Kupffer cell-independent acute hepatocellular oxidative stress and decreased bile formation in post-cold-ischemic rat liver

The purpose of this study was to examine distribution and time history of o xidative stress during the hyperacute period of reperfusion in the liver gr afts undergoing cold ischemia and to investigate roles of Kupffer cells as a potential oxidant source. Rat Livers were harvested at 4 degrees C in Uni versity of Wisconsin solution and followed by reperfusion with Krebs-Hensel eit buffer under monitoring bile excretion. To investigate oxidative change s, laser-confocal microfluorography was performed in reperfused livers prel oaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluor escence precursor sensing intracellular hydroperoxide generation. Livers un dergoing the 16-hour cold storage displayed an impaired recovery of bile ac id-dependent bile output concurrent with a marked increase in hydroperoxide generation in hepatocytes, which occurred as early as 5 minutes after the onset of reperfusion, whereas the status of lobular perfusion was well main tained. Pretreatment with liposome-encapsulated dichloromethylene diphospho nate, a Kupffer cell-depleting reagent, did neither alter the reperfusion-i nduced periportal oxidative changes nor improve the recovery of bile output in the graft. On the other hand, EPCK, a hepatotropic antioxidant composed of vitamin E phosphate ester bound to vitamin C, not only diminished the o xidative changes but also improved the reduction of bile acid-dependent bil e output. Furthermore, the reagent was capable of inhibiting H2O2-induced o xidative stress in cultured hepatocytes. These results suggest that hepatoc ytes constitute a major site of the oxidative insult triggered through Kupf fer cell-independent mechanisms and serve as an important cellular componen t to be protected by antioxidant therapeutics.