The Effect of H2O2 and Tertiary Butyl Hydroperoxide on an Immortal Lens Epithelial Cell Line, Alpha TN4-1

Abstract

Abstract: : Purpose: Recently this laboratory has conditioned a murine immortal lens epithelial cell line, αTN4-1, to live in concentrations of H₂O₂, the H line, or a lipid peroxide prototype, tertiary butyl hydroperoxide (TBHP), the T line, which can cause cataract in vitro. It was found that while T cells could survive H₂O₂ stress, the H cells could not survive a TBHP stress. Such observations suggest that there is a fundamental difference in the manner by which H₂O₂ and lipid peroxides kill cells. In order to obtain an understanding of the difference in the action of these peroxides, their effect on unconditioned αTN4-1 cells has been examined. Methods: Cells were stressed with either H₂O₂ or TBHP and the cell population was monitored by cell counting and Trypan blue staining. The effect of 3-aminobenzamide (3AB), a specific inhibitor of poly ADP ribose polymerase (PARP), was examined by determining PARP activity and NAD concentration. This enzyme is important in DNA repair and transfers ADP-ribose from NAD to acceptor sites. Results: Both H₂O₂ and TBHP cause a rapid loss of many of the standard markers of peroxide stress including GSH, ₁₄C-choline and ₈₆Rb uptake. A low level of lipid oxidation was also observed. However, no great difference between H₂O₂ and TBHP action was found with respect to the above parameters. Examination of single strand DNA breaks indicated that significant DNA damage occurred with H2O2 but was much less under comparable conditions with TBHP. It was found that 3AB had little effect on TBHP induced DNA damage or cell death rate. However, with H₂O₂ stress, even though a somewhat greater level of DNA damage was observed, surprisingly, the rate of cell death was significantly decreased. PARP activity was stimulated about 6 fold with H2O2 but only about 30% with TBHP. 3AB eliminated the PARP activation. Since PARP utilizes NAD as a substrate, it was measured and found to decrease 9 fold with H₂O₂ stress but not at all with TBHP. The H₂O₂ effect on NAD was eliminated with 3AB. Since the resynthesis of NAD requires 4 ATPs, ATP levels were measured. H₂O₂ stress caused a rapid drop in ATP which was prevented by 3AB. A much slower loss of ATP was observed with TBHP which was not affected by 3AB. H₂O₂ was also found to have a much greater effect on energy generating systems than TBHP. Conclusion: A significant difference in the impact of H₂O₂ and TBHP upon cell viability is caused by the precipitous loss of ATP observed with H₂O₂ stress. The ATP loss is related to its utilization to restore depleted NAD levels as well as for repair of oxidative damage.

Keywords: 504 oxidation/oxidative or free radical damage • 467 metabolism