Andrew T. Canada

The production of reactive oxygen species by dietary flavonols.

Flavonols are a group of naturally occurring compounds which are widely distributed in nature where they are found glycosylated primarily in vegetables and fruits. A number of studies have found both anti- and prooxidant effects for many of these compounds. The most widely studied because of their ubiquitous nature have been quercetin, a B-dihydroxylated and myricetin, a B-trihydroxylated flavonol. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. Studying a number of factors affecting autooxidation, we found the rate of autooxidation for both quercetin and myricetin to be highly pH dependent with no autooxidation detected for quercetin at physiologic pH. Both the addition of iron for the two flavonols and the addition of iron followed by SOD for quercetin at physiologic pH. Both the addistantially. Neither kaempferol, a monohydroxylated flavonol nor rutin, a glycosylated quercetin showed any ability to autooxidize. The results with rutin differ from what we expected based on the B-ring structural similarity to quercetin. The autooxidation of quercetin and myricetin was further studied by electron spin resonance spectroscopy (ESR). Whereas quercetin produced a characteristic DMPO-OH radical, it was not detected below a pH of 9. However, the addition of iron allowed the signal to be detected at a pH as low as 8.0. On the other hand, myricetin autooxidation yielded a semiquinone signal which upon the addition of iron, converted to a DMPO-OH signal detected at a pH of 7.5. In a microsome-NADPH system, quercetin produced an increase in oxygen utilization and with ESR, an ethanol-derived radical signal which could be completely suppressed by catalase indicating the dependence of the signal on hydrogen peroxide. These studies demonstrate that the extracellular production of active oxygen species by dietary flavonols is not likely to occur in vivo but the potential for intracellular redox cycling may have toxicologic significance.

Hydrogen Peroxide Production by Monoamine Oxidase during Ischemia-Reperfusion in the Rat Brain

Monoamine oxidase (MAO) as a source of hydrogen peroxide (H2O2) was evaluated during ischemiareperfusion in vivo in the rat brain. H2O2 production was assessed with and without inhibition of MAO during and after 15 min of ischemia. Metabolism of H2O2 by catalase during ischemia and reperfusion was measured in forebrain homogenates using aminotriazole (ATZ), an irreversible H2O2-dependent inhibitor of catalase. Catecholamine and glutathione concentrations in forebrain were measured with and without MAO inhibitors. During ischemia, forebrain blood flow was reduced to 8% of baseline and H2O2 production decreased as measured at the microperoxisome. During reperfusion, a rapid increase in H2O2 generation occurred within 5 min as measured by a threefold increase in oxidized glutathione (GSSG). The H2O2-dependent rates of ATZ inactivation of catalase between control and ischemia–reperfusion were similar, indicating that H2O2 was more available to glutathione peroxidase than to catalase in this model. MAO inhibitors eliminated the biochemical indications of increased H2O2 production and increased the catecholamine concentrations. Mortality was 67% at 48 h after ischemiareperfusion, and there was no improvement in survival after inhibition of MAO. We conclude that MAO is an important source of H2O2 generation early in brain reperfusion, but inhibition of the enzyme does not improve survival in this model despite ablating H2O2 production.

The protective role of glutathione in chlorothalonil-induced stoxicity to channel catfish

Abstract The purpose of this investigation was to examine the nature of the protective role of glutathione (GSH) in liver and gills of channel catfish (Ictalurus punctatus) exposed to chlorothalonil (2,4,5,6 tetrachloroiso-phthalonitrile). In an initial experiment, the acute toxicity of chlorothalonil was enhanced three-fold after channel catfish were depleted of liver and gill GSH by l -buthionine S.R-sulfoximine (BSO. 1000 mg/kg i.p.) and diethyl maleate (DEM, 0.6 ml/kg i.p.), indicating that GSH plays a critical role in mitigating acute chlorothalonil toxicity in this species. In a subsequent experiment, sublethal exposure to chlorothalonil elicited significant increases (p 0.05). These results indicate that the mechanism of GSH induction in liver and gills of catfish exposed to chlorothalonil involves increased cysteine uptake and also increased GCS activity. The results of this study support the hypothesis that simultaneous exposure to multiple environmental chemicals that utilize or deplete tissue GSH may predis pose fish to acute toxicity.

