to mutation-specific inhibitors underwent metabolic disruptions following acute exposure but partially recovered their basal metabolism in long-term exposure, most likely acquiring resistance skills. The protein microarrays inquired about the potential cellular mechanisms used by the resistant cells to escape drug treatment, by showing decreased levels of proteins linked to the drug efficacy, especially in the downstream part of the MAPK signaling pathway. Integrating metabonomic and proteomic findings revealed some metabolic pathways (i.e., glutaminolysis, choline metabolism, glutathione production, glycolysis, oxidative phosphorylation) and key proteins (i.e., EPHA2, DUSP4, and HIF-1A) as potential targets to discard drug resistance.

Patients with alcohol use disorder (AUD) present with important emotional, cognitive, and social impairments. The gut microbiota has been recently shown to regulate brain functions and behavior but convincing evidence of its role in AUD is lacking. Here, we show that gut dysbiosis is associated with metabolic alterations that affect behavioral (depression, sociability) and neurobiological (myelination, neurotransmission, inflammation) processes involved in alcohol addiction. By transplanting the gut microbiota from AUD patients to mice, we point out that the production of ethanol by specific bacterial genera and the reduction of lipolysis are associated with a lower hepatic synthesis of β-hydroxybutyrate (BHB), which thereby prevents the neuroprotective effect of BHB. We confirm these results in detoxified AUD patients, in which we observe a persisting ethanol production in the feces as well as correlations among low plasma BHB levels and social impairments, depression, or brain white matter alterations.

Doxorubicin (DOX) is an anticancer drug widely used in oncology. The main limitation to DOX treatments though is due to the cumulative dose that may lead to cardiotoxicity. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure consecutive to a progressive loss in cardiomyocytes due to cell necrosis and apoptosis induced by DOX. For many years, the cardiac oxidative stress caused by DOX was considered as its main toxic mechanism. Therefore, several clinical trials were carried out to assess the efficacy of various antioxidants as a cardioprotective strategy. Only dexrazoxane (DEX), did significantly reduce DOX cardiotoxicity. However, since other antioxidants used later on to counteract DOX cardiotoxicity were not as successful as DEX, DOX-induced oxidative stress and DEX antioxidant activity are not considered as the main feature anymore and this led the scientific world to suspect other involved mechanisms which are still unknown. The objective of the present work was to study from a metabolic point of view the side effects of DOX and the protective properties of DEX. In vitro 1H-NMR metabonomics was applied to the rat cardiomyoblastic H9C2 cell line. This strategy was used with the hope of unveiling possible new targets to cope with DOX cardiotoxicity. Another underlying goal was the validation of H9C2 in vitro model for metabolic investigations of DOX and DEX effects. For this purpose, several parameters, including oxidative stress, cell mortality, and apoptosis, were measured to assess the effects of DOX and DEX alone or in combination. The metabonomic study was carried out on cellular fluids collected after either 4 or 24 hours of DOX-exposure. Under such experimental conditions, both the major adverse effects reported in patients exposed to DOX and the protective effect of DEX were demonstrated in vitro, suggesting that the H9C2 in vitro model is relevant to investigate both DOX cardiotoxicity and putative cardioprotective strategies. In addition, the metabonomics findings highlighted several metabolic pathways involved in DOX cardiotoxicity and DEX cardioprotective effects as potential metabolic targets for cardioprotection: energy metabolism, redox balance, as well as phospholipids and proteins metabolism.

Obesity is an alarming yet increasing phenomenon worldwide, and more effective obesity management strategies have become essential. In addition to the numerous anti-adipogenic treatments promising a restauration of a healthy white adipose tissue (WAT) function, numerous studies reported on the critical role of nutritional parameters in obesity development. In a metabolic disorder context, a better control of nutrient intake is a key step in slowing down adipogenesis and therefore obesity. Of interest, the effect on WAT remodeling deserves deeper investigations. Among the different actors of WAT plasticity, the mitochondrial network plays a central role due to its dynamics and essential cellular functions. Hence, the present in vitro study, conducted on the 3T3-L1 cell line, aimed at evaluating the incidence of modulating the carbohydrates intake on adipogenesis through an integrated assessment of mitochondrial structure, dynamics, and functions-correlated changes. For this purpose, our experimental strategy was to compare the occurrence of adipogenesis in 3T3-L1 cells cultured either in a high-glucose (HG) medium (25 mM) or in a low-glucose (LG) medium (5 mM) supplemented with equivalent galactose (GAL) levels (20 mM). The present LG-GAL condition was associated, in differentiating adipocytes, to a reduced lipid droplet network, lower expressions of early and late adipogenic genes and proteins, an increased mitochondrial network with higher biogenesis marker expression, an equilibrium in the mitochondrial fusion/fission pattern, and a decreased expression of mitochondrial metabolic overload protein markers. Therefore, those main findings show a clear effect of modulating glucose accessibility on 3T3-L1 adipogenesis through a combined effect of adipogenesis modulation and overall improvement of the mitochondrial health status. This nutritional approach offers promising opportunities in the control and prevention of obesity.

