Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H.

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens. © 2019 International Society for Advancement of Cytometry.

Astragaloside IV Protects Against Oxidized Low-Density Lipoprotein (ox-LDL)-Induced Endothelial Cell Injury by Reducing Oxidative Stress and Inflammation.

BACKGROUND Endothelial injury is the main mechanism of atherosclerosis, and is caused by oxidized low-density lipoprotein (ox-LDL). Astragaloside IV (AS-IV) is the primary active ingredient of the Chinese herb Huangqi, and exhibits antioxidant and anti-inflammatory properties in cardiovascular diseases. This study investigated the protective effect of AS-IV in human umbilical vein endothelial cells (HUVECs). MATERIAL AND METHODS HUVEC cells were induced with ox-LDL to establish an in vitro atherosclerosis model. Then HUVECs were pretreated for 1 h with AS-IV at different concentrations (10, 20, and 50 µM) and then exposed to ox-LDL (100 µg/mL) for 48 h. The cell viability, lactate dehydrogenase (LDH) release, apoptosis, migration, intracellular reactive oxygen species (ROS), and NADPH oxidase activity of HUVECs were measured. qRT-PCR was performed to measure the mRNA expressions of Nrf2, HO-1, TNFalpha, and IL-6. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the supernatant contents of TNFalpha and IL-6. RESULTS Exposure of HUVECs to ox-LDL reduced cell viability and migration, induced apoptosis, and increased intracellular ROS production and NADPH oxidase. Pretreatment with AS-IV (10, 20, and 50 µM) significantly enhanced the cell viability and migration, suppressed LDH release, apoptosis, ROS production, and NADPH oxidase in HUVECs, in a concentration-dependent manner. The AS-IV (50 µM) alone did not show significant differences from control. AS-IV increased mRNA expressions of Nrf2 and HO-1 and decreased mRNA expressions of TNFalpha and IL-6 in the ox-LDL-HUEVC cells. Furthermore, AS-IV reduced supernatant contents of TNFalpha and IL-6. CONCLUSIONS Astragaloside IV prevents ox-LDL-induced endothelial cell injury by reducing apoptosis, oxidative stress, and inflammatory response.

Metabolic flux analysis and the NAD(P)H/NAD(P)+ ratios in chemostat cultures of Azotobacter vinelandii.

BACKGROUND: Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) + ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions. RESULTS: The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L-1 h-1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L-1 h-1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L-1 h-1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH). CONCLUSIONS: We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.

Erythropoietin Decreases the Occurrence of Myocardial Fibrosis by Inhibiting the NADPH-ERK-NF-x03BA;B Pathway.

OBJECTIVES: The aim of this study was to investigate the protective role of erythropoietin (EPO) against myocardial fibrosis (MF). METHODS: Pressure-overloaded rats were established by abdominal aortic constriction, the rats were randomly divided in a double-blind manner into 3 groups (n = 12 for each group): sham-operated rats (sham), operated rats receiving physiological saline (vehicle) and operated rats receiving 4,000 U/kg rhEPO (EPO group). The vehicle and drugs were administered to rats by intraperitoneal injection. In addition, cultured adult rat cardiac fibroblasts (CFs) were utilized to investigate the role of EPO in CF proliferation and collagen secretion. RESULTS: After 4 weeks, besides an increase in blood pressure, myocardial hypertrophy, collagen deposition in the myocardium and decreased cardiac function were observed in the pressure-overloaded rats. The expression of NADPH oxidase (Nox2 and Nox4) and inflammatory cytokines (CD45, F4/80 and MCP-1) was also significantly increased. All these alterations were prevented by EPO. TGF-ß promoted CF proliferation, collagen secretion, ROS production and Nox2/Nox4 expression, which was inhibited by EPO. In addition, the TGF-ß-induced increase of ERK1/2 phosphorylation and NF-x03BA;B expression were attenuated by EPO. CONCLUSION: EPO inhibited rat MF induced by pressure overload and improved myocardial function by decreasing CF proliferation and differentiation via inhibition of the NADPH-ERK-NF-x03BA;B pathway.

Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling.

BACKGROUND: Resistin, an adipocytokine, plays a potential role in cardiovascular disease and may contribute to increased atherosclerotic risk by modulating the activity of endothelial cells. A growing body of evidence suggests that aspirin is a potent antioxidant. We investigated whether aspirin mitigates resistin-induced endothelial dysfunction via modulation of reactive oxygen species (ROS) generation and explored the role that AMP-activated protein kinase (AMPK), a negative regulator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, plays in the suppressive effects of aspirin on resistin-induced endothelial dysfunction. METHODS: Human umbilical vein endothelial cells (HUVECs) were pretreated with various doses of aspirin (10-500 µg/mL) for 2 hours and then incubated with resistin (100 ng/mL) for an additional 48 hours. Fluorescence produced by the oxidation of dihydroethidium (DHE) was used to quantify the production of superoxide in situ; superoxide dismutase (SOD) and catalase activities were determined by an enzymatic assay; and protein levels of AMPK-mediated downstream signaling were investigated by Western blot. RESULTS: Treatment of HUVECs with resistin for 48 hours resulted in a 2.9-fold increase in superoxide production; however, pretreatment with aspirin resulted in a dose-dependent decrease in production of superoxide (10-500 µg/mL; n = 3 experiments; all P < .05). Resistin also suppressed the activity of superoxide dismutase and catalase by nearly 50%; that result, however, was not observed in HUVECs that had been pretreated with aspirin at a concentration of 500 µg/mL. The membrane translocation assay showed that the levels of NADPH oxidase subunits p47(phox)and Rac-1 in membrane fractions of HUVECs were threefold to fourfold higher in cells that had been treated with resistin for 1 hour than in untreated cells; however, pretreatment with aspirin markedly inhibited resistin-induced membrane assembly of NADPH oxidase via modulating AMPK-suppressed PKC-α activation. Application of AMPKα1-specific siRNA resulted in increased activation of PKC-α and p47(phox). In addition, resistin significantly decreased AMPK-mediated downstream Akt/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling and induced the phosphorylation of p38 mitogen-activated protein kinases, which in turn activated NF-κB-mediated inflammatory responses such as the release of interleukin (IL)-6 and IL-8, the overexpression of adhesion molecules, and stimulation of monocytic THP-1 cell attachment to HUVECs (2.5-fold vs control; n = 3 experiments). Furthermore, resistin downregulated eNOS and upregulated inducible NO synthase (iNOS) expression, thereby augmenting the formation of NO and protein nitrosylation. Pretreatment with aspirin, however, exerted significant cytoprotective effects in a dose-dependent manner (P < .05). CONCLUSIONS: Our findings suggest a direct connection between adipocytokines and endothelial dysfunction and provide further insight into the protective effects of aspirin in obese individuals with endothelial dysfunction.

Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells.

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo.

Oxidized phospholipid, 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (PON-GPC), produced in the lung due to cigarette smoking, impairs immune function in macrophages.

