Dynamics of H2O2 availability to ARPE-19 cultures in models of oxidative stress

Abstract

Oxidative injury to cells such as the retinal pigment epithelium (RPE) is often modeled using H 2 O 2 -treated cultures, but H 2 O 2 concentrations are not sustained in culture medium. Here medium levels of H 2 O 2 and cytotoxicity were analyzed in ARPE-19 cultures following H 2 O 2 delivery as a single pulse or with continuous generation using glucose oxidase (GOx). When added as a pulse, H 2 O 2 is rapidly depleted (within 2 hr); cytotoxicity at 24, determined by the MTT assay for mitochondrial function, is unaffected by medium replacement at 2 hr. Continuous generation of H 2 O 2 produces complex outcomes. At low GOx concentrations, H 2 O 2 levels are sustained by conditions in which generation matches depletion, but when GOx concentrations produce cytotoxic levels of H 2 O 2 , oxidant depletion accelerates. Acceleration results partly from the release of contents from oxidant damaged cells as indicated by testing depletion after controlled membrane disruption with detergents. Cytotoxicity analyses show that cells can tolerate short exposure to high H 2 O 2 doses delivered as a pulse but are susceptible to lower chronic doses. The results provide broadly applicable guidance for using GOx to produce sustained H 2 O 2 levels in cultured cells. This approach will be specifically useful for modeling chronic stress relevant for RPE aging and have wider value for studying cellular effects of sub-lethal oxidant injury and for evaluating antioxidants that may protect significantly against mild but not lethal stress.

PRDX6 Protects ARPE-19 Cells from Oxidative Damage via PI3K/AKT Signaling

“…We used H 2 O 2 and blue light to induce oxidative stress in these cells. H 2 O 2 -induced oxidative stress is a classic model by which researchers have detected susceptibility to oxidative stress or antioxidant efficiency in RPE cells [26][27][28]. Additionally, light exposure has been linked to the occurrence and development of AMD possibly through oxidative stress-related pathology [29,30].…”

Involvement of the Up-regulated FoxO1 Expression in Follicular Granulosa Cell Apoptosis Induced by Oxidative Stress

“…H 2 O 2 is commonly used to produce oxidative stress in cultured cells (31,34). However, few reports described applying H 2 O 2 to research oxidative stress in granulosa cells.…”

“…H 2 O 2 is widely used as a classical reagent to trigger oxidative stress in cultured cells, which may serve as a useful in vitro oxidative stress model (31). H 2 O 2 is an important form of active oxygen and is easily accessible across the cell membrane (32).…”

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

“…Hydrogen peroxide (H 2 O 2 ) treatment of cell cultures is a common model of oxidative stress induction in cells that in vivo are at high risk of oxidative damage, such as retinal pigment epithelium [23]. This small, uncharged molecule can easily cross cell membranes and localize in many sub-cellular compartments [24].…”

“…This small, uncharged molecule can easily cross cell membranes and localize in many sub-cellular compartments [24]. In the presence of redoxactive metal ions, H 2 O 2 decomposes to the highly reactive hydroxyl radical (  OH) [23,25,26] that is presumed to contribute to cytotoxic effects in cells. Even though photodynamic damage mediated by rose Bengal is usually thought to involve singlet oxygen ( 1 O 2 ) [27], in the presence of an appropriate electron donor and adequate concentration of iron ions, rose Bengal can also generate strongly oxidizing radicals [27][28][29].…”