Rex Munday

Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin

Aims/hypothesis. The role of selective uptake and alkylation in the diabetogenic action of streptozotocin was investigated in bioengineered RINm5F insulin-producing cells, with different expression levels of the glucose transporter GLUT2, by comparing the toxicity of streptozotocin with that of four chemically related alkylating compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N nitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). Methods. GLUT2 expressing RINm5F cells were generated through stable transfection of the rat glucose transporter GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results. Cells expressing the glucose transporter GLUT2 were much more susceptible to streptozotocin toxicity than control cells due to the uptake of streptozotocin by this specific glucose transporter. In contrast, the GLUT2 expression had no effect upon the toxicity of MNU, ENU, MMS or EMS. Although the latter substances are, like streptozotocin, cytotoxic through their ability to cause DNA alkylation, they are not diabetogenic because they are not taken up through the glucose transporter GLUT2. Conclusion/interpretation. Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research. [Diabetologia (2000) 43: 1528–1533]

Reduced glutathione in combination with superoxide dismutase as an important biological antioxidant defence mechanism

We propose that an important function of superoxide dismutase is to prevent radical-mediated chain oxidation of GSH, thereby enabling GSH to act physiologically as a free radical scavenger without concomitant oxidative stress to the cell. Through this action, the combination of SOD and GSH plays a significant role in intracellular antioxidant defence

Glutathione-mediated redox cycling of alloxan: Mechanisms of superoxide dismutase inhibition and of metal-catalyzed OH formation

The mechanism of the reaction between alloxan and GSH has been studied in the presence and absence of superoxide dismutase. Excess GSH reduced alloxan to dialuric acid, which underwent subsequent autoxidation, thus establishing a redox cycle in which O2 and GSH in excess of the alloxan concentration were consumed. The major reaction products were H2O2 and GSSG. At each cycle, a small fraction of the alloxan reacted with GSH to form a 305 nm-absorbing adduct that gradually accumulated. In the presence of SOD, alloxan was reduced by GSH, but increasing concentrations of GSH progressively inhibited redox cycling as shown by decreased rates of O2 uptake and GSH oxidation. With GSH: alloxan or dialuric acid molar ratios of greater than 8-10:1, redox cycling was almost completely suppressed. A mechanism based on known reactions of GSH and dialuric acid is proposed. Alloxan and GSH, with an iron chelate present as catalyst, caused the hydroxylation of salicylate, an indicator of hydroxyl radical production. Hydroxylation was inhibited by catalase but not by superoxide dismutase, and it is attributed to the Fenton reaction in which the ferric catalyst is reduced by dialuric acid.

Inhibition of Urinary Bladder Carcinogenesis by Broccoli Sprouts

Isothiocyanates are a well-known class of cancer chemopreventive agents, and broccoli sprouts are a rich source of several isothiocyanates. We report herein that dietary administration to rats of a freeze-dried aqueous extract of broccoli sprouts significantly and dose-dependently inhibited bladder cancer development induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. The incidence, multiplicity, size, and progression of bladder cancer were all inhibited by the extract, while the extract itself caused no histologic changes in the bladder. Moreover, inhibition of bladder carcinogenesis by the extract was associated with significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes that are important protectants against oxidants and carcinogens. Isothiocyanates are metabolized to dithiocarbamates in vivo, but dithiocarbamates readily dissociate to isothiocyanates. We found that >70% of the isothiocyanates present in the extract were excreted in the urine as isothiocyanate equivalents (isothiocyanates + dithiocarbamates) in 12 h after a single p.o. dose, indicating high bioavailability and rapid urinary excretion. In addition, the concentrations of isothiocyanate equivalents in the urine of extract-treated rats were 2 to 3 orders of magnitude higher than those in plasma, indicating that the bladder epithelium, the major site of bladder cancer development, is most exposed to p.o. dosed isothiocyanate. Indeed, tissue levels of isothiocyanate equivalents in the bladder were significantly higher than in the liver. In conclusion, broccoli sprout extract is a highly promising substance for bladder cancer prevention and the isothiocyanates in the extract are selectively delivered to the bladder epithelium through urinary excretion.

Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells

Aims/hypothesis. The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells. Methods. RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (SOD) were exposed to S-nitroso-N-acetyl-d,l-penicillamine (SNAP), sodium nitroprusside (SNP) and 3 morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay. Results. Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity. Conclusion/interpretation. The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both. [Diabetologia (1999) 42: 849–855]

Importance of the GLUT2 glucose transporter for pancreatic beta cell toxicity of alloxan

