Oxidative stress-induced DNA damage and repair in human peripheral blood mononuclear cells: protective role of hemoglobin.

BACKGROUND: DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response. AIM: To investigate the effect of red blood cells on H2O2-induced DNA damage and repair in human peripheral blood mononuclear cells. METHODS: DNA breaks were induced in peripheral blood mononuclear cells by H2O2 in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by (3)H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation. RESULTS: Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA. CONCLUSION: Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage

Dose-response curve of the effect of RBC hemolysate (% ^v/_v in medium) on ds-DNA ratio in H₂O₂-stimulated PBMC.

<p>p<0.001 for the effects of the 0.3, 0.6, 1 and 2% ^v/_v RBC hemolysate concentrations.</p

Dose-response curve of the effect of RBC hemolysate (% ^v/_v in medium) on H₂O₂-induced DNA repair (% of control) in PBMC.

<p>For the effects of 0.3, 0.6, 1 and 2% ^v/_v hemolysate, p = NS, p = NS, p<0.005 and p<0.005, respectively.</p

Correlation between ds-DNA ratio (control = 1.00) and DNA repair (% of control) in H₂O₂-stimulated PBMC, at concentrations of 0, 0.3, 0.6, 1 and 2% ^v/_v RBC hemolysate.

<p>For each point, N = 8. Coefficient r = −0.925, p<0.025. Regression line: y = −0.131x +2.432.</p

ds-DNA ratio in H₂O₂-stimulated PBMC: effect of purchased human hemoglobin (1.8 mg/ml) and RBC hemolysate (2% ^v/_v, mean hemoglobin content = 2.00±0.07 mg/ml).

<p>b vs a, p<0.001; c vs a, p<0.001; N = 8.</p

Comet assay – a representative experiment (N = 3).

<p>The left image depicts the control. The middle image depicts the effect of H₂O₂. The right image depicts the effect of H₂O₂ and RBC hemolysate.</p

Dose response curve of the effect of H₂O₂ on ds DNA.

<p>Effect of 10, 20, 50, 100 and 200 µmol/L H₂O₂ on PBMC was tested: 100 and 200 µmol/L reduced %ds DNA significantly (p<0.02, p<0.01, respectively).</p