Abstract 1149 Poster Session I, Saturday, 5/1 (poster 75)

Glutathione (GSH) plays vital roles in antioxidant defense mechanisms and in other physiological functions. Premature infants have lower plasma GSH concentrations than do healthy adults; evidence implicating oxidative stresses in diseases prevalent in premature infants indicates that the GSH status of these infants may represent a functional deficit. GSH synthesis begins with the rate-limiting, ATP-dependent addition of cysteine to glutamate by glutamate-cysteine ligase (GLCL), which exists as a dimer of non-identical subunits. The heavy or catalytic (glutamate-cysteine ligase catalytic, GLCLC) subunit contains the active site and the light or regulatory (glutamate-cysteine ligase regulatory, GLCLR) subunit modulates the activity of the GLCLC subunit. To determine whether GSH synthetic capacity could be augmented, we used Lipofectamine(Lf)-mediated transfer of the cDNAs for rat GLCLC and GLCLR subunits to transfect CHO cells and increased GLCL activities from 0.50+0.09 (mean±SD) mU/mg protein to 10.63±2.96 by transfection with the GLCLC subunit and to 0.96±0.12 with the GLCLR subunit (n=4-6). CHO cell GSH contents were increased to comparable levels by transfection with either the GLCLC or GLCLR cDNAs (GLCLC : 35.27±3.98 nmol/mg protein, GLCLR : 27.24±2.83 vs. native CHO : 17.51±2.17); however, treatment with Lf also increased cell GSH contents (28.6±2.56) despite no change in GLCL activities. To determine whether the enhanced GLCL activities noted in the above studies would afford protection against an oxidant challenge, CHO cells were plated (10,000/well) in a 96-well plate, transfected with the GLCLC cDNA, and exposed to tBuOOH for 6 hours. Cells were exposed to 106, 212 or 318 nmol tBuOOH/well, 3/4ths dose initially and 1/4th 2 h later. Toxicity was assessed as loss of viability as determined by MTT reduction assay 6 h after initial exposure to tBuOOH. Transfection with the GLCLC cDNA resulted in an attenuation of tBuOOH toxicity at all dosage groups. Prior treatment with Lf alone resulted in 26, 41 and 44% loss of viability, while prior treatment with Lf with vector resulted in 34, 38 and 37 % loss of viability; in contrast, prior treatment with Lf and GLCLC cDNA resulted in only 13, 25 and 24 % loss of viability. Conclusion: The present results indicate that transient transfection with GLCLC cDNA can afford measurable protection against tBuOOH-induced oxidant toxicity, and increased GSH contents without increases in GLCL activities, as produced by treatment with Lf provides only limited protection against oxidant stresses.

(Supported by HD27823).