INTRODUCTION: Pulmonary innate immunity is impaired in cigarette smokers, because the abundant oxidants present in cigarette smoke (CS) cause injury to lung cells. Pulmonary surfactant is a unique material that is important roles in reducing surface tension in the lung and defending against invading pathogens. Oxidants reportedly cleave surfactant phospholipids, resulting in the production of oxidized phospholipids, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (PON-GPC). Although oxidation of surfactant lipids is thought to be involved in the pathogenesis of smoking-related lung disease, there are no reports on the effect of oxidized surfactant lipid on the immune function of macrophages. We hypothesized that cigarette smoking elevates PON-GPC levels in the lung, and that PON-GPC impairs the innate immune function of macrophages. METHODS: The levels of PON-GPC in bronchoalveolar lavage fluid (BALF) recovered from mice exposed to CS for 2 weeks (n = 7) were measured by liquid chromatography with electrospray-ionization tandem mass spectrometry. The effects of PON-GPC on inducibility of tumor necrosis factor (TNF)-α, nitric oxide (NO), and nicotinamide adenine dinucleotide phosphate (NADP(+)) production, as well as bactericidal activity, were investigated in RAW264.7 cells or primary alveolar macrophages. RESULTS: The levels of PON-GPC in BALF of mice exposed to CS were significantly elevated, compared with those of control mice. PON-GPC attenuated TNF-α, NO, and NADP(+) production in macrophages on stimulation with LPS plus IFN-γ. PON-GPC treatment attenuated the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In addition, PON-GPC reduced the bactericidal activity of RAW264.7 cells. CONCLUSIONS: CS may attenuate innate immunity in the lungs through oxidization of surfactant phospholipids.

cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in Arabidopsis thaliana roots.

3',5'-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na(+)/K(+) ratio and a decrease in gene expression of the plasma membrane (PM) H(+)-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H(2)O(2) or CaCl(2) alleviated the NaCl-induced injury by maintaining a lower Na(+)/K(+) ratio and increasing the PM H(+)-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H(2)O(2) by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H(2)O(2) on ionic homeostasis was abolished when Ca(2+) was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, a Ca(2+) chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H(2)O(2) accumulation in salt stress, and Ca(2+) was necessary in the cGMP-mediated signaling pathway. H(2)O(2), as the downstream component of cGMP signaling pathway, stimulated PM H(+)-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.

Benzocaine-induced methaemoglobinaemia: a case study.

Benzocaine is a widely used topical anaesthetic and has been reported to cause toxic methaemoglobinaemia in otherwise healthy individuals with no predisposing risk factors. This article reports on a rare case of benzocaine-induced methaemoglobinaemia following adenotonsillectomy in a 5-year-old girl. Topical benzocaine was applied orally for the relief of postoperative wound pain on the eighth postoperative day. Two hours after application, generalized cyanosis, mild dyspnoea and some degree of agitation developed. The methaemoglobin level was 38.5%. Treatment with methylene blue was initiated immediately. Symptoms completely disappeared 4 hours after initiation of methylene blue therapy. The further course was uneventful. Therefore, all health professionals should be aware that topical anaesthetics after surgery can induce methaemoglobinaemia in children, even after a prolonged interval, and especially when applied on wound surfaces.

Targeting neuroinflammation to treat cerebral ischemia - The role of TIGAR/NADPH axis.

Cerebral ischemia is a disease of ischemic necrosis of brain tissue caused by intracranial artery stenosis or occlusion and cerebral artery embolization. Neuroinflammation plays an important role in the pathophysiology of cerebral ischemia. Microglia, astrocytes, leukocytes and other cells that release a variety of inflammatory factors involved in neuroinflammation may play a damaging or protective role during the process of cerebral ischemia. TP53-induced glycolysis and apoptotic regulators (TIGAR) may facilitate the production of nicotinamide adenine dinucleotide phosphoric acid (NADPH) via the pentose phosphate pathway (PPP) to inhibit oxidative stress and neuroinflammation. TIGAR can also directly inhibit NF-κB to inhibit neuroinflammation. TIGAR thus protect against cerebral ischemic injury. Exogenous NADPH can inhibit neuroinflammation by inhibiting oxidative stress and regulating a variety of signals. However, since NADPH oxidase (NOX) may use NADPH as a substrate to generate reactive oxygen species (ROS) to mediate neuroinflammation, the combination of NADPH and NOX inhibitors may produce more powerful anti-neuroinflammatory effects. Here, we review the cells and regulatory signals involved in neuroinflammation during cerebral ischemia, and discuss the possible mechanisms of targeting neuroinflammation in the treatment of cerebral ischemia with TIGAR/NADPH axis, so as to provide new ideas for the prevention and treatment of cerebral ischemia.

