Cold storage sensitizes hepatocytes to oxidative stress injury.

Abstract

Liver cold storage leads to oxygen free radical production and reperfusion injury. Antioxidants are effective in suppression reperfusion injury in rat livers when used in the reperfusion medium. However, in clinical liver transplantation their effectiveness is not clear, which may be due to the way they are used (in the recipient). In this study we compare the effectiveness of antioxidants when used in the reperfusion medium versus the cold storage solution in isolated hepatocytes and the isolated perfused liver. Hepatocytes were cold stored in UW solution for 24 h. Oxidative stress, induced by t-butyl hydroperoxide (tBHP), was measured in the presence of one of five different antioxidants--deferoxamine (DFO), dithiothreitol (DTT), trolox, tocopherol, dimethylthiourea (DMTU)--in the reperfusion buffer or UW solution. Efficacy was judged by reduction in membrane damage (LDH release) during rewarming. Also, rat livers were cold stored for 48 h in UW solution (+/- antioxidant) and reperfused (+/- antioxidants). Efficacy was judged by the effect on enzyme release and bile production. Cold storage of hepatocytes for 24 h sensitized them to oxidative stress. The concentration of tBHP required to induce maximal cell death (80%-90% LDH release) was reduced from 1.3 mM (fresh cells) to 0.37 mM (LD-50 values). All antioxidants except DMTU suppressed oxyradical-induced LDH release when used in the reperfusion medium, but only DFO was effective when used in the UW solution. In the isolated perfused liver, DFO, DTT, and trolox were effective and suppressed enzyme release when added to the reperfusion buffer, but none were effective when used in the UW solution. We conclude that cold storage sensitizes liver cells to oxidative stress. The most effective antioxidant was the iron chealator, DFO, which was effective in the reperfusion buffer (isolated perfused sliver or hepatocytes) but not in the UW solution when tested in the isolated perfused liver. Suppression of reperfusion injury in liver transplantation could be obtained by antioxidant therapy. However, it is unclear how best to deliver the antioxidants to the site of oxyradical generation.