Sulforaphane inhibits redox-regulated permeability transition and provides neuroprotection following cardiac arrest

Abstract

Exposure of mitochondria to oxidative stress and elevated Ca2+ promotes opening of the mitochondrial permeability transition pore, resulting in mitochondrial membrane depolarization, metabolic failure, and necrotic cell death. Pharmacologic activation of the Nrf2/ARE pathway of antioxidant gene expression by sulforaphane provides cytoprotection, but little is known about the effects of this pathway on mitochondrial responses to stress. This study tested two hypotheses: 1. Administration of sulforaphane increases the resistance of mitochondria from cells and tissues to permeability transition pore opening caused by oxidative stress. 2. Treatment of animals with sulforaphane following cardiac arrest reduces hippocampal neuronal death and improves neurologic outcome. Hypothesis 1. Sulforaphane or drug vehicle was administered to rats intraperitoneally 40 hr prior to isolation of brain or liver mitochondria. Cultured PC12 cells were exposed to sulforaphane or vehicle for 24 hr. Fluorescent measurements of mitochondrial Ca2+ uptake and subsequent release induced by the oxidant tert-butyl hydroperoxide were performed using isolated brain and liver mitochondria and digitonin permeabilized PC12 cells, while simultaneously monitoring NAD(P)H autofluorescence. Sulforaphane treatment substantially reduced the rate of cyclosporin A-sensitive Ca2+ release induced by tert-butyl hydroperoxide by 25% in brain mitochondria and 50% in both liver mitochondria and PC12 cells, but only inhibited NAD(P)H oxidation in liver mitochondria. Sulforaphane treatment significantly increased peroxide detoxification, glutathione, and expression of glutathione peroxidase, malic enzyme, and thioredoxin in liver mitochondria. Hypothesis 2. Anesthetized beagles were subjected to 10 min cardiac arrest followed by resuscitation and critical care. At 30 min reperfusion, dogs received either sulforaphane or drug vehicle, intravenously. At 23 hr animals were awakened and a neurologic deficit test performed. Animals were euthanized at 24 hr; hippocampal neurons and markers of oxidative stress were quantified. Sulforaphane treatment improved neurologic outcome and reduced neuronal death and DNA oxidation. We conclude that sulforaphane administration after cardiac arrest significantly reduces hippocampal neuronal death and oxidative stress and improves short-term neurologic outcome. As permeability transition pore opening plays an important role in many examples of acute cell death, the ability of sulforaphane to confer resistance of mitochondrial to permeability transition pore opening may contribute to the cytoprotective properties of sulforaphane.