Photochemistry of (Z)-Isovinylneoxanthobilirubic Acid Methyl Ester, a Bilirubin Dipyrrinone Subunit: Femtosecond Transient Absorption and Stimulated Raman Emission Spectroscopy.

Bilirubin (BR) is an essential metabolite formed by the catabolism of heme. Phototherapy with blue-green light can be applied to reduce high concentrations of BR in blood and is used especially in the neonatal period. In this work, we studied the photochemistry of (Z)-isovinylneoxanthobilirubic acid methyl ester, a dipyrrinone subunit of BR, by steady-state absorption, femtosecond transient absorption, and stimulated Raman spectroscopies. Both the (Z)- and (E)-configurational isomers of isovinylneoxanthobilirubic acid undergo wavelength-dependent and reversible photoisomerization. The isomerization from the excited singlet state is ultrafast (the lifetimes of (Z)- and (E)-isomers were found to be ∼0.9 and 0.1 ps, respectively), and its efficiencies increase with increased photon energy. In addition, we studied sensitized photooxidation of the dipyrrinone subunit by singlet oxygen that leads to the formation of propentdyopents. Biological activities of these compounds, namely, effects on the superoxide production, lipoperoxidation, and tricarboxylic acid cycle metabolism, were also studied. Finally, different photochemical and biological properties of this BR subunit and its structural analogue, (Z)-vinylneoxanthobilirubic acid methyl ester, studied before, are discussed.

Antiproliferative and Cytotoxic Activities of Fluorescein-A Diagnostic Angiography Dye.

Fluorescein is a fluorescent dye used as a diagnostic tool in various fields of medicine. Although fluorescein itself possesses low toxicity, after photoactivation, it releases potentially toxic molecules, such as singlet oxygen (1O2) and, as we demonstrate in this work, also carbon monoxide (CO). As both of these molecules can affect physiological processes, the main aim of this study was to explore the potential biological impacts of fluorescein photochemistry. In our in vitro study in a human hepatoblastoma HepG2 cell line, we explored the possible effects on cell viability, cellular energy metabolism, and the cell cycle. We observed markedly lowered cell viability (≈30%, 75-2400 µM) upon irradiation of intracellular fluorescein and proved that this decrease in viability was dependent on the cellular oxygen concentration. We also detected a significantly decreased concentration of Krebs cycle metabolites (lactate and citrate < 30%; 2-hydroxyglutarate and 2-oxoglutarate < 10%) as well as cell cycle arrest (decrease in the G2 phase of 18%). These observations suggest that this photochemical reaction could have important biological consequences and may account for some adverse reactions observed in fluorescein-treated patients. Additionally, the biological activities of both 1O2 and CO might have considerable therapeutic potential, particularly in the treatment of cancer.

New onset of diabetes mellitus and weight loss as a manifestation of pancreatic cancer.

The case report is about a 68-years-old man who developed a weight loss and diabetes mellitus as early symptoms of pancreatic cancer several months before the diagnosis. Unfortunately, the diagnosis was made too late, at the stage of generalized disease, when symptomatic treatment was the only possible way. The aim of the article is to warn about this possible way of pancreatic cancer manifestation, because only the diagnosis determined in time, when the tumor is localized, allows its resection - the only curative treatment method. For this reason, it is necessary to consider the presence of pancreatic cancer in the case of an atypical manifestation of diabetes.

Induction of fecal cholesterol excretion is not effective for the treatment of hyperbilirubinemia in Gunn rats.

BACKGROUND: Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations. METHODS: To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE. RESULTS: We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. CONCLUSIONS: These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia. IMPACT: Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions.

Diagnostic methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments.

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices. IMPACT: Key message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements. What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood. Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty.

Screening methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments.

