Ifosfamide - History, efficacy, toxicity and encephalopathy.

In this review we trace the passage of fundamental ideas through 20th century cancer research that began with observations on mustard gas toxicity in World War I. The transmutation of these ideas across scientific and national boundaries, was channeled from chemical carcinogenesis labs in London via Yale and Chicago, then ultimately to the pharmaceutical industry in Bielefeld, Germany. These first efforts to checkmate cancer with chemicals led eventually to the creation of one of the most successful groups of cancer chemotherapeutic drugs, the oxazaphosphorines, first cyclophosphamide (CP) in 1958 and soon thereafter its isomer ifosfamide (IFO). The giant contributions of Professor Sir Alexander Haddow, Dr. Alfred Z. Gilman & Dr. Louis S. Goodman, Dr. George Gomori and Dr. Norbert Brock step by step led to this breakthrough in cancer chemotherapy. A developing understanding of the metabolic disposition of ifosfamide directed efforts to ameliorate its side-effects, in particular, ifosfamide-induced encephalopathy (IIE). This has resulted in several candidates for the encephalopathic metabolite, including 2-chloroacetaldehyde, 2-chloroacetic acid, acrolein, 3-hydroxypropionic acid and S-carboxymethyl-L-cysteine. The pros and cons for each of these, together with other IFO metabolites, are discussed in detail. It is concluded that IFO produces encephalopathy in susceptible patients, but CP does not, by a "perfect storm," involving all of these five metabolites. Methylene blue (MB) administration appears to be generally effective in the prevention and treatment of IIE, in all probability by the inhibition of monoamine oxidase in brain potentiating serotonin levels that modulate the effects of IFO on GABAergic and glutamatergic systems. This review represents the authors' analysis of a large body of published research.

Short-Term Grape Consumption Diminishes UV-Induced Skin Erythema.

Over three million Americans are affected by skin cancer each year, largely as a result of exposure to sunlight. The purpose of this study was to determine the potential of grape consumption to modulate UV-induced skin erythema. With 29 human volunteers, we report that nine demonstrated greater resistance to UV irradiation of the skin after consuming the equivalent of three servings of grapes per day for two weeks. We further explored any potential relationship to the gut-skin axis. Alpha- and beta-diversity of the gut microbiome were not altered, but grape consumption modulated microbiota abundance, enzyme levels, and KEGG pathways. Striking differences in the microbiome and metabolome were discerned when comparing the nine individuals showing greater UV resistance with the 20 non-responders. Notably, three urinary metabolites, 2'-deoxyribonic acid, 3-hydroxyphenyl acetic and scyllo-inositol, were depressed in the UV-resistant group. A ROC curve revealed a 71.8% probability that measurement of urinary 2'-deoxyribonic acid identifies a UV skin non-responder. 2'-Deoxyribonic acid is cleaved from the DNA backbone by reactive oxygen species. Three of the nine subjects acquiring UV resistance following grape consumption showed a durable response, and these three demonstrated unique microbiomic and metabolomic profiles. Variable UV skin sensitivity was likely due to glutathione S-transferase polymorphisms. We conclude that a segment of the population is capable of demonstrating greater resistance to a dermal response elicited by UV irradiation as a result of grape consumption. It is uncertain if modulation of the gut-skin axis leads to enhanced UV resistance, but there is correlation. More broadly, it is reasonable to expect that these mechanisms relate to other health outcomes anticipated to result from grape consumption.

The gut microbiota - A vehicle for the prevention and treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) arises principally against a background of cirrhosis and these two diseases are responsible globally for over 2 million deaths a year. There are few treatment options for liver cirrhosis and HCC, so it is vital to arrest these pathologies early in their development. To do so, we propose dietary and therapeutic solutions that involve the gut microbiota and its consequences. Integrated dietary, environmental and intrinsic signals result in a bidirectional connection between the liver and the gut with its microbiota, known as the gut-liver axis. Numerous lifestyle factors can result in dysbiosis with a change in the functional composition and metabolic activity of the microbiota. A panoply of metabolites can be produced by the microbiota, including ethanol, secondary bile acids, trimethylamine, indole, quinolone, phenazine and their derivatives and the quorum sensor acyl homoserine lactones that may contribute to HCC but have yet to be fully investigated. Gram-negative bacteria can activate the pattern recognition receptor toll-like receptor 4 (TLR4) in the liver leading to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, which can contribute to HCC initiation and progression. The goal in preventing HCC should be to ensure a healthy gut microbiota using probiotic supplements containing beneficial bacteria and prebiotic plant fibers such as oligosaccharides that stimulate their growth. The clinical development of TLR4 antagonists is urgently needed to counteract the pathological effects of dysbiosis on the liver and other organs. Further nutrigenomic studies are required to understand better how the diet influences the gut microbiota and its adverse effects on the liver.

