The reactions of glutathione (GSH) with polychlorinated biphenyl (PCB) quinones having different degrees of chlorination on the quinone ring were examined. EPR spectroscopy and MS revealed 2 types of reactions yielding different products: (i) a nonenzymatic, nucleophilic displacement of chlorine on the quinone ring yielding a glutathiylated conjugated quinone and (ii) Michael addition of GSH to the quinone, a 2-electron reduction, yielding a glutathiylated conjugated hydroquinone. The pKa of parent hydroquinone decreased by 1 unit as the degree of chlorination increased. This resulted in a corresponding increase in the oxidizability of these chlorinated hydroquinones. The reaction with oxygen appears to be first-order each in ionized hydroquinone and dioxygen, yielding hydrogen peroxide stoichiometrically. The generation of semiquinone radicals, superoxide, and hydroxyl radicals was observed by EPR; however, the mechanisms and yields vary depending on the degree of the chlorination of hydroquinone/quinone and the presence or absence of GSH. Our discovery that chlorinated quinones undergo a rapid, nonenzymatic dechlorination upon reaction with GSH opens a different view on mechanisms of metabolism and the toxicity of this class of compounds.dechlorination ͉ EPR ͉ superoxide ͉ semiquinone ͉ hydrogen peroxide P olychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants (1, 2). Many PCBs are poorly biodegradable and thus accumulate and are amplified through the food chain (3). Several congeners, including lower chlorinated PCBs, act as tumor promoters (4). Mechanisms involving reactive metabolites have been proposed in the initiating action of PCBs. Lower-chlorinated biphenyls can be metabolized by cytochrome P450 1A1, 1A2, 2B1/2B2, via arene oxides to mono-and dihydroxylated intermediates and further to quinones (5, 6). Quinones are reactive electrophiles, which can readily undergo Michael addition with a multitude of intracellular nucleophiles, such as amino acids, glutathione (GSH), proteins, and nucleic acids. Quinones can also be reduced to highly reactive semiquinone radicals, which in turn, lead to the formation of reactive oxygen species (ROS), causing oxidative stress and toxicity (7).GSH is the major nonprotein sulfhydryl in cells (8). As a nucleophile, it will conjugate with electrophiles both enzymatically and nonenzymatically; conjugation with various xenobiotics and/or their metabolic intermediates typically converts them into less toxic products. These reactions can be complex because of the possible involvement of a variety of free radicals (9, 10). Here, we evaluate the redox properties and mechanisms of the reactions of GSH with PCB quinones. We have discovered that certain chlorinated quinones not only undergo Michael addition reactions with GSH, but also GSH can nonenzymatically displace chlorine on the quinone ring. Results and DiscussionGlutathione can react with quinone rings via Michael addition forming corresponding hydroquinones (11, 12), Q ϩ GSH 3 GS-H 2 Q.[1]These hydroquinon...

ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of Als2 ؊/؊ mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from Als2 ؊/؊ mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and Als2 ؊/؊ mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of Als2 ؊/؊ mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.ALS ͉ alsin ͉ knockout mouse ͉ motor neuron ͉ Rab5

Pharmacological ascorbate, via its oxidation, has been proposed as a pro-drug for the delivery of H2O2 to tumors. Pharmacological ascorbate decreases clonogenic survival of pancreatic cancer cells, which can be reversed by treatment with scavengers of H2O2. The goal of this study was to determine if inhibitors of intracellular hydroperoxide detoxification could enhance the cytotoxic effects of ascorbate. Human pancreatic cancer cells were treated with ascorbate alone or in combination with inhibitors of hydroperoxide removal including the glutathione disulfide reductase inhibitor 1,3 bis (2-chloroethyl)-1-nitrosurea (BCNU), siRNA targeted to glutathione disulfide reductase (siGR), and 2-deoxy-D-glucose (2DG), which inhibits glucose metabolism. Changes in the intracellular concentration of H2O2 were determined by analysis of the rate of aminotriazole-mediated inactivation of endogenous catalase activity. Pharmacological ascorbate increased intracellular H2O2 and depleted intracellular glutathione. When inhibitors of H2O2 metabolism were combined with pharmacological ascorbate the increase in intracellular H2O2 was amplified and cytotoxicity was enhanced. We conclude that inclusion of agents that inhibit cellular peroxide removal produced by pharmacological ascorbate leads to changes in the intracellular redox state resulting in enhanced cytotoxicity.

