Mechanisms of Liver Cell Death Due to Hypoxic Injury Under Conditions of “Chemical Hypoxia”
Chemical hypoxia occurs when liver cells are exposed to a combination of potassium cyanide, which blocks oxidative phosphorylation in the mitochondria, and iodoacetic acid, which blocks glycolysis in the cytosol. This effectively shuts off all energy production in the cell and leads to cell death. This “chemical hypoxia” model in some ways mimics the cell death due to ischemia/reperfusion injury. The precise mechanism is as yet unknown, but it appears to be linked to the release of free iron in the cytosol and subsequent uptake of that iron into the mitochondria. Our laboratory is studying various chemicals which show a protective effect in hepatocytes against this cell death in an effort to determine the precise mechanism and biochemical pathways responsible for cell death under this model. We hypothesize that chemicals like ethyl-3,4-dihydroxybenzoate (EDHB), 2,2'- Dipyridyl (DPD), and deferoxamine myselate (desferal, DFO) protect against hypoxic cell death by bonding to, or chelating, free iron in the cytosol, thereby preventing it from entering into the mitochondria where it increases the production of reactive oxygen species leading to cell death.Polarized mitochondria are imaged using tetramethyl rhodamine methylester (TMRM, red), and cytosol is imaged using calcein AM (green). Propidium iodide (PI, red) is used to indicate cell death. When TMRM fluorescence disappears, the mitochondria have become depolarized and are not functioning to produce energy in the cell. When the calcein fluorescence disappears, the cell membrane has become permeabilized and the cell is dying. When the cell membrane permeablizes, PI enters the cell and bonds with the nuclear material, producing red fluorescence. This indicates cell death.Figure 1 shows cells exposed to chemical hypoxia and treated with EDHB compared to untreated cells exposed to chemical hypoxia. Some cells treated with EDHB are still living after 120 min, while untreated cells have all died.
Figure 1. Cytoprotection of hepatocytes exposed to chemical hypoxia and treated
with EDHB. Treated cells remain alive after 120 min as opposed to 100% cell death
in cells not receiving treatment with EDHB