Metabolic Rewiring and the Characterization of Oncometabolites.

The study of low-molecular-weight metabolites that exist in cells and organisms is known as metabolomics and is often conducted using mass spectrometry laboratory platforms. Definition of oncometabolites in the context of the metabolic phenotype of cancer cells has been accomplished through metabolomics. Oncometabolites result from mutations in cancer cell genes or from hypoxia-driven enzyme promiscuity. As a result, normal metabolites accumulate in cancer cells to unusually high concentrations or, alternatively, unusual metabolites are produced. The typical oncometabolites fumarate, succinate, (2R)-hydroxyglutarate and (2S)-hydroxyglutarate inhibit 2-oxoglutarate-dependent dioxygenases, such as histone demethylases and HIF prolyl-4-hydroxylases, together with DNA cytosine demethylases. As a result of the cancer cell acquiring this new metabolic phenotype, major changes in gene transcription occur and the modification of the epigenetic landscape of the cell promotes proliferation and progression of cancers. Stabilization of HIF1α through inhibition of HIF prolyl-4-hydroxylases by oncometabolites such as fumarate and succinate leads to a pseudohypoxic state that promotes inflammation, angiogenesis and metastasis. Metabolomics has additionally been employed to define the metabolic phenotype of cancer cells and patient biofluids in the search for cancer biomarkers. These efforts have led to the uncovering of the putative oncometabolites sarcosine, glycine, lactate, kynurenine, methylglyoxal, hypotaurine and (2R,3S)-dihydroxybutanoate, for which further research is required.

Plasma fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxachola-4,6-dien-24-oic acid indicate severity of liver cirrhosis.

Two 3-oxo-Δ4 fetal bile acids, 3-oxachola-4,6-dien-24-oic acid (1) and 7α-hydroxy-3-oxochol-4-en-24-oic acid (2), occur normally in the human fetus but remain elevated in neonates and children with severe cholestatic liver disease due to an autosomal recessive inborn error of metabolism affecting Δ4-3-oxo-steroid 5ß-reductase (AKR1D1). Relatively little is known about 1 and 2 in adult patients with liver disease. The chemical synthesis of 1 and 2 is therefore described and their quantitation in plasma by ultrarapid chromatography-triple quadrupole mass spectrometry. Plasma concentrations of 1 and 2 were investigated in 25 adult patients with varying degrees of liver cirrhosis with and without hepatocellular carcinoma (HCC). Highly statistically significant correlations (P < 0.0001) were found between severity of liver cirrhosis, determined by the Child-Pugh and MELD scores, with plasma 1 and 2 concentrations, both alone and combined. The presence of HCC did not influence these correlations. Plasma cholic, chenodeoxycholic, deoxycholic, lithocholic or ursodeoxycholic acids, free and as their glycine or taurine conjugates, did not correlate with Child-Pugh or MELD score when corrected for multiple comparisons. These findings demonstrate that plasma levels of fetal bile acids 3-oxachola-4,6-dien-24-oic acid and 7α-hydroxy-3-oxochol-4-en-24-oic acid and likely deteriorating AKR1D1 activity indicate the severity of liver cirrhosis measured by the Child-Pugh and MELD scores.

