G3BP1 promotes DNA binding and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key sensor responsible for cytosolic DNA detection. Here we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for DNA sensing and efficient activation of cGAS. G3BP1 enhanced DNA binding of cGAS by promoting the formation of large cGAS complexes. G3BP1 deficiency led to inefficient DNA binding by cGAS and inhibited cGAS-dependent interferon (IFN) production. The G3BP1 inhibitor epigallocatechin gallate (EGCG) disrupted existing G3BP1-cGAS complexes and inhibited DNA-triggered cGAS activation, thereby blocking DNA-induced IFN production both in vivo and in vitro. EGCG administration blunted self DNA-induced autoinflammatory responses in an Aicardi-Goutières syndrome (AGS) mouse model and reduced IFN-stimulated gene expression in cells from a patient with AGS. Thus, our study reveals that G3BP1 physically interacts with and primes cGAS for efficient activation. Furthermore, EGCG-mediated inhibition of G3BP1 provides a potential treatment for cGAS-related autoimmune diseases.

CUE domain-containing protein 2 promotes the Warburg effect and tumorigenesis.

Cancer progression depends on cellular metabolic reprogramming as both direct and indirect consequence of oncogenic lesions; however, the underlying mechanisms are still poorly understood. Here, we report that CUEDC2 (CUE domain-containing protein 2) plays a vital role in facilitating aerobic glycolysis, or Warburg effect, in cancer cells. Mechanistically, we show that CUEDC2 upregulates the two key glycolytic proteins GLUT3 and LDHA via interacting with the glucocorticoid receptor (GR) or 14-3-3ζ, respectively. We further demonstrate that enhanced aerobic glycolysis is essential for the role of CUEDC2 to drive cancer progression. Moreover, using tissue microarray analysis, we show a correlation between the aberrant expression of CUEDC2, and GLUT3 and LDHA in clinical HCC samples, further demonstrating a link between CUEDC2 and the Warburg effect during cancer development. Taken together, our findings reveal a previously unappreciated function of CUEDC2 in cancer cell metabolism and tumorigenesis, illustrating how close oncogenic lesions are intertwined with metabolic alterations promoting cancer progression.

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia.

INTRODUCTION: Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics. OBJECTIVES: Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia. METHODS: Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC-MS based untargeted and stable isotope assisted metabolomic techniques. RESULTS: Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells. CONCLUSION: The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.

Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention.

Identification of differential metabolic characteristics between tumor and normal tissue from colorectal cancer patients by gas chromatography-mass spectrometry.

Colorectal cancer (CRC) is one of the most common human malignancies and encompasses cancers of the colon and rectum. Although the gold-standard colonoscopy screening method is effective in detecting CRC, this method is invasive and can result in severe complications for patients. The purpose of this study was to determine differences in metabolites between CRC and matched adjacent nontumor tissues from CRC patients, to identify potential biomarkers that may be informative and developed screening methods. Metabolomic analysis was performed on clinically localized CRC tissue and matched adjacent nontumor tissue from 20 CRC patients. Unsupervised analysis, supervised analysis, univariate analysis and pathway analysis were used to identify potential metabolic biomarkers of CRC. The levels of 25 metabolites in CRC tissues were significantly altered compared with the matched adjacent nontumor tissues. Four metabolites (lactic acid, alanine, phosphate and aspartic acid) demonstrated good area under the curve of receiver-operator characteristic with acceptable sensitivities and specificities, indicating their potential as important biomarkers for CRC. Alterations of amino acid metabolism and enhanced glycolysis may be major factors in the development and progression of CRC. Lactic acid, alanine, phosphate, and aspartic acid could be effective diagnostic indicators for CRC.

GC-MS-based plasma metabolomic investigations of morphine dependent rats at different states of euphoria, tolerance and naloxone-precipitated withdrawal.

Long-term or excessive application of morphine leads to tolerance and addiction, which hindered its conventional applications as a drug. Although tremendous progress has been made on the mechanisms of morphine, crucial evidence elaborating the neurobiological basis of tolerance and dependence is still lacking. To further explore the physiological adaptions during morphine's application, a systematic screening of small molecules in blood has been carried out. The plasma of morphine dependent rats was collected at different time points with or without naloxone treatment, and was analyzed by gas chromatography-mass spectrometry (GC-MS). Partial least squares discriminate analysis (PLS-DA) and the Student's t Tests with the false discovery rate (FDR) correction were conducted on the normalized data for the distinction of groups and the identification of the most contributed metabolites. Clear separation is observed between different treatments, and 29 out of 41 metabolites changes significantly compared with the corresponding controls. The concentration of threonine, glycine, serine, beta-d-glucose and oxalic acid are consistently changed in all morphine treated groups compared with controls. Through this experiment we find characteristic metabolites in different dependent states and discuss the possible compensation effects. The interpretation of these metabolites would throw light on the biological effects of morphine and reveal the possibilities to become marker of morphine addiction.

