Protection of Mitochondria Permeability Transition by Dihydroxybenzaldehyde against Hydroxyl Radicals

Abstract

Accumulated evidence strongly implicates the role of mitochondria in cell death that is often accompanied by a release of its components such as cytochrome c and apoptosis-inducing factor. The release of cytochrome c from the mitochondrial electron transport chain can cause the impairment of ATP generation, resulting in eventual cell death. Mitochondrial integrity and its Ca 2+ homeostasis are tightly regulated by Ca 2+ channels coupled with other ion permeability movements. Under increased oxidative stress, cells may undergo death because of dysregulation of [Ca 2+ ] i . A mitochondrial Ca 2+ megachannel, located in the inner membrane, may play a crucial role in the maintenance of Ca 2+ homeostasis through a mitochondria permeability transition (MPT) process. Because hydroxyl radicals are the most destructive oxidants, in the present study, we investigated the protection of 3,4-dihydroxybenzaldehyde (3,4-DB), isolated from Salvia miltiorrhiza and red algae, and its derivatives against hydroxyl radical attack on MPT. To monitor the cell viability by the protective effect of DB against hydroxyl radical-induced cell lysis, trypan blue exclusion was used. MPT activity was monitored by changes in optical density of suspended mitochondria in an appropriate media by spectrophotometry. Results found that treatment of mitochondria with 3,4-DB showed protection with increased cell viability in a dose-dependent manner. Mitochondrial ROS generation by hydroxyl radicals was also suppressed by 3,4-DB. It was found that hydroxyl radical challenge caused high [Ca 2+ ] i levels that were suppressed by 3,4-DB. Hydroxyl radicals caused the opening of the megachannel, allowing swelling of mitochondria. It was found that this structural alteration resulted in the release of cytochrome c from the inner membrane of mitochondria into the incubating media. Data showed that 3,4and 2,3-DB effectively suppressed MPT, and consequently prevented cytochrome c release. Since hydroxyl radicals deplete mitochondrial GSH levels, mitochondrial GSH content was measured. When mitochondria were treated with 3,4-and 2,3-DB, the GSH depletion was prevented, thus showing their sparing action of GSH. We conclude that DB protects mitochondria against hydroxyl radical attack by at least two processes: protection of the megachannel and sparing the mitochondrial GSH level. Further investigation on the precise mechanisms underlying the nature of protection of MPT, cytochrome c release, and sparing of GSH provides a better understanding of mitochondrial Ca 2+ homeostasis.