Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Authors

  • Shoichiro Shishido,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
    2. Second Department of Medicine, Fukushima Medical Collage, Fukushima, Japan
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  • Hironori Koga M.D.,

    Corresponding author
    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
    • F.J.S.I.M., Second Department of Medicine, and the Reseach Center for Innovative Cancer Therapy, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Fax: (81) 942 34 2623
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  • Masaru Harada,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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  • Hiroto Kumemura,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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  • Shinichiro Hanada,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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  • Eitaro Taniguchi,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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  • Ryukichi Kumashiro,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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  • Hiromasa Ohira,

    1. Second Department of Medicine, Fukushima Medical Collage, Fukushima, Japan
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  • Yukio Sato,

    1. Second Department of Medicine, Fukushima Medical Collage, Fukushima, Japan
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  • Masayoshi Namba,

    1. Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University School of Medicine, Okayama, Japan
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  • Takato Ueno,

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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  • Michio Sata

    1. Second Department of Medicine, and the Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
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Abstract

Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazone-treated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (ΔΨm) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2′,7′-dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electron-dense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the ΔΨm per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity.

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