Combination of biotransformation and metabolomics reveals tolfenpyrad-induced hepatocytotoxicity.

Tolfenpyrad (TFP) is an extensively used pesticide that inevitably leads to human exposure to both TFP and its transformation product residues. However, the biotransformation of TFP in humans has not been elucidated, and the toxicity of TFP along with its biotransformation products remains largely unknown. In this study, the biotransformation process of TFP was investigated using human liver microsomes and human hepatic cells. Endogenous metabolic changes in the cells were studied to investigate the hepatocytotoxicity of TFP at environmentally relevant concentrations. Fourteen phase I biotransformation products and four phase II TFP products were characterized, among which twelve products were identified for the first time. The oxidative product tolfenpyrad-benzoic acid (PT-CA) was particularly abundant and stable. Further hepatotoxicity assessments and metabolic studies demonstrated comparable metabolic profiles for TFP and PT-CA in HepG2 cells, with both significantly disrupting purine and glutathione metabolism. These processes are closely associated with oxidative stress, mitochondrial damage, and cell death. Our results provide novel perspectives on the biotransformation, metabolism, and hepatotoxicity of TFP, thereby highlighting the non-negligible toxicity of its crucial biotransformation product PT-CA in environmental risk assessments.

Metabonomic analysis of liver tissue from BALB/c mice with D-galactosamine/lipopolysaccharide-induced acute hepatic failure.

BACKGROUND: Compared with biofluids, target tissues and organs more directly reflect the pathophysiological state of a disease process. In this study, a D-galactosamine (GalN) / lipopolysaccharide (LPS)-induced mouse model was constructed to investigate metabonomics of liver tissue and directly characterize metabolic changes in acute liver failure (ALF). METHODS: After pretreatment of liver tissue, gas chromatography coupled to time-of-flight mass spectrometry (GC/TOFMS) was used to separate and identify the liver metabolites. Partial least squares--discriminant analysis models were constructed to separate the ALF and control groups and to find those compounds whose liver levels differed significantly between the two groups. RESULTS: Distinct clustering was observed between the ALF and control mice. Fifty-eight endogenous metabolites were identified. Compared with the control mice, many metabolites, including sugars, amino acids, fatty acids, and organic acids, underwent significant changes in the ALF group, some of which differed from changes observed in plasma. Significant reduction of some important intermediate metabolites indicates that production of ketone bodies, the tricarboxylic acid and urea cycles, gluconeogenesis, glycolysis and pentose phosphate pathways are inhibited after GalN/LPS administration. CONCLUSIONS: GC/TOFMS can be a powerful technique to perform metabonomic studies of liver tissue. GalN/LPS treatment can severely disturb substance metabolism in the liver, with different effects on metabolites compared with those observed in the plasma.

Extraction, derivatization, and determination of metabolome in human macrophages.

A GC/TOF-MS was applied to the determination of metabolites in human macrophages. The extraction conditions and quenching conditions were investigated and optimized. The results indicated that 0.9% w/v sodium chloride at 4°C was the most favorable condition to quench macrophage, 1 mL 50% ACN for 2 min in ice bath was the optimal condition to extract 5 × 10(6) cells. Two hundred six peaks could be detectable with peak area over 50 using this method. Among these peaks, 45 peaks with the similarity over 700 were identified using standard compounds for endogenous metabolites. Thirty-seven out of 45 metabolites could be quantified directly by this method. Twenty metabolites were selected randomly, and 15 amino acids were used for method validation. The correlation coefficients (r) ranging from 0.9902 to 0.9977 were obtained for 15 amino acids in the range of 2.35-150.20 µg/mL. The intraday and interday precisions were lower than 19.90% for the randomly selected 20 endogenous metabolites. Using this development method and multivariate statistical technique, several potential biomarkers were found from human macrophages infected by different Mycobacterium tuberculosis (M. tuberculosis) strains. The results suggest that the method could be applied to the investigation of the pathogenicity of tuberculosis.

A one-step solid-phase extraction with UHPLC-MS/MS for fast and accurate determination of multi-class veterinary drugs in animal muscles.