Propylene glycol and glycerol are e-cigarette constituents that facilitate liquid vaporization and nicotine transport. As these small hydrophilic molecules quickly cross the lung epithelium, we hypothesized that short-term cessation of vaping in regular users would completely clear aerosol deposit from the lungs and reverse vaping-induced cardiorespiratory toxicity. We aimed to assess the acute effects of vaping and their reversibility on biological/clinical cardiorespiratory parameters [serum/urine pneumoproteins, hemodynamic parameters, lung-function test and diffusing capacities, transcutaneous gas tensions (primary outcome), and skin microcirculatory blood flow]. Regular e-cigarette users were enrolled in this randomized, investigator-blinded, three-period crossover study. The periods consisted of nicotine-vaping (nicotine-session), nicotine-free vaping (nicotine-free-session), and complete cessation of vaping (stop-session), all maintained for 5 days before the session began. Multiparametric metabolomic analyses were used to verify subjects' protocol compliance. Biological/clinical cardiorespiratory parameters were assessed at the beginning of each session (baseline) and after acute vaping exposure. Compared with the nicotine- and nicotine-free-sessions, a specific metabolomic signature characterized the stop-session. Baseline serum club cell protein-16 was higher during the stop-session than the other sessions (P < 0.01), and heart rate was higher in the nicotine-session (P < 0.001). Compared with acute sham-vaping in the stop-session, acute nicotinevaping (nicotine-session) and acute nicotine-free vaping (nicotine-free-session) slightly decreased skin oxygen tension (P < 0.05). In regular e-cigarette-users, short-term vaping cessation seemed to shift baseline urine metabolome and increased serum club cell protein-16 concentration, suggesting a decrease in lung inflammation. Additionally, acute vaping with and without nicotine decreased slightly transcutaneous oxygen tension, likely as a result of lung gas exchanges disturbances.

In this study, metastatic melanoma, breast, and prostate cancer cell lines were analyzed using a 1H-NMR-based approach in order to investigate common features and differences of aggressive cancers metabolomes. For that purpose, 1H-NMR spectra of both cellular extracts and culture media were combined with multivariate data analysis, bringing to light no less than 20 discriminant metabolites able to separate the metastatic metabolomes. The supervised approach succeeded in classifying the metastatic cell lines depending on their glucose metabolism, more glycolysis-oriented in the BRAF proto-oncogene mutated cell lines compared to the others. Other adaptive metabolic features also contributed to the classification, such as the increased total choline content (tCho), UDP-GlcNAc detection, and various changes in the glucose-related metabolites tree, giving additional information about the metastatic metabolome status and direction. Finally, common metabolic features detected via 1H-NMR in the studied cancer cell lines are discussed, identifying the glycolytic pathway, Kennedy’s pathway, and the glutaminolysis as potential and common targets in metastasis, opening up new avenues to cure cancer.

Patients with alcohol use disorder (AUD) present with important emotional, cognitive and social impairments. The gut microbiota has been recently shown to regulate brain functions and behavior but convincing evidence of its role in AUD is lacking. Here, we show for the first time that the gut microbiota drives behavioral features of alcohol addiction including reduced sociability. By transplanting the gut microbiota from AUD patients to mice, we pointed out that the production of ethanol by specific bacterial genera, is associated with a lower hepatic synthesis of beta-hydroxybutyrate (BHB), and thereby prevent the protective effect of BHB on social behavior, neuroinflammation and neurotransmission. We confirmed this hypothesis detoxified AUD patients, in which we observed a persisting ethanol production in the feces as well as correlations between low plasma BHB levels and social impairments. Our data support new pathways involved in behavioral disorders associated with alcohol addiction, that could be considered for future treatments.