Severe neonatal hyperbilirubinemia (SNH) is a serious condition that occurs worldwide. Timely recognition with bilirubin determination is key in the management of SNH. Visual assessment of jaundice is unreliable. Fortunately, transcutaneous bilirubin measurement for screening newborn infants is routinely available in many hospitals and outpatient settings. Despite a few limitations, the use of transcutaneous devices facilitates early recognition and appropriate management of neonatal jaundice. Unfortunately, however, advanced and often costly screening modalities are not accessible to everyone, while there is an urgent need for inexpensive yet accurate instruments to assess total serum bilirubin (TSB). In the near future, novel icterometers, and in particular optical bilirubin estimates obtained with a smartphone camera and processed with a smartphone application (app), seem promising methods for screening for SNH. If proven reliable, these methods may empower outpatient health workers as well as parents at home to detect jaundice using a simple portable device. Successful implementation of ubiquitous bilirubin screening may contribute substantially to the reduction of the worldwide burden of SNH. The benefits of non-invasive bilirubin screening notwithstanding, any bilirubin determination obtained through non-invasive screening must be confirmed by a diagnostic method before treatment. IMPACT: Key message: Screening methods for neonatal hyperbilirubinemia facilitate early recognition and timely treatment of severe neonatal hyperbilirubinemia (SNH). Any bilirubin screening result obtained must be confirmed by a diagnostic method. What does this article add to the existing literature? Data on optical bilirubin estimation are summarized. Niche research strategies for prevention of SNH are presented. Impact: Transcutaneous screening for neonatal hyperbilirubinemia contributes to the prevention of SNH. A smartphone application with optical bilirubin estimation seems a promising low-cost screening method, especially in low-resource settings or at home.

Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment.

Metabolic transformation of cancer cells leads to the accumulation of lactate and significant acidification in the tumor microenvironment. Both lactate and acidosis have a well-documented impact on cancer progression and negative patient prognosis. Here, we report that cancer cells adapted to acidosis are significantly more sensitive to oxidative damage induced by hydrogen peroxide, high-dose ascorbate, and photodynamic therapy. Higher lactate concentrations abrogate the sensitization. Mechanistically, acidosis leads to a drop in antioxidant capacity caused by a compromised supply of nicotinamide adenine dinucleotide phosphate (NADPH) derived from glucose metabolism. However, lactate metabolism in the Krebs cycle restores NADPH supply and antioxidant capacity. CPI-613 (devimistat), an anticancer drug candidate, selectively eradicates the cells adapted to acidosis through inhibition of the Krebs cycle and induction of oxidative stress while completely abrogating the protective effect of lactate. Simultaneous cell treatment with tetracycline, an inhibitor of the mitochondrial proteosynthesis, further enhances the cytotoxic effect of CPI-613 under acidosis and in tumor spheroids. While there have been numerous attempts to treat cancer by neutralizing the pH of the tumor microenvironment, we alternatively suggest considering tumor acidosis as the Achilles' heel of cancer as it enables selective therapeutic induction of lethal oxidative stress.

Wavelength-Dependent Photochemistry and Biological Relevance of a Bilirubin Dipyrrinone Subunit.

Phototherapy is a standard treatment for severe neonatal jaundice to remove toxic bilirubin from the blood. Here, the wavelength-dependent photochemistry of vinylneoxanthobilirubic acid methyl ester, a simplified model of a bilirubin dipyrrinone subunit responsible for a lumirubin-like structural rearrangement, was thoroughly investigated by liquid chromatography and mass and absorption spectroscopies, with the application of a multivariate curve resolution analysis method supplemented with quantum chemical calculations. Irradiation of the model chromophore leads to reversible Z → E photoisomerization followed by reversible photocyclization to a seven-membered ring system (formed as a mixture of diastereomers). Both the isomerization processes are efficient (ΦZE ∼ ΦEZ ∼ 0.16) when irradiated in the wavelength range of 360-410 nm, whereas the E-isomer cyclization (Φc = 0.006-0.008) and cycloreversion (Φ-c = 0.002-0.004) reactions are significantly less efficient. The quantum yields of all processes were found to depend strongly on the wavelength of irradiation, especially when lower energy photons were used. Upon irradiation in the tail of the absorption bands (490 nm), both the isomers exhibit more efficient photoisomerization (ΦZE ∼ ΦEZ ∼ 0.30) and cyclization (Φc = ∼0.07). In addition, the isomeric bilirubin dipyrrinone subunits were found to possess important antioxidant activities while being substantially less toxic than bilirubin.

