Jordan R. Witmer

Nonenzymatic displacement of chlorine and formation of free radicals upon the reaction of glutathione with PCB quinones

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.

An assay for the rate of removal of extracellular hydrogen peroxide by cells

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.

Extracellular superoxide dismutase suppresses hypoxia-inducible factor-1α in pancreatic cancer.

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 seems 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 nonmitochondrial 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. Coexpression of the hydrogen peroxide-removing protein glutathione peroxidase 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 into preestablished 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.

Direct spectrophotometric measurement of supra-physiological levels of ascorbate in plasma

Background Supra-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. Methods Here 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. Results Direct 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. Conclusions This 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.

High trans but not saturated fat beverage causes an acute reduction in postprandial vascular endothelial function but not arterial stiffness in humans.

A diet high in trans-fatty acids (TFAs) is associated with a 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 an increase in arterial stiffness, in part from greater reductions in insulin sensitivity, compared with a beverage high in SFAs. Eleven healthy adults (aged 47±5 years) ingested a warm test beverage (520 kcal, 56 g total fat, 5 g carbohydrate, 1 g protein) high in either TFAs or SFAs in a randomized cross-over study. Ingestion of the beverage high in TFAs (p<0.01) but not high in SFAs (p=0.49) decreased endothelial function (brachial artery flow-mediated dilation, mmΔ) at 3–4 hours (p<0.01 for time; p=0.034 for interaction), but did not alter aortic stiffness or carotid β-stiffness. The 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 decreased insulin sensitivity, potentially explaining the higher risk of CVD with a diet high in TFAs.