Susceptibility To Oxidant Injury Decreases With Increasing Age In Rat Lung Fibroblasts † 1624

Abstract

The objective of this study was to evaluate the influence of fetal/postnatal age on reponse of rat lung fibroblasts to oxidative injuryin vitro. The effects of oxidant exposure on cell proliferation are reported to be dose dependent; lower doses enhance while higher doses decrease proliferation. Thus, the effects of 0.10-2.00 mM hydrogen peroxide (HP) were determined on viability and cell proliferation in fetal (E21) and postnatal(2, 5, and 9d) rat lung fibroblasts. first passage lung fibroblasts were grown to confluence. The 10% fetal bovine serum (FBS) medium was then replaced with 5% FBS medium. HP was added to achieve final mM concentrations of 0.10, 0.25, 0.50 and 2.00. Control flasks were incubated with 5% FBS medium. After 40 minutes, the medium was removed, the cell monolayer rinsed with Hank's Solution and fresh 10% FBS medium added. In addition, cell viability was determined 2 hrs after exposure to 2 mM HP by staining with trypan blue and counting on a hemocytometer. The lethality was 61% for E 21, 39% for 2d, 32% for 5d and 29% for9d fibroblasts. For cell proliferation studies, cells were cultured for 24 and 48 hrs. Cell number was then determined, by counting on a hemacytometer, and expressed as a% of same age control cells. The effect of HP on cell number varied with the age of the rats from which the cells were obtained [p=0.009, (ANOVA)]. Although there was no difference in sensitivity to HP at the lowest concentration tested, toxicity decreased with increasing postnatal age at higher HP concentrations. 24 hr after treatment with 0.25 mM HP, the number of E21 cells was less than the number of 9d cells (p=0.04), but not different than the number of 2d or 5d cells. After treatment with 0.5 mM HP, the number of E21 cells was less than the number of 5d and 9d cells(p<0.01). After treatment with 2mM HP, the number of E21 cells was less than the number of 2d, 5d and 9d cells (p=0.01). Between 24 and 48 hr post oxidant exposure, 2d, 5d and 9d fibroblasts continued to increase in number. In contrast, E21 fibroblasts decreased in number in flasks exposed to 0.25mM HP. During the first 48hr after oxidant injury, 9d cells proliferated to a greater extent than did either the 2d (p=0.0003) or the E21 cells (p=0.014). In conclusion, in vitro oxidant injury with HP reduces survival and proliferative capacity more in fetal than in postnatal rat lung fibroblasts.