The Extent of Intracellular Accumulation of Bilirubin Determines Its Anti- or Pro-Oxidant Effect.

BACKGROUND: Severe hyperbilirubinemia can cause permanent neurological damage in particular in neonates, whereas mildly elevated serum bilirubin protects from various oxidative stress-mediated diseases. The present work aimed to establish the intracellular unconjugated bilirubin concentrations (iUCB) thresholds differentiating between anti- and pro-oxidant effects. METHODS: Hepatic (HepG2), heart endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to increasing concentration of bilirubin. iUCB, cytotoxicity, intracellular reactive oxygen species (ROS) concentrations, and antioxidant capacity (50% efficacy concentration (EC50)) were determined. RESULTS: Exposure of SH-SY5Y to UCB concentration > 3.6 µM (iUCB of 25 ng/mg) and >15 µM in H5V and HK2 cells (iUCB of 40 ng/mg) increased intracellular ROS production (p < 0.05). EC50 of the antioxidant activity was 21 µM (iUCB between 5.4 and 21 ng/mg) in HepG2 cells, 0.68 µM (iUCB between 3.3 and 7.5 ng/mg) in SH-SY5Y cells, 2.4 µM (iUCB between 3 and 6.7 ng/mg) in HK2 cells, and 4 µM (iUCB between 4.7 and 7.5 ng/mg) in H5V cells. CONCLUSIONS: In all the cell lines studied, iUCB of around 7 ng/mg protein had antioxidant activities, while iUCB > 25 ng/mg protein resulted in a prooxidant and cytotoxic effects. UCB metabolism was found to be cell-specific resulting in different iUCB.

Hyperbilirubinemia in Gunn Rats is Associated with Decreased Inflammatory Response in LPS-Mediated Systemic Inflammation.

Decreased inflammatory status has been reported in subjects with mild unconjugated hyperbilirubinemia. However, mechanisms of the anti-inflammatory actions of bilirubin (BR) are not fully understood. The aim of this study is to assess the role of BR in systemic inflammation using hyperbilirubinemic Gunn rats as well as their normobilirubinemic littermates and further in primary hepatocytes. The rats were treated with lipopolysaccharide (LPS, 6 mg/kg intraperitoneally) for 12 h, their blood and liver were collected for analyses of inflammatory and hepatic injury markers. Primary hepatocytes were treated with BR and TNF-α. LPS-treated Gunn rats had a significantly decreased inflammatory response, as evidenced by the anti-inflammatory profile of white blood cell subsets, and lower hepatic and systemic expressions of IL-6, TNF-α, IL-1ß, and IL-10. Hepatic mRNA expression of LPS-binding protein was upregulated in Gunn rats before and after LPS treatment. In addition, liver injury markers were lower in Gunn rats as compared to in LPS-treated controls. The exposure of primary hepatocytes to TNF-α with BR led to a milder decrease in phosphorylation of the NF-κB p65 subunit compared to in cells without BR. In conclusion, hyperbilirubinemia in Gunn rats is associated with an attenuated systemic inflammatory response and decreased liver damage upon exposure to LPS.

Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds.

Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 g•L-1 [S. platensis], 60 µM [PCB], and 125 µM [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.

Hypoxia Induces Saturated Fatty Acids Accumulation and Reduces Unsaturated Fatty Acids Independently of Reverse Tricarboxylic Acid Cycle in L6 Myotubes.