STIMULATION OF SECRETION BY THE T84 COLONIC EPITHELIAL CELL LINE WITH DIETARY FLAVONOLS

Flavonols are dietary compounds widely distributed in plants and characterized by a 2-phenyl-benzo(alpha)pyrane nucleus possessing hydroxyl and ketone groups at positions 3 and 4, respectively. Kaempferol, quercetin, and myricetin are flavonols that are further mono-, di-, or trihydroxylated on the phenyl ring, respectively. To test whether these ingested flavonols might exert a direct secretory effect on intestinal epithelial cells, monolayers of the T84 colonocyte cell line were mounted in Ussing chambers and examined for ion transport response. Twenty minutes after addition of 100 microM quercetin to either the serosal or mucosal side, the short-circuit current change was maximal at 16.6 microA/cm2. Kaempferol was less potent than quercetin, while myricetin and glycosylated quercetin (rutin) did not induce secretion. The secretion induced by quercetin did not seem to be mediated by the reactive oxygen species generated by quercetin through auto-oxidation and/or redox cycling (superoxide, hydrogen peroxide, and the hydroxyl radical) because it was neither enhanced by iron, nor inhibited by desferroxamine B or catalase (alone or in combination with superoxide dismutase). Like vasoactive intestinal peptide, quercetin induced a secretory response that was inhibited by barium chloride and bumetanide, and which exhibited synergism with carbachol. Quercetin also stimulated a modest increase in intracellular cAMP levels and the phosphorylation of endogenous protein substrates for cAMP-dependent protein kinase. Thus, quercetin is a potent stimulus of colonocyte secretion that resembles secretagogues which act via a cAMP-mediated signaling pathway.

Catalase: its role in xenobiotic detoxification.

Catalase activity is found primarily in peroxisomes although in some species and in some organ systems, cytosolic catalase also may be involved in intracellular oxidant stress protection. Toxicology studies with repeat exposures to xenobiotics producing hydrogen peroxide either directly or indirectly generally indicate that the organisms develop resistance to the toxin (adaptation). This adaptation would result from induction of catalase activity in most target organs. The induction of hepatic peroxisomes accompanied by less than compensatory increase in catalase activity is now recognized as suggesting a potential for hepatotoxic and hepatocarcinogenic effects. Although these effects seem to also require mobilization of fatty acids, it is not clear if such mobilization is an absolute requirement. As would be expected, there are great differences among species in catalase activity thus making animal-human extrapolations difficult. Finally, with the exception of premature and neonatal animals, age-related variations in catalase activity do not seem to be large enough to have toxicological relevance. However, in old animals, their apparent inability to replace lost catalase activity after repeated stress may have major significance in explaining observed young-old differences in toxicity resulting from oxidant stress.

Modulation of human colonic T84 cell secretion by hydrogen peroxide

Hydrogen peroxide (H2O2) is a reactive oxygen species that can be produced in the digestive tract by inflammatory cells or during reperfusion following ischemia. To evaluate a possible direct effect of H2O2 on epithelial secretory cells, well-differentiated colonic T84 cells were grown to confluence on permeable membranes and studied in Ussing chambers. In this model, where the measured short-circuit current (Isc) reflects electrogenic secretion, we observed that H2O2 stimulated a concentration-dependent and transient secretory response: 5.5 mM H2O2 produced a peak Isc of 12.4 microA/cm2 after 4 min, 2.2 mM H2O2 a peak Isc of 7.9 microA/cm2 after 4 min, and 1.1 mM H2O2 a peak Isc of 5.5 microA/cm2 after 16 min (N = 5). When 97 experiments using 5.5 mM H2O2 were reviewed, the mean peak Isc response was 8.9 +/- 0.5 microA/cm2. A similar secretory response was elicited whether H2O2 was added to the serosal, to the mucosal, or simultaneously to both sides of the T84 cell monolayer. This secretory response reflected transcellular chloride secretion because it was inhibited by the depletion of chloride in the medium and by the suppression of the Na+,K+,2Cl- co-transporter activity necessary for the chloride gradient driving chloride secretion. When T84 cell monolayer resistance was studied, 5.5 mM H2O2 produced a transient decrease in resistance, reflecting transcellular chloride secretion, and a gradual decline in resistance (75% of the initial value after 55 min). The secretory response to H2O2 was increased 2-fold in T84 cells maximally stimulated with 10 nM vasoactive intestinal peptide (VIP), a neuropeptide which acts via cAMP, demonstrating synergism between the two agents. In contrast, the secretory responses produced by H2O2 and carbachol, which acts through the Ca2+ pathway, were additive. A late inhibitory effect of H2O2 was also observed: in cells previously treated with 5.5 mM H2O2, the subsequent secretory responses to either VIP or carbachol were partially inhibited. These secretory effects were specific for the oxidant properties of H2O2 because they were inhibited by 450 U/mL catalase and by 5 mM dithiothreitol, but were unaffected by 50 microM deferoxamine B or Fe3+. H2O2 may be a potential modulator of intestinal or colonic secretion in certain pathologic conditions such as inflammation or ischemia-reperfusion.

