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. 2010 Oct 19;5(10):e13501.
doi: 10.1371/journal.pone.0013501.

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

María G Mediavilla  1 , Gisela A Di Venanzio, Edgardo E Guibert, Claudio Tiribelli
Affiliations

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

María G Mediavilla et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cytotoxicity after 24 h exposure to H2O2.
(A) Cellular viability measure as LDH retention (n = 4 experiments, in triplicate). (B) Cellular metabolism measured as the reduction of MTT (n = 12 experiments, in triplicate). (•) Cos-7 cells, (○) Cos-7/pcDNA3 cells (▪), Cos-7/pFNR cells and (▴) Cos-7/pFld cells. a different from Cos-7 (p<0.05), b different from Cos-7/pcDNA3 (p<0.05), c different from Cos-7/pFNR (p<0.05).
Figure 2
Figure 2. Oxidative damage to lipids after 24 h exposure to H2O2.
(A) MDA levels. (B) Peroxide levels (FOX assay). (•) Cos-7 cells, (○) Cos-7/pcDNA3 cells, (▪) Cos-7/pFNR cells and (▴) Cos-7/pFld cells. a different from Cos-7 (p<0.05), b different from Cos-7/pcDNA3 (p<0.05), c different from Cos-7/pFNR (p<0.05), d different from Cos-7/pFld (p<0.05). n = 4 experiments, in triplicate.
Figure 3
Figure 3. Cytotoxicity after 48 h exposure to MV.
(A) Cellular metabolism (MTT reduction). (B) Lipid peroxidation levels (FOX assay). Black bars: Cos-7 cells, dark grey bars: Cos-7/pcDNA3 cells, light grey bars: Cos-7/pFNR cells and white bars: Cos-7/pFld cells. a different from Cos-7 (p<0.05), b different from Cos-7/pcDNA3 (p<0.05), c different from Cos-7/pFNR (p<0.05), d different from Cos-7/pFld (p<0.05). n = 4 experiments, in triplicate.
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References

    1. Carrillo N, Ceccarelli EA. Open questions in ferredoxin-NADP+ reductase catalytic mechanism. Eur J Biochem. 2003;270:1900–1915. - PubMed
    1. Krapp AR, Tognetti VB, Carrillo N, Acevedo A. The role of ferredoxin-NADP+ reductase in the concerted cell defense against oxidative damage – studies using Escherichia coli mutants and cloned plant genes. Eur J Biochem. 1997;249:556–563. - PubMed
    1. Pomposiello PJ, Demple B. Identification of SoxS-regulated genes in Salmonella enterica serovar typhimurium. J Bacteriol. 2000;182:23–29. - PMC - PubMed
    1. Krapp AR, Carrillo N. Functional complementation of the mvrA mutation of Escherichia coli by plant ferredoxin-NADP+ oxidoreductase. Arch Biochem Biophys. 1995;317:215–221. - PubMed
    1. Yousef N, Pistorius EK, Michel KP. Comparative analysis of idiA and isiA transcription under iron starvation and oxidative stress in Synechococcus elongatus PCC 7942 wild-type and selected mutants. Arch Microbiol. 2003;180:471–483. - PubMed

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