Obstructive sleep apnea syndrome, characterized by repetitive episodes of tissue hypoxia, is associated with several metabolic impairments. Role of fatty acids and lipids attracts attention in its pathogenesis for their metabolic effects. Parallelly, hypoxia-induced activation of reverse tricarboxylic acid cycle (rTCA) with reductive glutamine metabolism provides precursor molecules for de novo lipogenesis. Gas-permeable cultureware was used to culture L6-myotubes in chronic hypoxia (12%, 4% and 1% O2) with 13C labelled glutamine and inhibitors of glutamine uptake or rTCA-mediated lipogenesis. We investigated changes in lipidomic profile, 13C appearance in rTCA-related metabolites, gene and protein expression of rTCA-related proteins and glutamine transporters, glucose uptake and lactate production. Lipid content increased by 308% at 1% O2, predominantly composed of saturated fatty acids, while triacylglyceroles containing unsaturated fatty acids and membrane lipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositol) decreased by 20-70%. rTCA labelling of malate, citrate and 2-hydroxyglutarate increased by 4.7-fold, 2.2-fold and 1.9-fold in 1% O2, respectively. ATP-dependent citrate lyase inhibition in 1% O2 decreased lipid amount by 23% and increased intensity of triacylglyceroles containing unsaturated fatty acids by 56-80%. Lactate production increased with hypoxia. Glucose uptake dropped by 75% with progression of hypoxia from 4% to 1% O2. Protein expression remained unchanged. Altogether, hypoxia modified cell metabolism leading to lipid composition alteration and rTCA activation.

The Effects of Bilirubin and Lumirubin on the Differentiation of Human Pluripotent Cell-Derived Neural Stem Cells.

The 'gold standard' treatment of severe neonatal jaundice is phototherapy with blue-green light, which produces more polar photo-oxidation products that are easily excreted via the bile or urine. The aim of this study was to compare the effects of bilirubin (BR) and its major photo-oxidation product lumirubin (LR) on the proliferation, differentiation, morphology, and specific gene and protein expressions of self-renewing human pluripotent stem cell-derived neural stem cells (NSC). Neither BR nor LR in biologically relevant concentrations (12.5 and 25 µmol/L) affected cell proliferation or the cell cycle phases of NSC. Although none of these pigments affected terminal differentiation to neurons and astrocytes, when compared to LR, BR exerted a dose-dependent cytotoxicity on self-renewing NSC. In contrast, LR had a substantial effect on the morphology of the NSC, inducing them to form highly polar rosette-like structures associated with the redistribution of specific cellular proteins (ß-catenin/N-cadherin) responsible for membrane polarity. This observation was accompanied by lower expressions of NSC-specific proteins (such as SOX1, NR2F2, or PAX6) together with the upregulation of phospho-ERK. Collectively, the data indicated that both BR and LR affect early human neurodevelopment in vitro, which may have clinical relevance in phototherapy-treated hyperbilirubinemic neonates.

The Effects of Bilirubin and Lumirubin on Metabolic and Oxidative Stress Markers.

For severe unconjugated hyperbilirubinemia the gold standard treatment is phototherapy with blue-green light, producing more polar photo-oxidation products, believed to be non-toxic. The aim of the present study was to compare the effects of bilirubin (BR) and lumirubin (LR), the major BR photo-oxidation product, on metabolic and oxidative stress markers. The biological activities of these pigments were investigated on several human and murine cell lines, with the focus on mitochondrial respiration, substrate metabolism, reactive oxygen species production, and the overall effects on cell viability. Compared to BR, LR was found to be much less toxic, while still maintaining a similar antioxidant capacity in the serum as well as suppressing activity leading to mitochondrial superoxide production. Nevertheless, due to its lower lipophilicity, LR was less efficient in preventing lipoperoxidation. The cytotoxicity of BR was affected by the cellular glycolytic reserve, most compromised in human hepatoblastoma HepG2 cells. The observed effects were correlated with changes in the production of tricarboxylic acid cycle metabolites. Both BR and LR modulated expression of PPARα downstream effectors involved in lipid and glucose metabolism. Proinflammatory effects of BR, evidenced by increased expression of TNFα upon exposure to bacterial lipopolysaccharide, were observed in murine macrophage-like RAW 264.7 cells. Collectively, these data point to the biological effects of BR and its photo-oxidation products, which might have clinical relevance in phototherapy-treated hyperbilirubinemic neonates and adult patients.

Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma.

Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, we provide a background for vital mitochondrial production of 2HG in breast cancer cells with outcomes towards cancer biology and possible future diagnosis of breast carcinoma.

Fungal Gut Microbiota Dysbiosis and Its Role in Colorectal, Oral, and Pancreatic Carcinogenesis.