Excessive use of veterinary drugs in livestock growth poses a threat to food safety. It is, however, challenging to quantify these multi-class veterinary drugs within animal muscles, because of their varied physicochemical properties. In this work, we presented a simple, efficient and sensitive method for the simultaneous determination of multi-class veterinary drugs with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The method involves a highly efficient extraction using a EDTA (pH 7)-ACN (30:70, v/v) solvent system, followed by a one-step solid-phase extraction cleanup approach with PRiME HLB sorbent (Reversed-phase N-vinylpyrrolidone and divinylbenzene copolymer). For all the analytes, over a wide range of polarity, satisfactory recoveries were obtained between 70% and 120%, with relative standard deviations <15%. Excellent sensitivities were achieved with the limits of quantification ranging from 0.2 µg/kg to 3.0 µg/kg. This developed method provides a new targeted strategy for the analysis of multi-class veterinary drugs in muscle matrices.

Separation and structural elucidation of a novel vardenafil analogue as an adulterant in a natural health care dietary supplement.

A novel vardenafil analogue was identified in dietary supplement as an adulterant in herbal formulations. The structure of this analogue was elucidated using HRMS, NMR after extraction from the pulverized powder. It was named morphardenafil as a morpholine ring has replaced the N-ethyl piperazine ring in vardenafil. A tablet of this dietary supplement contained about 50 mg of unspecified morphardenafil, which is 2.5 - 20-times the prescriptive dosage of Levetra, the commercial formulation of the vardenafil monohydrochloride salt in the market and probably places unwary consumers at risk for potentially serious adverse effects or drug-drug interaction (DDI).

Metabolomics Analysis of Chronic Exposure to Dimethylarsenic Acid in Mice and Toxicity Assessment of Organic Arsenic in Food.

Dimethylarsenic acid is a natural organic arsenic in seafood and one of the important metabolites of inorganic arsenic, which is generally considered to have low or no toxicity. However, due to the controversy of the toxicity of organic arsenic, the food safety standard of organic arsenic has not been established until now, and the effects of organic arsenic on chronic toxicity and the overall metabolic level of animals are rarely reported. In our study, 64 female C57BL/6 mice were exposed to different concentrations of dimethylarsenic acid with water intake. Fifteen metabolites in serum were detected to be altered with the increase of arsenic concentration and exposure time. Dimethylarsenic acid exposure significantly affected the overall metabolic level of mice, and the related effects were not recovered shortly after the suspension of arsenic intake. Although arsenic was excreted largely in urine and feces, continued dimethylarsenic acid exposure could still lead to arsenic accumulation in the liver and kidneys and cause mild nephritis in mice.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry for the Analysis of Complex Compounds in Serum and Its Application in Accurate Detection of Early Arsenic Exposure.

With the acceleration of industrialization, environmental arsenic pollution is threatening human health. However, by the time clinical symptoms appear, arsenic toxicity has usually caused irreversible damage to the body, so it is important to establish a rapid and accurate screening method for early arsenic exposure. In this work, 32 female C57BL/6 mice were exposed to different concentrations of inorganic arsenic in drinking water for a week. By analyzing the changes in serum, more than 20 compounds were detected to increase or decrease with the increase of arsenic intake. The abnormal increase in inosine, xanthine, xanthosine, and hypoxanthine and the abnormal purine pathway were found at the same time. Dimethylarsenic acid, an important inorganic arsenic metabolite in the body, was also found in serum. Combined with statistical analysis, early arsenic exposure can be easily and quickly detected, and the potential health risks of short-term exposure can be revealed simultaneously.

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography-electrospray tandem mass spectrometry.

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra(®) MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r(2) = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.

Differential metabolic responses in breast cancer cell lines to acidosis and lactic acidosis revealed by stable isotope assisted metabolomics.

Extracellular acidosis is considered as a hallmark of most human tumors, which plays an important role in promoting tumor malignant and aggressive phenotype in tumorigenesis. Acidosis and lactic acidosis can induce different responses in tumors. Previous studies have associated the response to lactic acidosis of tumors with good survival outcomes. In this study, we investigated the metabolomic changes in triple negative and luminal subtype breast cancer cell lines in response to acidosis and lactic acidosis. Our results showed that acidosis results in the reduction of cell viability and glycolysis in breast cancer cells, which is reversely correlated with the malignancy of cell lines. Under lactic acidosis, this reduction is reversed slightly. Untargeted metabolomic profiling revealed that glutaminolysis and fatty acid synthesis in cancer cells under acidosis are increased, while TCA cycle and glycolysis are decreased. Under lactic acidosis, the pentose phosphate pathway and acetate release are increased in MDA-MB-231 cells. The current results uncovered the different metabolic responses of breast cancer cells to acidosis and lactic acidosis, demonstrating the power of combined untargeted and stable isotope assisted metabolomics in comprehensive metabolomic analysis.

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry.

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: (1)H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, beta-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF-MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.

