Prooxidative effects of green tea polyphenol (-)-epigallocatechin-3-gallate on the HIT-T15 pancreatic beta cell line.
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.
Suh
Kwang Sik
KS
Department of Endocrinology and Metabolism, Kyung Hee University School of Medicine, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, South Korea.
Chon
Suk
S
Oh
Seungjoon
S
Kim
Sung Woon
SW
Kim
Jin-Woo
JW
Kim
Young Seol
YS
Woo
Jeong-Taek
JT
D017209:Apoptosis / Q000187:drug effects; D002392:Catechin / Q000031:analogs & derivatives* / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000633:toxicity; D006861:Hydrogen Peroxide / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D016877:Oxidants / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D010636:Phenols / Q000633:toxicity; D059808:Polyphenols; D017382:Reactive Oxygen Species / Q000378:metabolism; D013662:Tea / Q000737:chemistry
D005419:Flavonoids; D016877:Oxidants; D010636:Phenols; D059808:Polyphenols; D017382:Reactive Oxygen Species; D013662:Tea; D002392:Catechin; D006861:Hydrogen Peroxide; C045651:epigallocatechin gallate