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.

Effect of immunosuppressive drugs on spontaneous DNA repair in human peripheral blood mononuclear cells.

INTRODUCTION: Immunosuppressive treatment increases the risk of post-transplant cancer. Cyclosporine reduced UV-induced DNA repair by peripheral blood mononuclear cells (PBMC) and increased cancer incidence in kidney transplant recipients. Calcineurin inhibitors (CNI), but not mammalian target of rapamycin (mTOR) inhibitors or mycophenolic acid, suppressed H2O2-induced DNA repair in human peripheral blood mononuclear cells (PBMC) in vitro at maintenance drug concentrations. DNA repair, when measured in quiescent cells, is named spontaneous DNA repair, and represents a basal ongoing DNA repair in response to endogenous DNA damage. The effect of immunosuppressive drugs on spontaneous DNA repair has not been investigated. AIM: To investigate the effect of currently used immunosuppressive drugs on spontaneous DNA repair. METHODS: Spontaneous DNA repair by human PBMC was tested in vitro in the presence of the CNI-cyclosporine and tacrolimus; mycophenolic acid (MPA); and the mTOR inhibitors-sirolimus and everolimus, at low to high nontoxic concentrations. RESULTS: Cyclosporine and tacrolimus suppressed spontaneous DNA repair throughout the tested dose range. In contrast, MPA, sirolimus and everolimus did so only at the high doses. CONCLUSION: A reduction in CNI dosage may lead to a decrease in the occurrence of post-transplant malignancy.

Effect of immunosuppressive drugs on DNA repair in human peripheral blood mononuclear cells.

INTRODUCTION: Cancer is a major cause of mortality among transplant recipients. Immunosuppressive treatment is a modifiable factor contributing to this phenomenon. Cyclosporine in kidney transplant recipients was associated with reduced UV-induced DNA repair by peripheral blood mononuclear cells (PBMC) and increased cancer rate. H(2)O(2) is a common cellular reactive oxygen species (ROS), which induces DNA damage followed by DNA repair. AIM: To investigate the effect of currently used immunosuppressive drugs on DNA repair. METHODS: H(2)O(2)-induced DNA repair by human PBMC was tested in vitro in the presence of the calcineurin inhibitors (CNI) cyclosporine and tacrolimus, mycophenolic acid (MPA), and the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus, at low to high non-toxic concentrations. The effect of combination therapy at maintenance levels was also tested. RESULTS: Cyclosporine and tacrolimus suppressed DNA repair throughout the tested dose range. In contrast, MPA, sirolimus and everolimus did so only at the high doses. Maintenance doses of a combination of tacrolimus and MPA, the most frequent treatment regimen, reduced DNA repair, while MPA with sirolimus or everolimus did not. CONCLUSION: In an attempt to reduce the risk of post-transplantation malignancy, treatment protocols may be modified by reducing CNI dose.

Inhibition of mitochondrial function reduces DNA repair in human mononuclear cells.

BACKGROUND: Mitochondria provide ATP and Ca(2+) needed for DNA repair, but also produce reactive oxygen species (ROS), which may damage DNA. AIM: To investigate the effect of mitochondrial function inhibition on DNA repair. METHOD: Five mitochondrial inhibitors acting at various sites of electron transport were studied. Human peripheral blood mononuclear cells, spontaneous and H(2)O(2)-induced DNA repair, as well as %-double-stranded-DNA, were measured. RESULTS: All mitochondrial inhibitors suppressed spontaneous and H(2)O(2)-induced DNA repair. However, their effect on %-double-stranded-DNA differed, which is partly related to ROS suppression. CONCLUSION: Mitochondrial inhibition may enhance efficacy and reduce toxicity of radiation and cytotoxic drugs therapy.

Spontaneous DNA repair increases during hemodialysis.

