Heterologous ferredoxin reductase and flavodoxin protect Cos-7 cells from oxidative stress.

BACKGROUND: Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro. PRINCIPAL FINDINGS: Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300-600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes. SIGNIFICANCE: The results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells

Subzero nonfreezing storage of rat hepatocytes using modified University of Wisconsin solution (mUW) and 1,4-butanediol. I-effects on cellular metabolites during cold storage

Various cryopreservation techniques have been investigated to extend the storage of isolated hepatocytes; however, most have a reduced viability after rewarming due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. In the present work we evaluated the viability and metabolic parameters of isolated rat hepatocytes preserved in subzero nonfreezing condition. Cell suspensions were maintained in modified University of Wisconsin (mUW) solution using 8% -1,4-butanediol as cryoprotectant, up to 120 h at -4°C. The time course evolution of hepatocytes viability were measured by LDH release and propidium iodide assay. The cellular concentrations of glutathione, ATP, glycogen and the lactate production during cold storage were also determined. Finally, results were compared with conventional hypothermic storage at 0 °C in mUW solution without cryoprotectant. After 5 days of subzero storage, we found an improvement in the ability of rat hepatocytes to maintain the metabolic resources in comparison with the cold preserved group

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes

In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 °C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 °C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0°C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups.In conclusion: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices