Predicting Valproate-Induced Liver Injury Using Metabolomic Analysis of Ex Ovo Chick Embryo Allantoic Fluid.

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 ß-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.

Protective Effect of Nebivolol against Oxidative Stress Induced by Aristolochic Acids in Endothelial Cells.

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 ß-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.

Targeting Metabolic Reprogramming to Improve Breast Cancer Treatment: An In Vitro Evaluation of Selected Metabolic Inhibitors Using a Metabolomic Approach.

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.

Investigation of Mitochondrial Adaptations to Modulation of Carbohydrate Supply during Adipogenesis of 3T3-L1 Cells by Targeted 1H-NMR Spectroscopy.

(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.

Influence of Nutritional Intake of Carbohydrates on Mitochondrial Structure, Dynamics, and Functions during Adipogenesis.

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.

Gut Microbiota-Induced Changes in ß-Hydroxybutyrate Metabolism Are Linked to Altered Sociability and Depression in Alcohol Use Disorder.

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 ß-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.

GPR91 Receptor Mediates Protection against Doxorubicin-Induced Cardiotoxicity without Altering Its Anticancer Efficacy. An In Vitro Study on H9C2 Cardiomyoblasts and Breast Cancer-Derived MCF-7 Cells.

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.

Metabolic Reprogramming in Metastatic Melanoma with Acquired Resistance to Targeted Therapies: Integrative Metabolomic and Proteomic Analysis.

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.

New Insights About Doxorubicin-Induced Toxicity to Cardiomyoblast-Derived H9C2 Cells and Dexrazoxane Cytoprotective Effect: Contribution of In Vitro 1H-NMR Metabonomics.

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.

Short halt in vaping modifies cardiorespiratory parameters and urine metabolome: a randomized trial.

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 nicotine-vaping (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.

A New Classification Method of Metastatic Cancers Using a 1H-NMR-Based Approach: A Study Case of Melanoma, Breast, and Prostate Cancer Cell Lines.

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.

Hepatic and Renal Toxicity Induced by TiO2 Nanoparticles in Rats: A Morphological and Metabonomic Study.

Titanium dioxide (TiO2) nanoparticles (NPs) are produced abundantly and are frequently used as a white pigment in the manufacture of paints, foods, paper, and toothpaste. Despite the wide ranges of uses, there is a lack of information on the impact of NPs on animal and human health. In the present study, rats were exposed to different doses of TiO2 nanoparticles and sacrificed, respectively, 4 days, 1 month, and 2 months after treatment. Dosage of TiO2 in tissues was performed by ICP-AES and revealed an important accumulation of TiO2 in the liver. The nanoparticles induced morphological and physiological alterations in liver and kidney. In the liver, these alterations mainly affect the hepatocytes located around the centrilobular veins. These cells were the site of an oxidative stress evidenced by immunocytochemical detection of 4-hydroxynonenal (4-HNE). Kupffer cells are also the site of an important oxidative stress following the massive internalization of TiO2 nanoparticles. Enzymatic markers of liver and kidney functions (such as AST and uric acid) are also disrupted only in animals exposed to highest doses. The metabonomic approach allowed us to detect modifications in urine samples already detectable after 4 days in animals treated at the lowest dose. This metabonomic pattern testifies an oxidative stress as well as renal and hepatic alterations.