Epigallocatechin-3-gallate (EGCG) is the main polyphenolic constituent in green tea and is believed to function as an antioxidant. However, increasing evidence indicates that EGCG produces reactive oxygen species (ROS) and subsequent cell death. In this study, we investigated the prooxidative effects of EGCG on the HIT-T15 pancreatic beta cell line. Dose-dependent cell viability was monitored with the cell counting kit-8 assay, while the induction of apoptosis was analyzed by a cell death ELISA kit and comet assay. Extracellular H(2)O(2) was determined using the Amplex Red Hydrogen Peroxide Assay Kit. Intracellular oxidative stress was measured by fluorometric analysis of 2',7'-dichlorofluorescin (DCFH) oxidation using DCFH diacetate (DA) as the probe. Treatment with EGCG (5-100 microM) decreased the viability of pancreatic beta cells, caused concomitant increases in apoptotic cell death, and increased the production of H(2)O(2) and ROS. Catalase, the iron-chelating agent diethylenetriaminepentaacetic acid, and the Fe(II)-specific chelator o-phenanthroline all suppressed the effects of EGCG, indicating the involvement of both H(2)O(2) and Fe(II) in the mechanism of action of EGCG. The antioxidant N-acetyl-cysteine and alpha-lipoic acid also suppressed the effects of EGCG. Furthermore, EGCG did not scavenge exogenous H(2)O(2), but rather, it synergistically increased H(2)O(2)-induced oxidative cell damage in pancreatic beta cells. Together, these findings suggest that in the HIT-T15 pancreatic beta cell line, EGCG mediated the generation of H(2)O(2), triggering Fe(II)-dependent formation of a highly toxic radical that in turn induced oxidative cell damage.

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During the progression of Type 2 diabetes, glucose toxicity is likely to contribute importantly to progressive beta cell failure. Oxidative stress is an important aspect of glucose toxicity in pancreatic beta cells, and reducing sugars, such as 2-deoxy-D-ribose (dRib), produce reactive oxygen species. Furthermore, many of the biological properties of flavonoids are likely to be related to their antioxidant and free-radical scavenging abilities. Accordingly, in the present study, we investigated whether kaempferol (a flavonol) protects beta cells from dRib-induced oxidative damage. HIT-T15 cells were cultured with various concentrations of dRib for 24h. Cell survivals, amounts of reactive oxygen species (ROS) generated, apoptosis, and lipid peroxidation were measured. dRib was found to dose-dependently reduce cell survival and to markedly increase intracellular ROS levels, apoptosis, and lipid peroxidation. However, kaempferol (10 microM) suppressed dRib (20 mM) induced intracellular ROS, apoptosis, and lipid peroxidation. So, we demonstrate that kaempferol reduces dRib-mediated beta cell damage interfering with ROS metabolism and protective effects against lipid peroxidation. Our findings indicate that kaempferol protects HIT-T15 cells from dRib-induced associated oxidative damage.

Several environmental contaminants have been linked to the development of diabetes and increased diabetes‑associated mortality. Perfluorooctanoic acid (PFOA) is a widely used perfluoroalkane found in surfactants and lubricants, and in processing aids used in the production of polymers. Furthermore, PFOA has been detected in humans, wildlife and the environment. The present study investigated the toxic effects of PFOA on rat pancreatic β‑cell‑derived RIN‑m5F cells. Cell viability, apoptosis, reactive oxygen and nitrogen species, cytokine release and mitochondrial parameters, including membrane potential collapse, reduced adenosine triphosphate levels, cardiolipin peroxidation and cytochrome c release were assessed. PFOA significantly decreased RIN‑m5F cell viability and increased apoptosis. Exposure to PFOA increased the formation of reactive oxygen species, mitochondrial superoxide, nitric oxide and proinflammatory cytokines. Furthermore, PFOA induced mitochondrial membrane potential collapse and reduced adenosine triphosphate levels, cardiolipin peroxidation and cytochrome c release. These results indicate that PFOA is associated with the induction of apoptosis in RIN-m5F cells, and induces cytotoxicity via increased oxidative stress and mitochondrial dysfunction.

Glucose toxicity contributes to progressive β-cell failure and the development of overt diabetes. Oxidative stress is an important aspect of glucose toxicity in pancreatic β-cells. We investigated whether the flavonoid apigenin protects pancreatic β-cells from 2-deoxy-D-ribose (dRib)-induced oxidative cell damage. HIT-T15 pancreatic β-cells were cultured with or without apigenin in the presence of dRib. Time-and dosedependent cell viability was monitored using a cell counting kit (CCK-8), while the induction of apoptosis was analyzed using a cell death enzyme-linked immunosorbent assay (ELISA) kit. Mitochondrial membrane potential (ΔΨ m ) was determined using the JC-1 kit. Intracellular oxidative stress was measured by fluorometric analysis of DCFH oxidation using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as the probe. In addition, the DNA binding activity of the oxidative stress-related transcriptional factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) were analyzed. dRib reduced cell survival and ΔΨ m , while it markedly increased intracellular levels of reactive oxygen species (ROS), apoptosis, and the activity of the oxidative stress-related transcription factors NF-κB and AP-1. However, pretreatment of cells with apigenin attenuated all the dRib-induced effects. The anti-oxidants, N-acetyl-L-cysteine (NAC) and alpha lipoic acid (ALA), also prevented both dRib-induced oxidative damage and activation of NF-κB and AP-1. Taken together, these results suggest that apigenin attenuates dRib-induced cell damage in pancreatic β-cells via oxidative stressrelated signaling. Key words oxidative stress; apigenin; 2-deoxy-D-ribose; diabetes; pancreatic β-cellOxidative stress is known to be a major contributor to the development of overt diabetes and its associated complications.1) Oxidative stress results from a persistent imbalance between antioxidant defenses and the production of highly reactive oxygen species (ROS).2) ROS are normal byproducts of cellular metabolism. However, a marked increase in glucose metabolism could lead to excessive ROS production, which could contribute to the development of diabetic complications. In particular, pancreatic β-cells express low levels of antioxidant enzymes and do not up-regulate these enzymes upon exposure to high concentrations of glucose.3) Thus, increased ROS production in the presence of low antioxidant defense levels could result in ROS accumulation, which would lead to the oxidative pancreatic β-cell damage that is partly responsible for diabetes. Nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) are 2 redox-sensitive transcription factors that can be regulated by oxidative stress. [4][5][6] By activating these transcription factors in isolated peripheral mononuclear cells of diabetic patients, ROS are also involved in the pathogenesis of diabetic nephropathy. 7) In addition, activation of NF-κB correlates with the quality of glycemic control and has been shown to be reduced by treatment with antioxidants in a diabetic animal model and diabetic pa...