ToxSci Advance Access originally published online on February 27, 2007
Toxicological Sciences 2007 97(2):562-568; doi:10.1093/toxsci/kfm033
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Reduced 293T Cell Susceptibility to Acrolein Due to Aldose Reductase–like-1 Protein Expression
* Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 North Rutledge Street, Springfield, Illinois 62702
Division of Pharmacoproteomics, Institute of Pharmacy and Pharmacology, Nanhua University School of Life Science and Technology, 28 Changshengxi Road, Hengyang, Hunan, China 421001
1 To whom correspondence should be addressed. Fax: (217) 545-9718. E-mail: dcao{at}siumed.edu.
Received December 12, 2006; accepted February 15, 2007
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Abstract |
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Acrolein is a highly reactive 
,ß-unsaturated aldehyde produced endogenously during lipid peroxidation and naturally distributed pervasively in living environments, posing serious threats to human health if not properly metabolized. In this study, we report aldose reductase–like-1 (ARL-1) as a novel enzyme that catalyzes the reduction of acrolein and protects cells from their toxicity. Using purified ARL-1 protein, we determined its enzymatic activity in response to acrolein and defined its steady-state kinetics with Km and Vmax at 0.110 ± 0.012mM and 3122.0 ± 64.7 nmol/mg protein/min, respectively. By introducing a functional Enhanced Green Fluorescent Protein (EGFP)/ARL-1 fusion protein into 293T cells, we demonstrated that plating efficiency in liquid culture and focus formation in soft agar increased by more than 60% (p < 0.05), compared to the vector control cells. More significantly, at a low dose of 5µM acrolein, EGFP/ARL-1 expression enhanced both plating efficiency and focus formation by more than threefold, and the foci (in soft agar) of 293T cells expressing EGFP/ARL-1 were significantly larger than those of the vector control cells. At high concentrations of acrolein (25 and 50µM), EGFP/ARL-1 protein prevented oncotic death of 293T cells induced by acrolein. In summary, our data demonstrated for the first time that the ARL-1 protein protects 293T cells from acrolein toxicity. Due to the high toxicity and wide distribution of acrolein, this finding is important to the understanding of its detoxification mechanisms.
Key Words: aldose reductase–like-1; aldo-keto reductase family 1 B10; lactate dehydrogenase; clonogenic growth; acrolein.
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