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Mechanism of Cell Death Induced by in Vitro Hypoxia/Reoxygenation Using Glucose Oxidase in Human Cells
Author(s) -
Dubick Michael,
Barr Johnny,
Bowman Phillip
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.977.3
Subject(s) - programmed cell death , propidium iodide , hypoxia (environmental) , apoptosis , chemistry , in vitro , hela , glucose oxidase , in vivo , mitochondrion , cell culture , pharmacology , microbiology and biotechnology , biochemistry , biology , oxygen , enzyme , organic chemistry , genetics
Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke and heart attack. This study investigated the mode of cell death induced in an vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce I/RI. The usual method of subjecting cells to reduced oxygen (O 2 ) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O 2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O 2 level (< 0.4 µM) within 5 min with only minor reductions in glucose levels. Human umbilical vein endothelial cells (HUVEC) and HeLa cells were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. Cells loaded with tetramethylrhodamine ethyl ester (TMRE) and subjected to hypoxia showed that the mitochondrial permeability transition pore was compromised by 3 hrs and mitochondria were losing the ability to retain TMRE. Significant propidium iodide (PI)‐positive HUVEC cells, an indicator of cell death,were evident by 6 hrs. Similar responses were observed in HeLa cells indicating a generalized necroptotic cell death. When catalase was included in the medium following GOX‐induced hypoxia, cell death was prevented implicating hydrogen peroxide as an important contributor to this injury. This in vitro system can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo. Funded by US Army Medical Research Materiel Command.