Release of reactive oxygen by hepatocytes on reoxygenation: three phases and role of mitochondria.

Reoxygenation of isolated hepatocytes in primary culture resulted in a three-phase response in the release of reactive oxygen species (ROS) as determined by peroxidase-dependent luminol chemiluminescence. Release of ROS within the first and second phase correlated well with the extent of reoxygenation injury, both being most significant after approximately 4 h of hypoxic incubation. During the third phase, some of the ROS were released by already nonviable cells. Both antimycin A and rotenone significantly increased release of ROS, indicating severe alterations of the mitochondrial respiratory chain caused by hypoxia and suggesting that the altered mitochondrial respiratory chain represents an important source for the release of ROS on reoxygenation. Generation of ROS rose sharply when the O2 content was increased from 0 to 2%, whereas a further increase in the O2 content, of up to 95%, resulted in only small but steady increases in the formation of ROS. The latter suggests that, in addition to enzymatic sources such as the mitochondrial respiratory chain, nonenzymatic reactions may also contribute to the formation of ROS on reoxygenation.