(2R,3S)-Dihydroxybutanoic Acid Synthesis as a Novel Metabolic Function of Mutant Isocitrate Dehydrogenase 1 and 2 in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) frequently harbors mutations in isocitrate 1 (IDH1) and 2 (IDH2) genes, leading to the formation of the oncometabolite (2R)-hydroxyglutaric acid (2R-HG) with epigenetic consequences for AML proliferation and differentiation. To investigate if broad metabolic aberrations may result from IDH1 and IDH2 mutations in AML, plasma metabolomics was conducted by gas chromatography-mass spectrometry (GC-MS) on 51 AML patients, 29 IDH1/2 wild-type (WT), 9 with IDH1R132, 12 with IDH2R140 and one with IDH2R172 mutations. Distinct metabolic differences were observed between IDH1/2 WT, IDH1R132 and IDH2R140 patients that comprised 22 plasma metabolites that were mainly amino acids. Only two plasma metabolites were statistically significantly different (p < 0.0001) between both IDH1R132 and WT IDH1/2 and IDH2R140 and WT IDH1/2, specifically (2R)-hydroxyglutaric acid (2R-HG) and the threonine metabolite (2R,3S)-dihydroxybutanoic acid (2,3-DHBA). Moreover, 2R-HG correlated strongly (p < 0.0001) with 2,3-DHBA in plasma. One WT patient was discovered to have a D-2-hydroxyglutarate dehydrogenase (D2HGDH) A426T inactivating mutation but this had little influence on 2R-HG and 2,3-DHBA plasma concentrations. Expression of transporter genes SLC16A1 and SLC16A3 displayed a weak correlation with 2R-HG but not 2,3-DHBA plasma concentrations. Receiver operating characteristic (ROC) analysis demonstrated that 2,3-DHBA was a better biomarker for IDH mutation than 2R-HG (Area under the curve (AUC) 0.861; p < 0.0001; 80% specificity; 87.3% sensitivity). It was concluded that 2,3-DHBA and 2R-HG are both formed by mutant IDH1R132, IDH2R140 and IDH2R172, suggesting a potential role of 2,3-DHBA in AML pathogenesis.

Metabolomic and Lipidomic Biomarkers for Premalignant Liver Disease Diagnosis and Therapy.

In recent years, there has been a plethora of attempts to discover biomarkers that are more reliable than α-fetoprotein for the early prediction and prognosis of hepatocellular carcinoma (HCC). Efforts have involved such fields as genomics, transcriptomics, epigenetics, microRNA, exosomes, proteomics, glycoproteomics, and metabolomics. HCC arises against a background of inflammation, steatosis, and cirrhosis, due mainly to hepatic insults caused by alcohol abuse, hepatitis B and C virus infection, adiposity, and diabetes. Metabolomics offers an opportunity, without recourse to liver biopsy, to discover biomarkers for premalignant liver disease, thereby alerting the potential of impending HCC. We have reviewed metabolomic studies in alcoholic liver disease (ALD), cholestasis, fibrosis, cirrhosis, nonalcoholic fatty liver (NAFL), and nonalcoholic steatohepatitis (NASH). Specificity was our major criterion in proposing clinical evaluation of indole-3-lactic acid, phenyllactic acid, N-lauroylglycine, decatrienoate, N-acetyltaurine for ALD, urinary sulfated bile acids for cholestasis, cervonoyl ethanolamide for fibrosis, 16α-hydroxyestrone for cirrhosis, and the pattern of acyl carnitines for NAFL and NASH. These examples derive from a large body of published metabolomic observations in various liver diseases in adults, adolescents, and children, together with animal models. Many other options have been tabulated. Metabolomic biomarkers for premalignant liver disease may help reduce the incidence of HCC.

A Novel Anti-Hepatitis C Virus and Antiproliferative Agent Alters Metabolic Networks in HepG2 and Hep3B Cells.

A series of novel diflunisal hydrazide-hydrazones has been reported together with their anti-hepatitis C virus and antiproliferative activities in a number of human hepatoma cell lines. However, the mechanisms underlying the efficacy of these agents remain unclear. It was chosen to investigate the lead diflunisal hydrazide-hydrazone, 2',4'-difluoro-4-hydroxy-N'- [(pyridin-2-yl)methylidene]biphenyl-3-carbohydrazide (compound 3b), in two cultured human hepatoma cell lines-HepG2 and Hep3B-using a metabolomic protocol aimed at uncovering any effects of this agent on cellular metabolism. One sub-therapeutic concentration (2.5 µM) and one close to the IC50 for antimitotic effect (10 µM), after 72 h in cell culture, were chosen for both compound 3b and its inactive parent compound diflusinal as a control. A GCMS-based metabolomic investigation was performed on cell lysates after culture for 24 h. The intracellular levels of a total of 42 metabolites were found to be statistically significantly altered in either HepG2 or Hep3B cells, only eight of which were affected in both cell lines. It was concluded that compound 3b affected the following pathways-purine and pyrimidine catabolism, the glutathione cycle, and energy metabolism through glycolysis and the pentose phosphate pathway. Although the metabolomic findings occurred after 24 h in culture, significant cytotoxicity of compound 3b to both HepG2 and Hep3B cells at 10 µM were reported not to occur until 72 h in culture. These observations show that metabolomics can provide mechanistic insights into the efficacy of novel drug candidates prior to the appearance of their pharmacological effect.