Abstract Aims/hypothesis. We investigated the importance of the low affinity GLUT2 glucose transporter in the diabetogenic action of alloxan in bioengineered RINm5F insulin-producing cells with different expressions of the transporter. Methods. GLUT2 glucose transporter expressing RINm5F cells were generated through stable transfection of the rat GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results. Cells expressing the GLUT2 transporter were susceptible to alloxan toxicity due to the uptake of alloxan by this specific glucose transporter isoform. The extent of the toxicity of alloxan was dependent upon the GLUT2 protein expression in the cells.The lipophilic alloxan derivative, butylalloxan, was toxic also to non-transfected control cells. Expression of the GLUT2 glucose transporter caused only a marginal increase in the toxicity of this substance. Butylalloxan, unlike alloxan itself, is not diabetogenic in vivo although, like the latter substance, it is beta-cell toxic in vitro through its ability to generate free radicals during redox cycling with glutathione. Conclusion/interpretation. Our results are consistent with the central importance of selective uptake of alloxan through the low affinity GLUT2 glucose transporter for the pancreatic beta-cell toxicity and diabetogenicity of this substance. Redox cycling and the subsequent generation of oxygen free radicals leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as alloxan diabetes in experimental diabetes research.

Relative activities of organosulfur compounds derived from onions and garlic in increasing tissue activities of quinone reductase and glutathione transferase in rat tissues

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is due to the organosulfur compounds in these vegetables and that these substances act through induction of phase II detoxification enzymes. In the present studies, we have compared the ability of diallyl sulfide, diallyl disulfide, and diallyl trisulfide, compounds that are derived from garlic, to increase the activity of the phase II enzymes quinone reductase and glutathione transferase in a variety of rat tissues. We have also examined the onion-derived substances, dipropyl sulfide, dipropyl disulfide, dipropenyl sulfide, and dipropenyl disulfide, under identical conditions. Diallyl trisulfide and diallyl disulfide were potent inducers of the phase II enzymes. Dipropenyl disulfide was much less active, while little effect on enzyme activity was seen in animals dosed with dipropyl disulfide. Diallyl sulfide and dipropyl sulfide were weak inducers of quinone reductase and glutathione transferase, but dipropenyl sulfide was very active, with an effect similar to that of diallyl disulfide. It is possible that diallyl disulfide and diallyl trisulfide are important in the anticancer action of garlic, while dipropenyl sulfide could be involved in the beneficial action of onions.

Isolation, structural determination and acute toxicity of pinnatoxins E, F and G.

Pinnatoxins and pteriatoxins are a group of cyclic imine toxins that have hitherto only been isolated from Japanese shellfish. As with other cyclic imine shellfish toxins, pinnatoxins cause rapid death in the mouse bioassay for lipophilic shellfish toxins, but there is no evidence directly linking these compounds to human illness. We have identified the known pinnatoxins A (1) and D (6), and the novel pinnatoxins E (7), F (8) and G (5), in a range of shellfish and environmental samples from Australia and New Zealand using LC-MS. After isolation from the digestive glands of Pacific oysters, the structures of the novel pinnatoxins were determined by mass spectrometry and NMR spectroscopy, and their LD(50) values were evaluated by ip administration to mice. Examination of the toxin structures, together with analysis of environmental samples, suggests that pinnatoxins F and G are produced separately in different dinoflagellates. Furthermore, it appears probable that pinnatoxin F (8) is the progenitor of pinnatoxins D (6) and E (7), and that pinnatoxin G (6) is the progenitor of both pinnatoxins A-C (1 and 2) and pteriatoxins A-C (3 and 4), via metabolic and hydrolytic transformations in shellfish.

Low Doses of Diallyl Disulfide, a Compound Derived From Garlic, Increase Tissue Activities of Quinone Reductase and Glutathione Transferase in the Gastrointestinal Tract of the Rat

Diallyl disulfide (DADS), a substance that is formed from the organosulfur compounds present in garlic, is known to increase tissue activities of the phase II detoxification enzymes quinone reductase (QR) and glutathione transferase (GT) in animals. In previous experiments, however, high doses of DADS were employed and only a limited range of tissues were examined. In the present studies, increased activities of QR and GT were recorded in the forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon, liver, kidneys, spleen, heart, lungs, and urinary bladder of rats given DADS over a wide range of dose levels. Large variations in response were recorded among the different organs, with forestomach, duodenum, and jejunum being the most sensitive to enzyme induction by DADS. In these organs, significant increases in QR activity were observed at a dose of only 0.3 mg/kg/day. Such a dose level is close to that which may be achieved through human consumption of garlic, suggesting that induction of phase II enzymes may contribute to the protection that is afforded by this vegetable against cancer of the gastrointestinal tract in humans.

Dialuric acid autoxidation: effects of transition metals on the reaction rate and on the generation of active oxygen species

The autoxidation of dialuric acid, a process which is believed to be of crucial importance in the diabetogenic action of alloxan, was found to be strongly catalysed by copper, iron and manganese. Superoxide radical and hydrogen peroxide were generated in both the uncatalysed and the metal-catalysed reactions. In contrast, hydroxyl radical was formed during dialuric acid autoxidation only in the presence of added iron salts. Production of the latter radical was strongly inhibited by catalase but only weakly by superoxide dismutase, implying that the metal-catalysed Haber-Weiss reaction is of comparatively little importance in hydroxyl radical generation from dialuric acid.