Short and long term modulation of oxidant induced ATP depletion--implications for effective treatment.

Exposure of mammalian cells to oxygen radicals formed by the reduction of molecular oxygen may result in cell dysfunction and death. In endothelial cells, one of the earliest and most consistent features of oxidant attack is a dramatic depletion in intracellular ATP content. The mechanisms involved in this depletion include inhibition of ADP phosphorylation, inactivation of the glycolytic pathway and activation of poly (ADP-ribose) polymerase due to DNA damage which leads to a consumption of NAD and culminates in a decrease in ATP levels [1,2]. Depletion of ATP, by whichever pathway, will ultimately lead to a perturbation in cellular energy requiring processes. Effective treatment against oxidant damage, in addition to protecting cellular targets from attack, must be able to preserve cellular function. Maintenance of cellular ATP levels may be a good indicator of antioxidant efficacy, however, numerous reports [3,4] have demonstrated that depletion of ATP in itself, is not sufficient to cause cell death and also agents which do not maintain ATP levels have been shown to prevent cell death [ 31. In this study we investigated the relationship between early ATP depletion and eventual cell death in human endothelial cells. The ability of various agents to preserve both ATP levels and cell viability, in response to hydrogen peroxide (H2@), over a short treatment time was compared with their ability to sustain these effects over a longer 'recovery' period. The agents used included catalase, the classic H202 scavenger, dimethylthiourea (DMTU) which scavenges both the hydroxyl radical ('OH) and H2Q, phenanthroline, an iron chelator, and 3-aminobenzamide (3-AB), an inhibitor of p l y (ADP-ribose) polymerase. Human endothelial cells were isolated and cultured as previously described [S]. Confluent cell monolayers were washed with a Hank's balanced salt solution containing 0.5% bovine serum albumin (HBSS + A) and treated with H2@ * scavenger in lml of HBSS + A. After a 5h exposure, HBSS + A was replaced with complete growth media and cells were incubated for a further 24h. ATP was determined by a lucin-luciferase assay [6] and cell viability by the lactate dehydrogenase (LD) leakage assay [7]. After a 5h exposure, H2@ caused a depletion in ATP levels to 5% of control values (Fig 1). Catalase and DMTU completely prevented this depletion (Fig 1) with a concommitant prevention of cell lysis (results not shown). Similar effects were observed with 3-AB. In contrast, phenanthroline prevented cell lysis but only partially preserved ATP ( 40% of control values). After 24h, no recovery of ATP was observed in cells exposed to H2& alone. Catalase and DMTU treatment maintained ATP content and viability at control levels. In contrast, the short term protective effects of 3-AB were not sustained. Cells treated with phenanthroline did however recover ATP to control levels after 24h. The different effects of the agents examined illustrates the number of pathways involved in oxidant induced ATP depletion. Scavenging of H2@ completely abolished ATP depletion whilst inhibiting 'OH formation, which is thought to be the major DNA damaging species, by preventing iron-dependent Fenton reactions only partially prevented ATP depletion. However the ability of phenanthroline treated cells to recover ATP levels indicates that such H202 catalase DMTU Phcn 3-AB