Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2

The aim of this study was to evaluate melatonin cytotoxicity by measuring i ts effects on various cellular targets. Cell viability, intracellular reduc ed glutathione (GSH) level, and reactive oxygen species (ROS) production we re assessed in the human liver cell line (HepG2), after incubation with inc reasing melatonin concentrations (0.1-10,000 muM). The incubation times tes ted were 24, 72, and 96 h for cell viability and intracellular GSH level, a nd 15 and 45 minutes for ROS production. Cellular target evaluations were p ossible in living cells by means of a new microplate cytofluorimeter. This technology was suitable for the assessment of cell viability, GSH level, an d ROS overproduction with, respectively, neutral red, monochlorobimane (mBC l), and 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probes. At the lowest melatonin concentrations (0.1-10 muM) and for a relatively shor t incubation time (24 h), the antioxidant effect of melatonin was revealed by an increased intracellular GSH level, associated to cell viability impro vement. In contrast, after longer incubation (96 h), cell viability signifi cantly decreased with these lowest melatonin concentrations (0.1-10 muM). M oreover, high melatonin concentrations (1,000-10,000 muM) induced GSH deple tion. This oxidative stress is associated with ROS overproduction from 10 m uM after only 15 minutes of incubation. This dual effect is strong evidence that, in vitro, melatonin can be both antioxidant and prooxidant on the hu man liver cell line, depending on the concentration and incubation time. (C ) 2000 Elsevier Science Inc. All rights reserved.