The effect of tert-butylhydroquinone-induced oxidative stress in MDBK cells using XTT assay: implication of tert-butylhydroquinone-induced NADPH generating enzymes.

Tetrazolium salts such as XTT and MTT are widely used to produce formazan for cell proliferation and cytotoxicity assays through bioreductase activity. However, the XTT assay showed significant increase in MDBK cell viability when cells were treated with both 50 and 100 muM of the pro-oxidant, tert-butylhydroquinone (t-BHQ), although the crystal violet assay showed no cytotoxic effect with these concentrations, and the induction of lipid peroxidation was not observed. We investigated the mechanism of enhancement of XTT substrate reduction after treatment of MDBK cells with t-BHQ, leading to apparent increase in cell viability. t-BHQ caused an increase in absorbance at 340 nm in culture medium, suggesting that t-BHQ increases cellular production and release of NADH and/or NADPH. Although t-BHQ did not change the NADH concentration in cell culture medium, the addition of NADP(+)-dependent glutathione reductase decreased the XTT reduction to the control level, indicating cellular release of NADPH. t-BHQ also increased intracellular glucose-6-phosphate dehydrogenase activity, producing NADPH. Taken together, our findings indicate that t-BHQ treatment activates NADPH generating enzymes such as glucose-6-phosphate dehydrogenase followed by release of NADPH in the cell culture medium, resulting in direct XTT reduction by NADPH.

NADPH oxidase1 inhibition leads to regression of central sensitization during formalin induced acute nociception via attenuation of ERK1/2-NFκB signaling and glial activation.

Role of NADPH oxidase1 in the development of inflammatory pain has been demonstrated by gene knockout studies. Nevertheless, pharmacological inhibition of NOX1 is a requisite approach for therapeutic utility. Recently, we have reported the anti-nociceptive effect of newly identified NOX1 specific inhibitor ML171 (2-acetylphenothiazine). Inhibition of NOX1 resulted in attenuation of nociceptive sensitization during acute inflammatory pain via inhibition of ROS generation and its downstream ERK1/2 activation. However, glial activation accompanying inflammation is closely related to the initiation and maintenance of pain. Peripheral nociceptive inputs activate the primary afferents via release of various chemical mediators which are potentially capable of mediating signals from neuron to glia in DRG and subsequently in spinal cord dorsal horn. The subsequent interactions between neuron and glia contribute to pain hypersensitivity. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in a mouse model of formalin induced acute nociception. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in DRG and dorsal horn of the spinal cord of lumbar region (L3-L5) in a mouse model of formalin induced acute nociception. Intraperitoneal administration of ML171 decreased nociceptive behavioral responses, i.e. the flinch and lick counts, in formalin induced nociceptive mice. Immunofluorescence and Western blot analysis demonstrated decreased levels of nociceptive mediators like p-ERK1/2, p-NFκB p65, Iba1 and GFAP in DRG as well as in spinal cord dorsal horn; supporting anti-nociceptive potential of ML171. Further, co-localization studies showed the neuron-glia crosstalk in tissue dependent manner. ERK1/2 was found to be activated in glia and NFκB in neurons in DRG; whereas in case of spinal cord ERK1/2 was activated in neurons and NFκB in astrocytes. Decrease in nociceptive behavioral response and activation of nociceptive mediators after intraperitoneal administration of ML171 strongly advocate anti-nociceptive potential of ML171. This is the first report demonstrating modulation of ERK1/2-NFκB signaling pathway, glial activation and regulation of neuron-glia crosstalk by NADPH oxidase1 inhibition towards its anti-nociceptive action.

mu-opioid receptor-stimulated synthesis of reactive oxygen species is mediated via phospholipase D2.

We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.

Study of aerobic granulocyte functional activity in the presence of a radiosensitizer (metronidazole).