Cells have a wide range of capacities to remove extracellular hydrogen peroxide. At higher concentrations of extracellular H2O2 (micromolar) the rate of removal can be approximated by a rate equation that is first-order in the concentration of H2O2 and cell density. Here we present a method to determine the observed rate constant for the removal of extracellular H2O2 on a per cell basis. In the cells examined, when exposed to 20 μM H2O2, these rate constants (kcell) range from 0.46×10−12 s−1 cell−1 L for Mia-PaCa-2 cells (human pancreatic carcinoma) to 10.4×10−12 s−1 cell−1 L for U937 cells (human histiocytic lymphoma). For the relatively small red blood cell kcell=2.9×10−12 s−1 cell−1 L. These rate constants, kcell, can be used to compare the capacity of cells to remove higher levels of extracellular H2O2, as often presented in cell culture experiments. They also provide a means to estimate the rate of removal of extracellular H2O2, rate=−kcell [H2O2] (cells L−1), and the half-life of a bolus of H2O2. This information is essential to optimize experimental design and interpret data from experiments that expose cells to extracellular H2O2.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that governs cellular responses to reduced oxygen availability by mediating crucial homeostatic processes and is a major survival determinant for tumor cells growing in a low oxygen environment. Clinically, HIF-1α appears to be important in pancreatic cancer, as HIF-1α correlates with metastatic status of the tumor. Extracellular superoxide dismutase (EcSOD) inhibits pancreatic cancer cell growth by scavenging non-mitochondrial superoxide. We hypothesized that EcSOD overexpression leads to changes in the O2•−/H2O2 balance modulating the redox status affecting signal transduction pathways. Both transient and stable overexpression of EcSOD suppressed the hypoxic accumulation of HIF-1α in human pancreatic cancer cells. This suppression of HIF-1α had a strong inverse correlation with levels of EcSOD protein. Co-expression of the hydrogen peroxide-removing protein, glutathione peroxidase (GPx), did not prevent the EcSOD-induced suppression of HIF-1α, suggesting that the degradation of HIF-1α observed with high EcSOD overexpression is possibly due to a low steady-state level of superoxide. Hypoxic induction of vascular endothelial growth factor (VEGF) was also suppressed with increased EcSOD. Intratumoral injections of an adenoviral vector containing the EcSOD gene (AdEcSOD) into pre-established pancreatic tumors suppressed both VEGF levels and tumor growth. These results demonstrate that the transcription factor HIF-1α and its important gene target VEGF can be modulated by the antioxidant enzyme EcSOD.

BackgroundSupra-physiological concentrations of ascorbate, vitamin C, in blood, greater than 1 mM, achieved through intravenous administration (IV), are being tested in clinical trials to treat human disease, e.g. cancer. These trials need information on the high levels of ascorbate achieved in blood upon IV administration of pharmacological ascorbate so appropriate clinical decisions can be made.MethodsHere we demonstrate that in the complex matrix of human blood plasma supra-physiological levels of ascorbate can be quantified by direct UV spectroscopy with use of a microvolume UV–vis spectrophotometer.ResultsDirect quantitation of ascorbate in plasma in the range of 2.9 mM, lower limit of detection, up to at least 35 mM can be achieved without any sample processing, other than centrifugation.ConclusionsThis approach is rapid, economical, and can be used to quantify supraphysiological blood levels of ascorbate associated with the use of IV administration of pharmacological ascorbate to treat disease.

A diet high in trans-fatty acids (TFAs) is associated with higher risk of cardiovascular disease (CVD) than a diet high in saturated fatty acids (SFAs) but the mechanisms remain unclear. We hypothesized that a beverage high in TFAs would cause a larger reduction in postprandial endothelial function and increase in arterial stiffness in part from greater reductions in insulin sensitivity compared with a beverage high in SFAs. Eleven healthy adults (age=47±5 yrs) ingested a warm test beverage (520kcal, 56g total fat, 5g carbohydrate, 1g protein) high in either TFAs or SFAs in a randomized cross-over study. Ingestion of the high TFAs (P<0.01), but not SFAs (P=0.49) beverage decreased endothelial function (brachial artery flow-mediated dilation, mmΔ) at 3 hours (P<0.01 for time; p=0.034 for interaction), but did not alter aortic stiffness or carotid β-stiffness. Homeostasis model of insulin resistance (interaction P=0.062) tended to decrease after SFAs but not TFAs. A beverage high in TFAs but not SFAs results in a postprandial reduction in endothelial function and a trend for insulin sensitivity potentially explaining the higher risk of CVD with a diet high in TFAs.

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