A variety of mechanisms have been identified as responsible for the onset of diabetes mellitus. Alloxan shows a selective cytotoxicity on pancreatic b-cells and thus causes insulin-dependent (type I) diabetes mellitus. Although the mechanism of alloxan cytotoxicity is not yet clearly understood, several researchers 1,2) demonstrated that the diabetogenic action was initiated by the generation of reactive oxygen species (ROS). Alloxan is a mild oxidant and is easily reduced to alloxan radicals (A· Ϫ ) by GSH or ascorbic acid. 3) Our previous study 4) demonstrated that A· Ϫ can directly reduce O 2 and ferric ions to superoxide anion radical (O 2 · Ϫ ) and ferrous ions, respectively. The extreme and indiscriminate reactivity of ROS could easily explain the killing of b-cells by alloxan. In support of this hypothesis alloxan diabetes is prevented by HO · scavengers, 5-7) superoxide dismutase (SOD) 2) and vitamin E. 8) In addition, on the basis of in vivo and in vitro studies using rats and isolated islet cells, respectively, it was proposed that the process of alloxan toxicity involved the generation of HO · by which the DNA strands break of pancreatic islets is attacked to produce. 9-11) These findings indicate that DNA strand breaks are involved in the events of alloxan-diabetogenesis. However, the complex sequence of events that eventually leads to the DNA strand breaks and death of pancreatic b-cells in alloxan diabetes mellitus is not clear.Cell death might occur by one of two fundamental processes, necrosis or apoptosis. The biochemical hallmarks of apoptosis are the distribution of phosphatidylserine (PS) at the external surface of the plasma membrane (PS exposure) in the early stages, and the cleavage of chromosomal DNA into nucleosomal units, which appears to be the final blow in the cell death process. Recent studies have shown that different trigger such as IL-1b 12) and streptozotocin 13) can induce apoptosis in pancreatic b-cells. Although detailed studies indicate that alloxan can induce insulin-dependent diabetes mellitus, nothing is known about apoptotic action or the sequence of cellular events of alloxan on the b-cells.According to current understanding, mitochondrial damage seems to occur in the early phase of apoptosis and to participate in the control of apoptosis. 14,15) Release of cytochrome c and apoptosis induced factor from mitochondria into cytoplasm during mitochondrial permeability transition cause to active caspase family, leading to the completion of apoptosis. 15,16) Isolated liver mitochondria incubated with alloxan alters respiration, decreases in the concentration of adenine nucleotides, causes Ca 2ϩ release, and changes in the Dy, 17,18) indicating that alloxan directly induces the mitochondrial damage. However, the relationship between diabetes, apoptosis and mitochondrial damage has remained elusive.INS-1 cells were established by X-ray-induced rat transplantable insulinoma, and have retained the similar ability of glucose-stimulated insulin release of native b-cells. 19) INS-1 ...

Baicalein decreased the production of thiobarbituric acid reactive substances, the rate of oxygen consumption and iron reduction in the reaction system of ascorbic acid with FeCl3. Superoxide dismutase, catalase and hydroxyl radical scavengers had no significant effect. Ironchelators had an inhibitory effect similar to that of baicalein. The production of thiobarbituric acid reactive substances of baicalein‐treated microsomes obtained by centrifugation after incubation with baicalein was not observed in the reaction system, but was stimulated by adding iron with increases in concentration. The amount of bound iron to microsomal membranes increased by increasing both the concentration of baicalein and iron. The amount of baicalein bound to microsomal membranes increased with increasing concentration of added baicalein. These results suggest that baicalein bound to microsomal membranes inhibits lipid peroxidation by formating an iron‐baicalein complex.

In addition to their critical function in energy metabolism, mitochondria contain a permeability transition pore, which is regulated by adenine nucleotides. We investigated conditions required for ATP to induce a permeability transition in mammalian mitochondria. Mitochondrial swelling associated with mitochondria permeability transition (MPT) was initiated by adding succinate to a rat liver mitochondrial suspension containing alloxan, a diabetogenic agent. If alloxan was added immediately with or 5 min after adding succinate, MPT was strikingly decreased. MPT induced by alloxan was inhibited by EGTA and several agents causing thiol oxidation, suggesting that alloxan leads to permeability transition through a mechanism dependent on Ca 2؉ uptake and sulfhydryl oxidation. Antimycin A and cyanide, inhibitors of electron transfer, carbonyl cyanide m-chlorophenylhydrazone, and oligomycin all inhibited MPT. During incubation with succinate, alloxan depleted ATP in mitochondria after an initial transient increase. However, in a mitochondrial suspension containing EGTA, ATP significantly increased in the presence of alloxan to a level greater than that of the control. These results suggest the involvement of energized transport of Ca 2؉ in the MPT initiation. Addition of exogenous ATP, however, did not trigger MPT in the presence of alloxan and had no effect on MPT induced by alloxan. We conclude that alloxan-induced MPT requires mitochondrial energization, oxidation of protein thiols, and matrix ATP to promote energized uptake of Ca 2؉ .Mitochondrial permeability transition (MPT) 1 is associated with an increase in the permeability of the mitochondrial inner membrane to solutes with a molecular mass of 1.5 kDa or lower and can disturb mitochondrial functions in several ways (1-3). It is generally accepted that MPT is due to the opening of a so-called permeability transition pore (PTP), which accompanies a loss of transmembrane potential (⌬), a release of Ca 2ϩ and other cations, and mitochondrial swelling, causing ATP synthesis to stop in mitochondria (2-6). The PTP is thought to have a role in cellular Ca 2ϩ homeostasis, in import of mitochondrial protein, in thermal regulation in mammalian mitochondria, and as a common mediator of cell death (7,8).We focused on the role of adenine nucleotides in the modulation of MPT. The sensitivity of MPT to [Ca 2ϩ ] is greatly increased by ATP depletion (9). Matrix ADP is an important modulator of PTP opening and acts by decreasing the sensitivity of the calcium trigger site to Ca 2ϩ (10). The opening of PTP may be caused by well known membrane constituents, including the adenine nucleotide translocator (ANT), porin molecules, and the complex forming the peripheral benzodiazepine receptor (11,12). Two ADP binding sites may exist, one with a high affinity with the ANT that is blocked by the inhibitor atractyloside and the other site with a lower affinity (5, 13). These findings suggest that adenine nucleotides may be negative regulators of MPT.Mitochondrial damage seems to occur ...

The diabetogenic action of alloxan is thought to be initiated by generation of reactive oxygen species (ROS). Ascorbate can be an antioxidant in a predominantly aqueous environment, such as plasma and extracellular ‰uids. We have investigated the generation of ROS in the interaction of alloxan with ascorbate. Rapid oxygen consumption was observed in the reaction system of alloxan with ascorbate. The oxygen consumption was suppressed by superoxide dismutase and catalase, suggesting that superoxide and hydrogen peroxide could be generated in the reaction system. In addition, the generation of alloxan radical, an electron reductance of alloxan, and ascorbate free radical (AFR), an electron oxidant of ascorbate, was observed using electron spin resonance (ESR). Under anaerobic conditions, the ESR signal intensity of alloxan radical was signiˆcantly increased in comparison with that under aerobic conditions, whereas the intensity of AFR was signiˆcantly decreased. These results suggest that alloxan radical and AFR were generated in the reaction system of alloxan with ascorbate, and that the alloxan radical but not AFR reacted with molecular oxygen, resulting in the generation of ROS.

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