The in vitro effects of metronidazole on the production of reactive oxygen species by polymorphonuclear (PMN) cells were studied by means of nitroblue tetrazolium and luminol-dependent chemiluminescence. At therapeutic doses of metronidazole (4.98-24.86 microg/mL) significant inhibition of the production of reactive oxygen species was noted in both methods. The inhibitory effect was in a dose-dependent pattern. The data suggest a scavenging mechanism of metronidazole on reactive oxygen species generated by PMN.

Glucose metabolism is accelerated by exposure to t-butylhydroperoxide during NADH consumption in human erythrocytes.

Several mechanisms have been proposed to underlie the events that occur during oxidative damage in red blood cells (RBCs) exposed to reactive oxygen species. This work explores what happens when metabolites related to redox regulation in human RBCs are oxidized to form alkoxyl radical and peroxyl radical as a result of exposure to tert-buthylhydroperoxide (BHP). During exposure to BHP, the glutathione level and the ratio of NADPH to total nicotinamide adenine dinucleotide phosphate (NADPH plus NADP(+)) were significantly decreased. Although alteration in the concentration of monosaccharides metabolized in the pentose phosphate pathway (PPP) was not observed, exposing RBCs to BHP caused the formation of methemoglobin (metHb) and a significant decrease in NADH. Moreover, we detected a significant increase in one of the peaks during BHP exposure by using HPLC with dansyl hydrazine as a prelabel reagent. A complete enzymatic conversion procedure was used to identify the peak as pyruvate based on comparison with standards. These results suggest that the rapid recovery in the level of glutathione and the formation of metHb by BHP require NADPH and NADH consumption. Subsequently, glucose metabolism accelerates to reproduce NADPH and NADH, which results in pyruvate accumulation. Our findings indicate that the level of pyruvate markedly increases upon exposure to a radical-generating oxidant capable of forming metHb. Methemoglobin reductase requires NADH as a co-factor, and oxidized form (NHADP(+)) is reduced via the glycolytic reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. Thus, the overall acceleration of glycolysis induced by BHP is strongly dependent on the NADH reproducing pathway. In addition, the decrease in NADH enhances the increase in pyruvate by inhibiting the conversion of pyruvate to lactate in the presence of lactate dehydrogenase.

Synthesis and biological activity of a 3,4,5-trimethoxybenzoyl ester analogue of epicatechin-3-gallate.

Despite presenting bioavailability problems, tea catechins have emerged as promising chemopreventive agents because of their observed efficacy in various animal models. To improve the stability and cellular absorption of tea polyphenols, we developed a new catechin-derived compound, 3- O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), which has shown significant antiproliferative activity against several cancer cell lines, especially melanoma. The presence of methoxy groups in its ester-bound gallyl moiety drastically decreased its antioxidant and prooxidant properties without affecting its cell-antiproliferative effects, and the data indicated that the 3-gallyl moiety was essential for its biological activity. As regards its action mechanism, we demonstrated that TMECG binds efficiently to human dihydrofolate reductase and down-regulates folate cycle gene expression in melanoma cells. Disruption of the folate cycle by TMECG is a plausible explanation for its observed biological effects and suggests that, like other antifolate compounds, TMECG could be of clinical value in cancer therapy.

Effects of homocysteine on metabolic pathways in cultured astrocytes.

Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes.

Benidipine, an anti-hypertensive drug, inhibits reactive oxygen species production in polymorphonuclear leukocytes and oxidative stress in salt-loaded stroke-prone spontaneously hypertensive rats.