The association between bacterial as well as viral gut microbiota imbalance and carcinogenesis has been intensively analysed in many studies; nevertheless, the role of fungal gut microbiota (mycobiota) in colorectal, oral, and pancreatic cancer development is relatively new and undiscovered field due to low abundance of intestinal fungi as well as lack of well-characterized reference genomes. Several specific fungi amounts are increased in colorectal cancer patients; moreover, it was observed that the disease stage is strongly related to the fungal microbiota profile; thus, it may be used as a potential diagnostic biomarker for adenomas. Candida albicans, which is the major microbe contributing to oral cancer development, may promote carcinogenesis via several mechanisms, mainly triggering inflammation. Early detection of pancreatic cancer provides the opportunity to improve survival rate, therefore, there is a need to conduct further studies regarding the role of fungal microbiota as a potential prognostic tool to diagnose this cancer at early stage. Additionally, growing attention towards the characterization of mycobiota may contribute to improve the efficiency of therapeutic methods used to alter the composition and activity of gut microbiota. The administration of Saccharomyces boulardii in oncology, mainly in immunocompromised and/or critically ill patients, is still controversial.

A novel accurate LC-MS/MS method for quantitative determination of Z-lumirubin.

Although phototherapy (PT) is a standard treatment for neonatal jaundice, no validated clinical methods for determination of bilirubin phototherapy products are available. Thus, the aim of our study was to establish a such method for clinical use. To achieve this aim, a LC-MS/MS assay for simultaneous determination of Z-lumirubin (LR) and unconjugated bilirubin (UCB) was conducted. LR was purified after irradiation of UCB at 460 nm. The assay was tested on human sera from PT-treated neonates. Samples were separated on a HPLC system with a triple quadrupole mass spectrometer detector. The instrument response was linear up to 5.8 and 23.4 mg/dL for LR and UCB, respectively, with submicromolar limits of detection and validity parameters relevant for use in clinical medicine. Exposure of newborns to PT raised serum LR concentrations three-fold (p < 0.01), but the absolute concentrations were low (0.37 ± 0.16 mg/dL), despite a dramatic decrease of serum UCB concentrations (13.6 ± 2.2 vs. 10.3 ± 3.3 mg/dL, p < 0.01). A LC-MS/MS method for the simultaneous determination of LR and UCB in human serum was established and validated for clinical use. This method should help to monitor neonates on PT, as well as to improve our understanding of both the kinetics and biology of bilirubin phototherapy products.

SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2.

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.

Chlorophyll-Mediated Changes in the Redox Status of Pancreatic Cancer Cells Are Associated with Its Anticancer Effects.

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10-125 µmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.

Hyperbilirubinemia Protects against Aging-Associated Inflammation and Metabolic Deterioration.

Mild constitutive hyperbilirubinemia is associated with a reduced risk of cardiovascular diseases, diabetes, and cancer. Since these pathologies are associated with aging, inflammation, and oxidative stress, we investigated whether hyperbilirubinemia interferes with ROS homeostasis in cell cultures and with inflammation, senescence, and mitochondrial dysfunction in aged rats. Human embryonic kidney cells and rat primary fibroblasts showed a dose-dependent decrease in the ratio of oxidized/reduced glutathione, intracellular H2O2 levels, and mitochondrial ROS production, with increasing bilirubin concentrations in the culture media. Compared to their normobilirubinemic siblings, aged hyperbilirubinemic Gunn rats showed significantly smaller amounts of visceral fat, better glucose tolerance, and decreased serum levels of proinflammatory cytokines TNFα, IL-1ß, and IL-18. Simultaneously, livers from Gunn rats showed decreased expression of senescence markers and cell cycle inhibitors p21 and p16. Mitochondria from aged Gunn rats showed higher respiration and lower H2O2 production compared to controls. In conclusion, we demonstrated that mildly elevated serum bilirubin is generally associated with attenuation of oxidative stress and with better anthropometric parameters, decreased inflammatory status, increased glucose tolerance, fewer signs of cellular senescence, and enhanced mitochondrial function in aged rats.