The Effect of tert-Butylhydroquinone-Induced Oxidative Stress in MDBK Cells using XTT Assay: Implication of tert-Butylhydroquinone-Induced NADPH Generating Enzymes

Journal of Veterinary Medical Science

Naoko SHIBUYA¹  Tomoo NAOI²  Sakura MITA¹  Koichi ORINO¹  Shigeki KOBAYASHI²  Haruka INOUE¹  Kiyotaka WATANABE¹ 

[1] Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University;Laboratory of Animal Nutritio, School of Veterinary Medicine, Kitasato University

关键词: NADPH;    pro-oxidant;    reduction;    tert-butylhydroquinone;    XTT assay;   

DOI  :  10.1292/jvms.09-0346

学科分类：兽医学

来源: Japanese Society of Veterinary Science

【 摘 要 】

References(25)Cited-By(3)Tetrazolium salts such as XTT and MTT are widely used to produce formazan for cell proliferation and cytotoxicity assays through bioreductase activity. However, the XTT assay showed significant increase in MDBK cell viability when cells were treated with both 50 and 100 μM of the pro-oxidant, tert-butylhydroquinone (t-BHQ), although the crystal violet assay showed no cytotoxic effect with these concentrations, and the induction of lipid peroxidation was not observed. We investigated the mechanism of enhancement of XTT substrate reduction after treatment of MDBK cells with t-BHQ, leading to apparent increase in cell viability. t-BHQ caused an increase in absorbance at 340 nm in culture medium, suggesting that t-BHQ increases cellular production and release of NADH and/or NADPH. Although t-BHQ did not change the NADH concentration in cell culture medium, the addition of NADP+-dependent glutathione reductase decreased the XTT reduction to the control level, indicating cellular release of NADPH. t-BHQ also increased intracellular glucose-6-phosphate dehydrogenase activity, producing NADPH. Taken together, our findings indicate that t-BHQ treatment activates NADPH generating enzymes such as glucose-6-phosphate dehydrogenase followed by release of NADPH in the cell culture medium, resulting in direct XTT reduction by NADPH.

【 授权许可】

Unknown