Oxidative stress is associated with exacerbation of renal injuries in hypertension. In clinical studies benidipine hydrochloride (benidipine), a dihydropyridine calcium channel blocker with antioxidant activity, reduced oxidative stress. However, the mechanism of suppression of oxidative stress remains to be fully characterized. Reactive oxygen species production by polymorphonuclear leukocyte plays important pathological roles in hypertension. Therefore, we examined the effects of benidipine both on reactive oxygen species production of human polymorphonuclear leukocytes and oxidative stress of an animal model. Human peripheral polymorphonuclear leukocytes or polymorphonuclear leukocyte-like differentiated HL-60 cells were used to examine effects of benidipine (0.1-30 microM) on formyl-Met-Leu-Phe-induced reactive oxygen species production, calcium mobilization, NADPH oxidase activation and phosphorylation of protein kinase C substrates. High-salt (8% NaCl) loaded stroke-prone spontaneously hypertensive rats were treated with or without benidipine (1, 3, 10 mg/kg/day) for 2 weeks, and thiobarbituric acid reactive substances, a plasma oxidative stress marker, and renal expression of oxidative stress-induced genes were measured. Benidipine concentration-dependently suppressed formyl-Met-Leu-Phe-induced reactive oxygen species production in polymorphonuclear leukocytes more potently than other calcium channel blockers such as amlodipine, azelnidipine, nitrendipine and nifedipine. Benidipine partially inhibited all of intracellular Ca(2+) elevation, protein kinase C activation and NADPH oxidase activation. Salt loading in stroke-prone spontaneously hypertensive rats augmented plasma thiobarbituric acid reactive substances levels; renal dysfunction; and renal expression of transforming growth factor-beta, collagen I and collagen III mRNAs; which were attenuated by benidipine treatment. These results indicate that benidipine prevents the polymorphonuclear leukocyte-derived reactive oxygen species production, which is due at least in part to its antioxidant action and inhibition of Ca(2+)/protein kinase C/NADPH oxidase signaling. The attenuation of reactive oxygen species production might contribute to the drug's reduction of oxidative stress and renal injuries in hypertension.

Increase in intracellular free/bound NAD[P]H as a cause of Cd-induced oxidative stress in the HepG(2) cells.

The present study shows the use of confocal autofluorescence spectroscopy coupled with the time-resolved fluorescence decay analysis to measure changes in FAD/NAD[P]H and free/bound NAD[P]H in HepG(2) cells at 0.5, 1.5, 3 and 4.5h after exposure to cadmium chloride (Cd). These changes were compared to changes in GSSG/GSH and production of reactive oxygen radicals (ROS) production. The results demonstrated that both FAD/NAD[P]H and GSSG/GSH increased significantly upon exposure to Cd. The change in GSSG/GSH occurred as early as 1.5h after treatment while the change in FAD/NAD[P]H did not occur until 3h after exposure. Production of ROS was also increased at 1.5h. The ratio of free/bound NAD[P]H was studied. It was demonstrated that free/bound NAD[P]H increased significantly as early as 0.5h and remained elevated until 4.5h after treatment with Cd. The present study provides novel data to show that changes in NAD[P]H metabolism precedes the increase in ROS production and cellular oxidative stress (increase GSSG/GSH, FAD/NAD[P]H). It is suggested that Cd causes a release of NAD[P]H, an important cofactor for electron transfer, from its normal protein binding sites. This may result in a disruption of the activity of the enzyme and proteins, and may lead to the subsequent toxic events.

Inhibition of palmityl carnitine oxidation in rat liver mitochondria by tert-butyl hydroperoxide.

Mitochondria as an energy generating cell device are very sensitive to oxidative damage. Our previous findings obtained in hepatocytes demonstrated that Complex I of the respiratory chain is more sensitive to oxidative damage than other respiratory chain complexes. We present additional data on isolated mitochondria showing that palmityl carnitine oxidation is strongly depressed at a low (200 microM) tert-butyl hydroperoxide (tBHP) concentration, while oxidation of the flavoprotein-dependent substrate-succinate is not affected and neither is ATP synthesis inhibited by tBHP. In the presence of tBHP, the respiratory control index for palmityl carnitine oxidation is strongly depressed, but when succinate is oxidized the respiratory control index remains unaffected. Our findings thus indicate that flavoprotein-dependent substrates could be an important nutritional factor for the regeneration process in the necrotic liver damaged by oxidative stress.