Metabolomic Analysis of Mice Exposed to Gamma Radiation Reveals a Systemic Understanding of Total-Body Exposure.

Diagnostic markers are needed for accidental or deliberate radiation exposure that could cause acute and chronic radiation toxicity. Biomarkers of temporal, dose-dependent, aging-attenuated and multiple radiation exposures have been previously described by others. However, the physiological origin and biochemical networks that generate these biomarkers and their association at the molecular level have yet to be explored. Hence, the discovery and identification of total-body-irradiation-induced tissue specific biomarkers remains an enormous challenge within radiation biodosimetry research. To determine the tissue level response of total-body exposure (6 Gy), metabolomics analysis was carried out on radiosensitive tissues bone marrow, ileum, liver, muscle and lung as well as serum and on urine within 12 h postirradiation. Differences in the metabolic signatures between the sham and gamma-irradiated groups were analyzed by hydrophilic interaction liquid chromatography (HILIC)-based ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS). A panel of 67 biomarkers identified in radiosensitive tissues and biofluids (serum and urine) at a 6 Gy dose. Among the identified biomarkers, 3-methylglutarylcarnitine (3-MGC) was found to be a novel metabolite in liver, serum and urine that could potentially be an early radiation response marker. The degree of metabolic changes among different tissues showed perturbations in pathways including DNA methylation, energy, nucleic acid, amino acid, glutathione and bile acid metabolism. These results highlight metabolomics as a potential novel approach to understand functional alterations in the metabolome that could be adapted for use in the rapid assessment of radiation exposure and triage protocols in the case of nuclear incidents.

Metabolic profiling by gas chromatography-mass spectrometry of energy metabolism in high-fat diet-fed obese mice.

A novel, selective and sensitive single-ion monitoring (SIM) gas chromatography-mass spectrometry (GCMS) method was developed and validated for the determination of energy metabolites related to glycolysis, the tricarboxylic acid (TCA) cycle, glutaminolysis, and fatty acid ß-oxidation. This assay used N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) containing 1% tert-butyldimethylchlorosilane (TBDMCS) as derivatizing reagent and was highly reproducible, sensitive, specific and robust. The assay was used to analyze liver tissue and serum from C57BL/6N obese mice fed a high-fat diet (HFD) and C57BL/6N mice fed normal chow for 8 weeks. HFD-fed mice serum displayed statistically significantly reduced concentrations of pyruvate, citrate, succinate, fumarate, and 2-oxoglutarate, with an elevated concentration of pantothenic acid. In liver tissue, HFD-fed mice exhibited depressed levels of glycolysis end-products pyruvate and lactate, glutamate, and the TCA cycle intermediates citrate, succinate, fumarate, malate, and oxaloacetate. Pantothenate levels were 3-fold elevated accompanied by a modest increased gene expression of Scl5a6 that encodes the pantothenate transporter SLC5A6. Since both glucose and fatty acids inhibit coenzyme A synthesis from pantothenate, it was concluded that these data were consistent with downregulated fatty acid ß-oxidation, glutaminolysis, glycolysis, and TCA cycle activity, due to impaired anaplerosis. The novel SIM GCMS assay provided new insights into metabolic effects of HFD in mice.

The plasma lipidome in acute myeloid leukemia at diagnosis in relation to clinical disease features.

BACKGROUND: Early studies established that certain lipids were lower in acute myeloid leukemia (AML) cells than normal leukocytes. Because lipids are now known to play an important role in cell signaling and regulation of homeostasis, and are often perturbed in malignancies, we undertook a comprehensive lipidomic survey of plasma from AML patients at time of diagnosis and also healthy blood donors. METHODS: Plasma lipid profiles were measured using three mass spectrometry platforms in 20 AML patients and 20 healthy blood donors. Data were collected on total cholesterol and fatty acids, fatty acid amides, glycerolipids, phospholipids, sphingolipids, cholesterol esters, coenzyme Q10 and eicosanoids. RESULTS: We observed a depletion of plasma total fatty acids and cholesterol, but an increase in certain free fatty acids with the observed decline in sphingolipids, phosphocholines, triglycerides and cholesterol esters probably driven by enhanced fatty acid oxidation in AML cells. Arachidonic acid and precursors were elevated in AML, particularly in patients with high bone marrow (BM) or peripheral blasts and unfavorable prognostic risk. PGF2α was also elevated, in patients with low BM or peripheral blasts and with a favorable prognostic risk. A broad panoply of lipid classes is altered in AML plasma, pointing to disturbances of several lipid metabolic interconversions, in particular in relation to blast cell counts and prognostic risk. CONCLUSIONS: These data indicate potential roles played by lipids in AML heterogeneity and disease outcome. GENERAL SIGNIFICANCE: Enhanced catabolism of several lipid classes increases prognostic risk while plasma PGF2α may be a marker for reduced prognostic risk in AML.

The metabolomic profile of gamma-irradiated human hepatoma and muscle cells reveals metabolic changes consistent with the Warburg effect.

The two human cell lines HepG2 from hepatoma and HMCL-7304 from striated muscle were γ-irradiated with doses between 0 and 4 Gy. Abundant γH2AX foci were observed at 4 Gy after 4 h of culture post-irradiation. Sham-irradiated cells showed no γH2AX foci and therefore no signs of radiation-induced double-strand DNA breaks. Flow cytometry indicated that 41.5% of HepG2 cells were in G2/M and this rose statistically significantly with increasing radiation dose reaching a plateau at ∼47%. Cell lysates from both cell lines were subjected to metabolomic analysis using Gas Chromatography-Mass Spectrometry (GCMS). A total of 46 metabolites could be identified by GCMS in HepG2 cell lysates and 29 in HMCL-7304 lysates, most of which occurred in HepG2 cells. Principal Components Analysis (PCA) showed a clear separation of sham, 1, 2 and 4 Gy doses. Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA) revealed elevations in intracellular lactate, alanine, glucose, glucose 6-phosphate, fructose and 5-oxoproline, which were found by univariate statistics to be highly statistically significantly elevated at both 2 and 4 Gy compared with sham irradiated cells. These findings suggested upregulation of cytosolic aerobic glycolysis (the Warburg effect), with potential shunting of glucose through aldose reductase in the polyol pathway, and consumption of reduced Glutathione (GSH) due to γ-irradiation. In HMCL-7304 myotubes, a putative Warburg effect was also observed only at 2 Gy, albeit a lesser magnitude than in HepG2 cells. It is anticipated that these novel metabolic perturbations following γ-irradiation of cultured cells will lead to a fuller understanding of the mechanisms of tissue damage following ionizing radiation exposure.

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver.

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint(-/-)) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1(-/-) mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1(-/-) mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1(-/-) mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1(-/-) mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.

Metabolic profiling of praziquantel enantiomers.

Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24h after dosing, indicating that the major metabolic reactions encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity toward the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in substrate location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel.

Noninvasive urinary metabolomic profiling identifies diagnostic and prognostic markers in lung cancer.

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

The metabolomic window into hepatobiliary disease.

The emergent discipline of metabolomics has attracted considerable research effort in hepatology. Here we review the metabolomic data for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), alcoholic liver disease (ALD), hepatitis B and C, cholecystitis, cholestasis, liver transplantation, and acute hepatotoxicity in animal models. A metabolomic window has permitted a view into the changing biochemistry occurring in the transitional phases between a healthy liver and hepatocellular carcinoma or cholangiocarcinoma. Whether provoked by obesity and diabetes, alcohol use or oncogenic viruses, the liver develops a core metabolomic phenotype (CMP) that involves dysregulation of bile acid and phospholipid homeostasis. The CMP commences at the transition between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral hepatitis (Phase 1). This CMP is maintained in the presence or absence of cirrhosis (Phase 2) and whether or not either HCC or CCA (Phase 3) develops. Inflammatory signalling in the liver triggers the appearance of the CMP. Many other metabolomic markers distinguish between Phases 0, 1, 2 and 3. A metabolic remodelling in HCC has been described but metabolomic data from all four Phases demonstrate that the Warburg shift from mitochondrial respiration to cytosolic glycolysis foreshadows HCC and may occur as early as Phase 1. The metabolic remodelling also involves an upregulation of fatty acid ß-oxidation, also beginning in Phase 1. The storage of triglycerides in fatty liver provides high energy-yielding substrates for Phases 2 and 3 of liver pathology. The metabolomic window into hepatobiliary disease sheds new light on the systems pathology of the liver.

