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Cell Biology International (2012) 36, 183–188 (Printed in Great Britain)

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Cytoprotective effect of trolox against oxidative damage and apoptosis in the NRK-52e cells induced by melamine

Chengzhi Guo*, Zuping He†‡¹, Lixin Wen*, Li Zhu*, Yin Lu*, Sijun Deng*, Yang Yang*, Qiang Wei* and Hui Yuan*¹

*College of Veterinary Medicine, Hunan Agricultural University, 19 East Lake Road, Changsha, Hunan 410128, Peoples Republic of China, †Clinical Stem Cell Research Center, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, Peoples Republic of China, and ‡Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20057, U.S.A.

An outbreak of urinary stones associated with consumption of melamine-tainted milk products occurred in 2008 in China, leading to serious illness of many infants and even death. However, the toxicity of melamine in kidney epithelial cells remains unclear. We have explored the effects of melamine and trolox on renal NRK-52e (normal rat kidney 52e) cells. The IC₅₀ of melamine was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Total SOD (superoxide dismutase) was determined by NBT (Nitro Blue Tetrazolium) staining method. GSH-Px (glutathione peroxidase) activity was detected by UV colorimetric assay, and MDA (malondialdehyde) content was determined by thiobarbituric acid assay. Apoptosis induced by melamine was determined by flow cytometry. The IC₅₀ increased when NRK-52e cells were treated with both melamine and trolox compared with melamine only. SOD and GSH-Px activities were decreased, but MDA content was increased by melamine in a dose-dependent manner. Trolox significantly enhanced SOD and GSH-Px activity in melamine-treated NRK-52e cells, but it decreased their MDA content. LDH (lactate dehydrogenase) activity and the level of ROS (reactive oxygen species) of the NRK-52e cells were enhanced by melamine compared with the control. Furthermore, the apoptosis rate increased in NRK-52e cells treated with melamine, whereas trolox was protective. These results show that melamine has an obvious adverse effect on proliferation of NRK-52e cells, causing oxidative damage and apoptosis, thus providing a novel insight into renal cytotoxicology of melamine. Trolox ameliorates the effect on melamine toxicity.

Key words: melamine, trolox, NRK-52e cells, oxidative damage, cell apoptosis

Abbreviations: DCF, 2′,7′-dichlorofluorescein, DCFH-DA, 2′,7′-dichlorofluorescein diacetate, DMEM, Dulbecco's modified Eagle's medium, FBS, fetal bovine serum, GSH-Px, glutathione peroxidase, LDH, lactate dehydrogenase, MDA, malondialdehyde, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, NBT, Nitro Blue Tetrazolium, NRK, normal rat kidney, PI, propidium iodide, ROS, reactive oxygen species, SOD, superoxide dismutase

¹Correspondence may be addressed to either of these authors (email zupinghe@gmail.com or yuanhui7269@yahoo.com.cn).

Received 16 January 2011/23 May 2011; accepted 22 September 2011

Published as Cell Biology International Immediate Publication 22 September 2011, doi:10.1042/CBI20110036

© The Author(s) Journal compilation © 2012 Portland Press Limited