Am. J. Respir. Cell Mol. Biol., Volume 20, Number 2, February, 1999 256-263

Gene Transfer of Mitochondrially Targeted Glutathione Reductase Protects H441 Cells from t-Butyl Hydroperoxide-Induced Oxidant Stresses

Donough J. O'Donovan, Julie P. Katkin, Toshiya Tamura, Richard Husser, Xudong Xu, Charles V. Smith, and Stephen E. Welty

Department of Pediatrics, Baylor College of Medicine, Houston, Texas

Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 ± 1.7; DOS 16 ± 1.8; CON 16.6 ± 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 ± 0.4; DOS 47 ± 1; CON 52.6 ± 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 ± 0.01; DOS 0.08 ± 0.01; CON 0.07 ± 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.

This article has been cited by other articles:

				N. Watanabe, D. A. Dickinson, D. M. Krzywanski, K. E. Iles, H. Zhang, C. J. Venglarik, and H. J. Forman A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L726 - L736. [Abstract] [Full Text] [PDF]

				L. W. Velsor, A. van Heeckeren, and B. J. Day Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L31 - L38. [Abstract] [Full Text] [PDF]

				D. J. O'Donovan, J. P. Katkin, T. Tamura, C. V. Smith, and S. E. Welty Attenuation of Hyperoxia-Induced Growth Inhibition in H441 Cells by Gene Transfer of Mitochondrially Targeted Glutathione Reductase Am. J. Respir. Cell Mol. Biol., June 1, 2000; 22(6): 732 - 738. [Abstract] [Full Text]

				I. Rahman and W. MacNee Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease Am J Physiol Lung Cell Mol Physiol, December 1, 1999; 277(6): L1067 - L1088. [Abstract] [Full Text] [PDF]

				Y. Abe, C. W. Smith, J. P. Katkin, L. M. Thurmon, X. Xu, L. H. Mendoza, and C. M. Ballantyne Endothelial {alpha}2,6-Linked Sialic Acid Inhibits VCAM-1- Dependent Adhesion Under Flow Conditions J. Immunol., September 1, 1999; 163(5): 2867 - 2876. [Abstract] [Full Text] [PDF]