Glutathione Reductase (GR) targeted to the mitochondria (LGR) protects H441 cells from t-BuOOH induced oxidant stresses

Reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants. GSH-dependent antioxidants protect against ROS in the cytoplasm and mitochondria, and regenerating GSH from GSSG by GR is essential. Previously, we described a vector for human GR cDNA that contains a synthetic mitochondrial targeting signal, and used liposomal gene transfer to increase GR in Chinese hamster ovary (CHO) cells. CHO cells stably transformed with LGR showed greater resistance to tBuOOH. The present studies were to determine whether adenoviral-mediated gene transfer could increase GR activities in H441 cells, and whether such cells would be more resistant to tBuOOH. Cells transfected with LGR showed increased cellular GR activities and were protected from tBuOOH cytotoxicity, as indicated by lower %LDH release, than did cells transfected with an irrelevant gene (DOS=SOD in reverse orientation; a1AT=alpha-1 antitrypsin) or cells not infected with virus (C). Total GR activities were higher (LGR 458±13: a1AT 42±4; C 41±4 mU/mg pro). Cytosolic GR activities were higher (LGR 377±3 ; a1AT 57±0.3; C 58±2), while mitochondrial GR activities were enhanced (LGR 231±67;a1AT 21±7; C 23±1). Cells with enhanced GR activities released less LDH when exposed to tBuOOH (LGR 6.6±1.7; DOS 16±1-8;C 16.6±0.7). In addition, cells transfected with LGR and exposed to t-BuOOH, had higher GSH/GSSG ratios (LGR 66±0.4; DOS 47±1; C 53±2) and less cellular GSSG efflux (LGR 0.05±0.01; DOS 0.08±0.01; C 0.07±0.01 nmol/mg pro). In conclusion, adenoviral-mediated gene transfer of GR protected cells from oxidant stresses.