Original Contributions
Effect of superoxide dismutase, catalase, chelating agents, and free radical scavengers on the toxicity of alloxan to isolated pancreatic islets in vitro

https://doi.org/10.1016/S0891-5849(98)00325-6Get rights and content

Abstract

The effect of superoxide dismutase, catalase, metal-chelating agents and hydroxyl radical scavengers on the toxicity of alloxan to isolated ob/ob mouse pancreatic islets in vitro has been compared with the reported ability of such substances to protect against alloxan diabetes in vivo. Superoxide dismutase and catalase protected β-cells of isolated pancreatic islets against alloxan cytotoxicity, as did the hydroxyl radical scavengers dimethyl sulfoxide (DMSO) and butanol. However, 1,3-dimethylurea and thiourea, that are recognised as effective hydroxyl radical scavengers and that protect animals against the diabetogenic effects of alloxan, were without effect. Similarly, desferrioxamine, that inhibits hydroxyl radical formation from alloxan in chemically defined systems, did not protect against alloxan toxicity. Diethylenetriamine pentaacetic acid, which does not inhibit hydroxyl radical formation from alloxan, also gave no significant protection. The results indicate a role for superoxide radical and hydrogen peroxide in the mechanism of toxicity of alloxan but do not support the involvement of the hydroxyl radical in this process. Alternative explanations must be sought for the ability of hydroxyl radical scavengers and metal-chelating agents to protect against alloxan toxicity in vivo.

Introduction

The pyrimidine derivative, alloxan, causes diabetes in animals through its ability to destroy the insulin-producing β-cells of the pancreas [1], [2], [3], [4]. The mechanism of alloxan diabetes has been the subject of many investigations, and it is now generally accepted that free radicals are involved in the initiation of the damage that ultimately leads to β-cell death [4].

Free radical production from alloxan has been unequivocally demonstrated in vitro. Alloxan is reduced by biological reducing agents such as cysteine, glutathione and ascorbate to dialuric acid; the latter readily autoxidizes, establishing a redox cycle for generation of superoxide radical and hydrogen peroxide [5], [6], [7]. Furthermore, in the presence of an iron catalyst, hydroxyl radical is formed during dialuric acid autoxidation and in the reaction between alloxan and glutathione [5], [8], [9], [10]. There is also evidence that free radicals are produced during the interaction of alloxan with isolated β-cells in vitro. Chemiluminescence, an indicator of oxygen free radical formation, has been observed in such cells incubated with alloxan [11].

It has been suggested [12], [13] that the free radical species responsible for alloxan toxicity is the hydroxyl radical, formed via the metal-catalysed Haber–Weiss reaction. In this process, ferric iron is reduced by superoxide, with subsequent oxidation of ferrous iron by hydrogen peroxide forming hydroxyl radical: Fe3++O2•−→Fe2++O2 Fe2++H2O2→Fe3++OH+OH If this is true, it would be expected that destruction of superoxide radical or hydrogen peroxide by superoxide dismutase (SOD) or catalase (CAT) would ameliorate alloxan toxicity, as would substances able to scavenge the hydroxyl radical. Furthermore, metal chelating agents able to inactivate the iron essential for hydroxyl radical production from alloxan would also protect.

Many experiments have been conducted on the effects of radical scavengers and chelating agents on alloxan toxicity in vitro and in vivo. Both SOD and CAT protected against alloxan toxicity in dispersed pancreatic β-cells [14] and SOD diminished the toxicity of alloxan to β-cells in isolated islets [15]. Furthermore, SOD, when given to animals before challenge with alloxan, decreased the severity of the induced diabetes [16], [17]. Many compounds that are recognised as hydroxyl radical scavengers, such as dimethyl sulphoxide (DMSO), aliphatic alcohols and urea derivatives, have similarly been shown to decrease the diabetogenicity of alloxan in vivo [12], [13], [18], [19], [20], [21], [22]. Among chelating agents, DTPA [23] and 1,10-phenanthroline [24] have been reported to protect, as has EDTA in some experiments [22] but not in others [19]. The potent iron chelator, desferrioxamine, has been claimed in one report to decrease alloxan toxicity [25], although in another study [23] an increased severity of diabetes was seen in animals pre-treated with this substance.

