Abstract
Premature birth and its medical management subjects human infants to oxygen gas tensions far in excess of the intrauterine environment for which they are prepared developmentally. Glutathione (GSH) is important in cellular protection against oxidants and premature infants have low plasma GSH concentrations, suggesting a possible functional deficit. To determine whether enhanced GSH synthetic capacities would augment antioxidant defense functions and protect cells, CHO cell lines with stably enhanced GGCS activities(GGCS-h) were developed by transfection with vectors containing the cDNA for the rat GGCS heavy subunit and hygromycin/neomycin resistance genes. Southern analyses indicate the presence of the heavy chain cDNA in the genomic DNA of the GGCS-h cells, and Northern analyses indicate transcription of the transfected GGCS gene. Native CHO and GGCS-h cells (4.5 × 105 per plate) were seeded and grown initially in room air for 18 h, and subsequently in room air or hyperoxia (>85% oxygen) for a period of 72 h. Cells grown in hyperoxia exhibited slower cell growth than those grown in room air, as evidenced by lower cellular protein concentrations (0.69±0.10 vs. 1.68±0.26 mg pro/plate in CHO cells, and 0.73±0.08 vs. 1.54±0.09 in GGCS-h cells; mean±SD, n=4). Despite greater GSH contents (41.7±5.2 vs. 16.8±1.6 nmol/mg pro; mean±SD, n=4) and GGCS activities (8.49±1.06 vs. 0.26±0.08 mU/mg pro) in the GGCS-h cells than in the parent CHO cells, the GGCS-h cells displayed similar growth inhibition as the CHO cells (52.88% vs. 59.23%).Conclusion: The failure of overexpression of GGCS to protect against the growth inhibitory effects of hyperoxia suggests that cellular GSH synthesis is not rate-limiting for all the toxicities that arise from exposure to elevated oxygen tensions and while enhancement of GSH synthesis may prove to be a significant component of therapies for amelioration of the adverse effects of hyperoxia, the present data suggest that additional mechanisms and therapies need to be addressed. (Supported by Wyeth Neonatology Research Grants Program and GM44263 and HD27823).
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