Superoxide dismutase: Its role in xenobiotic detoxification

Since the discovery of superoxide dismutase in 1969, the role of this enzyme in modulating cellular toxicity of superoxide has been well established. Experimentally, cellular damage from compounds or exposures which produce superoxide extracellularly can be prevented or modified by pretreating a cell or organ system with SOD. Likewise, induction of intracellular SOD by exposing the cell system to various types of nonlethal stress will impart resistance or tolerance to further exposures to oxidant and nonoxidant stresses which would normally be toxic. The differences in intracellular SOD activity based on species, age, and organ variability can have a major impact on the interpretation of toxicology data, particularly extrapolation to human toxicology. An awareness of the importance of SOD to the toxicity of xenobiotics which produce superoxide, either directly or indirectly, will enable those conducting toxicology studies to better understand and interpret their results.

Long-term propofol infusion for refractory postoperative nausea : a case report with quantitative propofol analysis

P ropofol possesses direct antiemetic properties at subanesthetic doses. Low-dose infusions of propofol have been successfully used to treat chemotherapy-induced nausea and vomiting refractory to conventional therapies (1). Bolus doses of propofol have been effective in treating postoperative nausea and vomiting in the recovery room (2). Serum samples were not obtained for quantitative propofol analysis in either of these reports. The authors obtained a serum propofol level in a patient with severe postoperative nausea and vomiting who was treated with a subanesthetic propofol infusion for 5 days.

Glutathione depletion increases the cytotoxicity of melphalan to PC-3, an androgen-insensitive prostate cancer cell line

SummaryProstate cancer that is androgen-insensitive is unresponsive to a wide spectrum of cytotoxic agents, including all of the alkylating agents. Since a major pathway for the detoxification of the alkylating agents is conjugation with glutathione (GSH), GSH depletion has proved to be effective as a technique to restore melphalan sensitivity in melphalan-resistant cancer cell lines. However, the effect of GSH depletion has not been widely studied in tumor cell lines that have not developed resistance due to previous exposure to alkylating agents. Thus, we decided to investigate GSH depletion as a technique to increase melphalan cytotoxicity to PC-3 cells, an androgen-insensitive prostate cancer line. After 2 and 6 h incubation with 0.25–5 μm melphalan, virtually no effect was observed on either clonogenic lethality or MTT viability until 5 μm exposures. A 24-h incubation of the cells with 100 μm buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced the GSH content by 70%–75%. Following GSH depletion, an increase in clonogenic lethality and a decrease in MTT viability occurred after exposure to concentrations as low as 0.25 μm. The dose modification factor ranged from 2.9 after 2 h incubation to 4.5 at 6 h. These results provide support for additional studies in prostate cancer for further investigation of GSH depletion as a technique to induce sensitivity to alkylating agents in this chemotherapy-resistant tumor.

The evaluation of Escherichia coli as a model for oxidant stress in mammalian hepatocytes: Role of glutathione

Among bacteria, Escherichia coli are unique because they contain an amount of glutathione (GSH) comparable to that of mammalian cells. Thus, this bacterium has been suggested as a model for oxidant stress in mammalian systems. Two common strains of E. coli, ATCC 29682, a B strain, and AB 1157, a K-12 strain, were exposed to paraquat (PQ) or t-butyl hydroperoxide (TBH) and the effect on GSH, growth, and lethality was assessed. Exposure of both strains to 5 mM PQ resulted in an 80% decrease in GSH. Exposure to 5 mM TBH resulted in a 31% decrease in GSH in the K-12 strain and an 80% decrease in the B strain. No correlation was found between the GSH decrease in either strain and the PQ or TBH growth inhibitory effects. TBH exposures increased oxidized GSH (GSSG) export. However, no increase in intracellular GSSG or protein-mixed disulfides was found after exposure to either oxidant nor was GSSG secreted following PQ. After failing to inhibit GSH synthesis with buthionine sulfoximine, a B strain GSH-deficient mutant [RCI-1] was constructed. There was no difference in the growth and lethality responses to the oxidants between GSH-deficient and -sufficient strains. GSH supplementation with N-acetylcysteine or L-2-oxothiazolidine decreased the sensitivity of the E. coli B strain to the growth inhibitory but not the lethality effects of TBH. The lack of a correlation of changes in GSH with either oxidant-induced growth inhibitory or lethality effects, the presence of catalase in the cytoplasm not peroxisomes, and the absence of glutathione peroxidase are limitations to the value of this bacterium as a model for mammalian oxidant stress.