Metabolomics reveals aging-associated attenuation of noninvasive radiation biomarkers in mice: potential role of polyamine catabolism and incoherent DNA damage-repair.

Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sublethal irradiation was examined in mice using UPLC-ESI-QTOF mass spectrometry. Aging attenuated postexposure elevation in excretions of DNA damage biomarkers as well as N(1)-acetylspermidine. Although N(1)-acetylspermidine and 2'-deoxyuridine elevation was highly correlated in all age groups, xanthine and N(1)-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N(1)-acetylspermidine, 2'-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2'-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2'-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sublethal radiation exposure.

Metabolomics reveals trichloroacetate as a major contributor to trichloroethylene-induced metabolic alterations in mouse urine and serum.

Trichloroethylene (TCE)-induced liver toxicity and carcinogenesis is believed to be mediated in part by activation of the peroxisome proliferator-activated receptor α (PPARα). However, the contribution of the two TCE metabolites, dichloroacetate (DCA) and trichloroacetate (TCA) to the toxicity of TCE, remains unclear. The aim of the present study was to determine the metabolite profiles in serum and urine upon exposure of mice to TCE, to aid in determining the metabolic response to TCE exposure and the contribution of DCA and TCA to TCE toxicity. C57BL/6 mice were administered TCE, TCA, or DCA, and urine and serum subjected to ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based global metabolomics analysis. The ions were identified through searching metabolomics databases and by comparison with authentic standards, and quantitated using multiple reactions monitoring. Quantitative polymerase chain reaction of mRNA, biochemical analysis, and liver histology were also performed. TCE exposure resulted in a decrease in urine of metabolites involved in fatty acid metabolism, resulting from altered expression of PPARα target genes. TCE treatment also induced altered phospholipid homeostasis in serum, as revealed by increased serum lysophosphatidylcholine 18:0 and 18:1, and phosphatidylcholine metabolites. TCA administration revealed similar metabolite profiles in urine and serum upon TCE exposure, which correlated with a more robust induction of PPARα target gene expression associated with TCA than DCA treatment. These data show the metabolic response to TCE exposure and demonstrate that TCA is the major contributor to TCE-induced metabolite alterations observed in urine and serum.

Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the commonest causes of death from cancer. A plethora of metabolomic investigations of HCC have yielded molecules in biofluids that are both up- and down-regulated but no real consensus has emerged regarding exploitable biomarkers for early detection of HCC. We report here a different approach, a combined transcriptomics and metabolomics study of energy metabolism in HCC. A panel of 31 pairs of HCC tumors and corresponding nontumor liver tissues from the same patients was investigated by gas chromatography-mass spectrometry (GCMS)-based metabolomics. HCC was characterized by ∼2-fold depletion of glucose, glycerol 3- and 2-phosphate, malate, alanine, myo-inositol, and linoleic acid. Data are consistent with a metabolic remodeling involving a 4-fold increase in glycolysis over mitochondrial oxidative phosphorylation. A second panel of 59 HCC that had been typed by transcriptomics and classified in G1 to G6 subgroups was also subjected to GCMS tissue metabolomics. No differences in glucose, lactate, alanine, glycerol 3-phosphate, malate, myo-inositol, or stearic acid tissue concentrations were found, suggesting that the Wnt/ß-catenin pathway activated by CTNNB1 mutation in subgroups G5 and G6 did not exhibit specific metabolic remodeling. However, subgroup G1 had markedly reduced tissue concentrations of 1-stearoylglycerol, 1-palmitoylglycerol, and palmitic acid, suggesting that the high serum α-fetoprotein phenotype of G1, associated with the known overexpression of lipid catabolic enzymes, could be detected through metabolomics as increased lipid catabolism. CONCLUSION: Tissue metabolomics yielded precise biochemical information regarding HCC tumor metabolic remodeling from mitochondrial oxidation to aerobic glycolysis and the impact of molecular subtypes on this process.