Although the above evidence has been taken as support for the involvement of the hydroxyl radical in the initiation of alloxan diabetes, it must be accepted that there are many anomalies. In vitro, hydroxyl radical formation during autoxidation of dialuric acid or via redox cycling of alloxan in the presence of glutathione does not involve reduction of iron by superoxide, and SOD has no effect on hydroxyl radical formation in these systems [5], [8]. DTPA and EDTA do not prevent hydroxyl radical formation via the metal-catalysed Haber–Weiss reaction [26] and these substances actually promote hydroxyl radical formation from dialuric acid and alloxan in vitro [5], [8]. The equivocal results with desferrioxamine also argue against a role for iron in the induction of alloxan diabetes, because this substance strongly inhibits iron-mediated free radical formation [25], including the generation of hydroxyl radical from dialuric acid [8]. Furthermore, if iron was important in promoting alloxan toxicity, it would be expected that prior administration of salts of this metal would increase the severity of its harmful effects. This is not the case [27].

The interpretation of in vivo experiments is often complicated by artefacts resulting from the metabolism of administered compounds or from physiological effects that would not be recognised in vitro. Indeed, there is good evidence that the protection given by butanol and dimethylthiourea against alloxan-induced diabetes is mediated not by their reactivity toward the hydroxyl radical but by their ability to increase plasma levels of glucose in vivo [28], [29]. Glucose gives excellent protection against alloxan diabetes [30], [31].

In order to test the free radical hypothesis, an appropriate in vitro test system, that accurately mimics alloxan toxicity in vivo, would be of great value, because such a system would not be subject to the hyperglycaemic or other interfering effects of the test compounds.

The most relevant in vitro parameter for studies related to alloxan diabetes is obviously pancreatic β-cell death. We have recently developed a method for quantitating β-cell death in isolated pancreatic islets and shown that this can give valuable information on the mechanism by which sugars protect against alloxan toxicity [32]. In the present experiments, we have used the same test system to investigate the effects of a number of free radical scavengers and chelating agents on alloxan toxicity in vitro, and the ability of these substances to protect against alloxan toxicity to β-cells in isolated islets has been compared with their reported activities in ameliorating the diabetogenicity of alloxan in vivo.

Section snippets

Materials

Collagenase and SOD (from bovine erythrocytes) were from Boehringer Mannheim (Germany). HEPES, alloxan monohydrate, diethylenetriamine pentaacetic acid (DTPA) dimethylsulfoxide (DMSO), 1,3-dimethylurea, EDTA, 1,10-phenanthroline, and catalase (from bovine liver) were from Sigma (St. Louis, MO, USA), bovine serum albumin (fraction V) from Miles Laboratories, (Elkart, IN, USA). Desferrioxamine methanesulphonate was from Ciba-Geigy (Basle, Switzerland), butanol, thiourea and all other reagents

Results

β-cells in control islets were well preserved, with no degenerative changes being observed in any of the islet cell types. In islets exposed to 5 mM alloxan, however, almost all the β-cells in the islets were necrotic, with both the cell membranes and the membranes of organelles such as the secretory granules and the cisternae of the rough endoplasmic reticulum and Golgi complex being ruptured as described before [32]. Damage was confined to β-cells alone, with the integrity of other cell types

Discussion

We have previously demonstrated the value of an isolated pancreatic islet system for study of the mechanism of alloxan toxicity [32]. As in the whole animal, the toxic effects of alloxan in this system are confined to β-cells; other endocrine cells and the exocrine parenchymal cells are not damaged, even in the presence of high concentrations of the test substance. Furthermore, the morphological alterations induced in β-cells in vitro were identical to the cellular changes seen in animals dosed

Acknowledgements

We gratefully acknowledge the skillful technical assistance of D. Lischke and F. Hurkuck. This work was supported by a grant from the Waikato Medical Research Foundation (R.M.)

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