Rapid detection of conotoxin SO(3) in serum using Cu-chelated magnetic beads coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A novel method based on Cu-chelated magnetic beads (Cu-Magbeads) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the rapid detection of peptide toxins in serum. The peptides in the serum were efficiently adsorbed by the Cu-Magbeads, eluted with methanol solution, and assayed by MALDI-TOF-MS. Specific peptides were identified according to their characteristic mass-to-charge ratio values. Conotoxin SO(3), a synthesized peptide, was used as a model to evaluate the method. Conotoxin SO(3) was detected in human serum, as well as bovine and murine serum, with a detection sensitivity in the low femtomole range. The assay was performed within 40 min, without the need for a specific antibody or an expensive reagent. It shows potential for future use in clinical and emergency rescue practice because of its simplicity, high speed, and high sensitivity.

Dynamic metabonomic analysis of BALB/c mice with different outcomes after D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure.

Fulminant hepatic failure (FHF) is one of the most challenging gastrointestinal emergencies encountered in clinical practice. Early identification of patients with FHF who need liver transplantation is very important. To construct a prediction model for the early diagnosis and prognosis of FHF, we studied the dynamics of metabolic intermediates and metabolic profiles with a D-galactosamine (GalN)/lipopolysaccharide (LPS)-treated BALB/c mouse model of FHF. Levels of plasma metabolites were quantified with gas chromatography/time-of-flight mass spectrometry, and data were processed with partial least squares discriminant analysis (PLS-DA). Distinct clustering differences were observed 5 and 6 hours after GalN/LPS treatment between mice that survived and those that died, but there were no differences between these groups 4 hours after treatment. Five hours after treatment, plasma levels of some metabolites differed significantly between the survival, dead, and control groups. Ketogenesis and the tricarboxylic acid cycle were inhibited in both the survival and dead groups, but in the dead group, the urea cycle was also inhibited, and glycolysis was elevated. PLS-DA indicated that principal component weighting was greatest for plasma levels of phosphate, beta-hydroxybutyrate, urea, glucose, and lactate. The Y-predicted scatter plot in the partial least squares (PLS) model assigned samples to the survival or dead groups with an a priori cutoff of 0.10 with 100% sensitivity and specificity. Similar results were observed in 11 FHF patients with different outcomes. In conclusion, the PLS model based on metabonomic analysis can be used to predict outcomes well, and plasma levels of phosphate, beta-hydroxybutyrate, urea, glucose, and lactate may constitute a set of markers for the early diagnosis and prognosis of FHF.

Metabonomic study on the toxicity of Hei-Shun-Pian, the processed lateral root of Aconitum carmichaelii Debx. (Ranunculaceae).

The purpose of this paper was to study the effects of Hei-Shun-Pian, the processed lateral root of Aconitum carmichaelii Debx. (Ranunculaceae), on the metabolic profile of rats, to discuss the mechanism of toxicology and to find out the potential biomarkers of the toxic effects. Twenty male Wistar rats were divided into four groups (n=5) and each group were administered orally with the decoction of Hei-Shun-Pian (88.1g/kg per day, 35.6g/kg per day, 17.6g/kg per day) or equal volume of drinking water respectively for 14 days. Urine of every 24-h and the plasma of the last day were collected for NMR experiments, and then analyzed by multivariate analysis methods. Decreases in urinary excretion of taurine and trimethylamine-N-oxide (TMAO) and increases in urinary levels of citrate, 2-oxoglutarate (2-OG), succinate and hippurate were observed in the high and medium dosed groups at the early stage of the dosing period. Taurine level increased at the later stage of the dosing period to the normal value, and then even to a value higher than that of the control group at the end of the experiment. No metabolic differences were observed between low dosed and control groups until the later stage of the dosing period when a slight increase in urinary taurine level was observed, suggesting a cumulative effect. These results suggest the toxic effect of Hei-Shun-Pian on rat heart in a dose dependent manner.

Quantitative determination of quinocetone and its metabolites in chicken plasma by liquid chromatography/tandem mass spectrometry.

A new method has been established for the simultaneous determination of the new veterinary drug quinocetone (3-methyl-2-cinnamoyl-N-1,4-dioxyquioxaline; QTN) and its 2 metabolites de-monoxy-quinocetone (3-methyl-2-cinnamoyl-N-1-monoxyquinoxaline; DMO-QTN) and de-dioxy-quinocetone (3-methyl-2-cinnamoyl-N-quinoxaline; DDI-QTN) in chicken plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The detection was performed using electrospray ionization in multiple reaction monitoring mode. The analysis of the linearity, accuracy, and precision of the method is described. The limits of detection and quantification of the LC/MS/MS method used for testing QTN, DMO-QTN, and DDI-QTN were 0.002 and 0.008 microg/mL; 0.002 and 0.008 microg/mL; and 0.003 and 0.010 microg/mL, respectively. The method was validated and can be used in future pharmacokinetic studies.