Evidence for the involvement of oxygen free radicals in the ethanol-induced late cellular injury in mouse myeloma cells

Abstract

In this study, we analysed the ethanol-induced long term cell injury on a general cell model (Sp2/0-Ag14 cell line). Cells were incubated in 1, 5, 10, 15 and 20% of ethanol (EtOH) for 5 min. After washing cell viability was tested by the Trypan Blue exclusion test in 5, 60 min, 4 and 24 h after EtOH exposure. Free radicals were monitored every 30 min by electron spin resonance (ESR) with alpha-phenyl-N-tert-butylnitrone (PBN) spin trapping technique. Scavenger compounds such as glutathione (GSH), dimethyl sulfoxide (DMSO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were applied for 24 h incubation after EtOH exposure. EtOH concentration dependently decreased the cell viability immediately after 5 min exposure, but with 4 and 24 h, a secondary cell destruction was found. Using ESR-spin trapping technique, an increased free radical activity could be detected. DMPO, DMSO and GSH significantly, but in different period protected the cells against free-radical induced cellular damage. EtOH produces an early (immediately after EtOH exposure) and a late (in about 4 h) cellular damage on Sp2/0-Ag14 cells. The oxygen free radicals can be detected in a short time after EtOH exposure, its biological effect manifested as a secondary cell destruction at 4 and 24 h. This phenomenon can be prevented by scavenger compounds.

Introduction

Oxygen radicals are involved in the pathogenesis of ethanol (EtOH)-induced tissue injury in vivo. Besides its lipid solvent property, ethanol generates hydrogen peroxide which can be a damaging molecular species toward biomolecules, e.g. lipoperoxides [5]. However, lipid peroxidation is one of the major causative factor in cell injury produced by the free radical processes. There are many experimental evidence regarding the role of EtOH-induced tissue injury mediated by free radicals in the liver [1], in the brain [6] and in the gastrointestinal tract [3], [7], [10].

These in vivo mechanisms of tissue injury differ from the direct cellular damage, because many other factors might influence and modify the effect of ethanol on the cells. Regarding these facts, the exact mechanisms of the injury at the level of the cells can only be analysed by in vitro systems. Experimental data were reported on the effect of EtOH on primary cultures of gastrointestinal cells [8], [9], [11]. These kind of examinations on oxygen free radicals were carried out within a short time after EtOH administration, and these conclusions reflect relatively acute events.

The aim of our study was to analyse the long-term (24 h) effect of different concentration (1–20%) of EtOH after 5 min exposure time on a general cell model (Sp2/0-Ag14 cell line). By the help of electron spin resonance-spin trapping technique, a possible free-radical activity was determined in the cell suspension, in addition scavenger compounds were applied to prevent the ethanol-induced long term cell injury.