Melatonin improves mitochondrial function in rat liver after cold storage and normothermic reoxygenation.

Restoring function after cold preservation is a critical issue in liver transplantation. Oxidative stress and mitochondria dysfunction are major effectors of the ischemia-reperfusion (I/R) injury. Melatonin (N-acetyl-5-methoxytryptamine) has a great potential to help the liver overcoming I/R stress. It is non toxic even at high concentration, is a wide-spectrum antioxidant, and increases electron transfer in the respiratory chain while protecting mitochondria from oxidative stress. Gastro-intestinal-melatonin is thought to control hepato-biliary functioning and bile-melatonin to protect biliary and intestinal cells from oxidative stress. Melatonin showed to protect the liver from warm I/R damage. To test melatonin against cold I/R damage we used the isolated and perfused rat liver model and compared two preservation solutions, the conventional University of Wisconsin (UW) as opposed to Celsior solution. Livers preserved for 20h at 4°C were reperfused for 2h with normothermic, oxygenated KHB without or with 100M melatonin. Reperfusion with melatonin increased bile production and liver ATP respect to the reperfusion without melatonin, especially when the cold preservation was made with Celsior solution. No significant effect was found for melatonin on lipid peroxidation (TBARS), which was very low for both solutions even when the reperfusion was made without melatonin, neither on the tissue GSH/GSSG ratio. These results suggested that melatonin was effective since it improved hepatocyte respiration rather than behaving as an antioxidant. To test this we demonstrated reactive oxygen species (ROS) generation with Kerver et al.’s DAB-Mn2+-Co2+ method (1997) which visualizes mainly superoxide anion leaking from mitochondria electron chain or being produced as a cytotoxic agent during NADPH oxidase activity in phagocytic cells. In control rat liver, the ROS reaction was strong in portal and mid-zonal hepatocytes, reflecting their intense aerobic metabolism; several sinusoidal cells were positive as well. After cold preservation and reperfusion without melatonin the livers were edematous (especially for Celsior preservation), and ROS staining was restricted to a narrow hepatocyte layer, suggesting poor metabolic activity. The absence of reaction in sinusoidal cells was in keeping with the low TBARS reaction in the perfusate. By contrast, when reperfusion was made with melatonin, liver morphology was markedly improved and the ROS reaction was located in a wider area of hepatocytes for both preservation solutions. In conclusion, the histochemical data confirm that melatonin improves electron transfer in mitochondria and support the usefulness of the indole in liver transplantation. (COFIN 2001,04; FAR-UniPv)