Study of Ferredoxin NADP(H) Oxidoreductase effect in Cos-7 cells injured by hypothermia

PUCCI MOLINERIS, M.; MAMPRIN, M. AND MEDIAVILLA, M.

Rosario

Congreso; 49th Annual Meeting of the Society for Cryobiology; 2012

CAIC- Society for Cryobiology

 Mammalian cellular injury produced by cold incubation is mediated, at least in part, by an increase in the intracellular chelatable iron pool. Such increment results in an elevation of reactive oxygen species (ROS), especially hydroxyl radicals (OH?-) and related ferril species. These react with a number of cellular components, especially lipids that propagate the process to close molecules causing functional disruption and finally cellular death when temperature is raised again to physiological levels. Lipoperoxidation and the strong inhibition exerted by hypoxia, antioxidants, radical scavengers and iron chelators clearly demonstrate the participation of ROS in the damage produced by cold exposure. There exists evidence that Ferredoxin NADP(H) Oxidoreductase (FNR) of Pisum sativum is cytoprotective under oxidative stress conditions when damage is mediated by a raise of iron. This enzyme has demonstrated antioxidant activity in a number of bacterial and plant models and also Cos-7 cells submitted to oxidative insults (Mediavilla et al. PLoS ONE 2010). Hence, we studied FNR capability to protect Cos-7 cells from cold damage and characterize the phenomenon occurring. Cells expressing FNR (Cos-7/FNR), and control cells transfected with the empty plasmid, were incubated (4, 24h) under hypothermia (4ºC) in homemade Histidine-Tryptophan-Ketogutarate (HTK) and modified University of Wisconsin (mUW) preservation solutions, in Krebs-Henseleit buffer or in complete DMEM. For comparison, a set of cultures was submitted to iron overload (4.5 µM). We studied viability (MTT reduction) and malondialdehyde (MDA) levels. Also, we evaluated the effect of 10 mM DFO (iron chelator) and 10% Me2SO (OH?- scavenger) on cell viability. n=3 independent experiments in duplicate. Saline solutions and mUW were discarded because the firsts killed the totality of the cells at 24h and mUW caused almost no damage (70% at 48h). When exposed to hypothermia in HTK solution no significant differences were found between Cos-7/FNR and control viabilities (Cos-7/FNR:  94.2±0.5% at 4h, 43.8±2.8% at 24h; Cos-7: 93.6±9% at 4 h, 46.2±0.4% at 24h). The same observation was found for a Fe overload (at 24h 31.3±0.6% for Cos-7/FNR and 40.8±7.6% for Cos-7). MDA levels did not rise significantly albeit viabilities were reduced. After 24h of HTK/DFO treatment, MTT reductions were 50.0±3.1% for Cos-7/FNR and 53.7±1.1% for Cos-7. After 24h of HTK/Me2SO treatment MTT results were 59.2±1.7% for Cos-7/FNR and 61.1±2.2% for Cos-7. For Fe-treated cells, DFO completely reverted the damage (at 24h 100.4±2.3% for Cos-7/FNR and 94.8±2.1% for controls). We did not observed protection from FNR against cold injury or Fe overload, in contradiction of previous observations. In Cos-7 cells the mechanisms of action of this enzyme must be distinct from what observed in bacteria and plants. The use of Me2SO partially reverted the damage provoked by hypothermia in a similar extent for both groups of cells. DFO avoided the deleterious effect caused by Fe as expected and demonstrating that the dose used was appropriated. So, the damage generated by cold exposure in this model is only slightly mediated by an elevation of iron and, then, ROS of different origin must be involved.