BACKGROUND: Hemodialysis (HD) patients are subjected to increased oxidative stress. Oxidative stress causes DNA damage, which may be repaired by a DNA repair system. 'Spontaneous DNA repair' expresses DNA repair of in vitro unstimulated cells. The aim of the study was to evaluate the effect of one HD session on spontaneous DNA repair in peripheral blood mononuclear cells (PBMC). METHODS: PBMC were separated from blood samples for the determination of spontaneous DNA repair, measured by (3)H-thymidine incorporation, before and immediately after one HD session. Percent double-stranded DNA (ds-DNA) was measured by the fluorometric assay of DNA unwinding (FADU). RESULTS: DNA repair increased significantly following HD. To examine if this increase was caused by newly produced DNA damage, we studied the effect of HD on percent ds-DNA in PBMC. HD significantly reduced percent ds-DNA, indicating increased DNA breakage. By repeating FADU in the presence of formamidopyrimidine-DNA glycosylase (Fpg), which nicks DNA at oxidized purine sites, we could show that the increased DNA damage was caused by oxidation. CONCLUSION: Spontaneous DNA repair increases during HD in response to an increase in DNA damage induced by oxidative stress.

Spontaneous DNA repair in human mononuclear cells is calcium-dependent.

Spontaneous DNA repair in peripheral blood mononuclear cells (PBMC) has been recently described. The aim of this study was to evaluate whether spontaneous DNA repair is Ca(2+)-dependent, as in vitro-stimulated DNA repair. Spontaneous DNA repair in PBMC was measured in a 1mM Ca2+ medium. The effect of extracellular Ca2+ chelation by EGTA, intracellular Ca2+ chelation by bapta-AM, and Ca2+ loading by the ionophore A23187 was examined. The signal transduction pathway was evaluated by inhibiting protein tyrosine kinase with genistein, calmodulin with W7, and calcineurin with cyclosporin A and tacrolimus. Extracellular Ca2+ chelation had no effect on spontaneous DNA repair, while both intracellular chelation and calcium overloading inhibited the DNA repair. Inhibition of protein tyrosine kinase, calmodulin or calcineurin reduced DNA repair. In conclusion, spontaneous DNA repair is mainly Ca(2+)-dependent at a narrow range of intracellular Ca2+ concentrations. The signal transduction cascade includes protein tyrosine kinase, calmodulin, and calcineurin.

Spontaneous DNA repair in human peripheral blood mononuclear cells.

DNA molecules are constantly damaged during mitosis and by oxygen-free radicals produced by either cellular metabolism or by external factors. Populations at risk include patients with cancer-prone disease, patients under enhanced oxidative stress, and those treated with immunosuppressive/cytotoxic therapy. The DNA repair process is crucial in maintaining the genomal DNA integrity. The aim of this study was to evaluate spontaneous DNA repair capacity of peripheral blood mononuclear cells (PBMC) from normal blood donors. PBMC DNA repair ability represents DNA repair by other tissues as well. It is shown in the present study that in vitro incorporation of [3H]thymidine in non-stimulated PBMC expresses the ability of the cells to repair DNA damage. This method was validated by double-stranded DNA measurements. Both catalase and Fe2+ increased DNA repair, the former by preventing re-breakage of newly repaired DNA and the latter by introducing additional DNA damage, which enhanced DNA repair. Better understanding of DNA repair processes will enable to minimize DNA damage induced by oxidative stress.

DNA repair in mononuclear cells: role of serine/threonine phosphatases.

Treatment with cyclosporin A (CsA) in kidney-transplant recipients is associated with reduced DNA repair and enhanced cancer incidence. CsA is an inhibitor of the serine/threonine phosphatase calcineurin, also termed PP2B, which is a Ca(2+)/calmodulin-dependent phosphatase. In this study we sought to elucidate the role of calcineurin in DNA repair using CsA and tacrolimus; examine whether UV-induced DNA repair is associated with dephosphorylation; and investigate whether phosphatases other than calcineurin are active in DNA repair, in light of the fact that calcineurin inhibition only partially suppressed DNA repair. Peripheral blood mononuclear cells from healthy donors were used. In vitro, we assayed UV-induced DNA repair by measuring the incorporation of tritiated thymidine in UV-irradiated cells. We gauged phosphatase activity indirectly by measuring free inorganic phosphate (Pi) excreted into the medium. The phosphatase assay was performed under the same conditions and in parallel to the DNA-repair assay. Tacrolimus, like CsA, inhibited DNA repair in a dose-dependent fashion. DNA repair was associated with production of Pi, which correlated with the number of cells performing DNA repair. Phosphatase activity increased after UV irradiation. DNA repair correlated directly with phosphatase activity, whereas CsA reduced both DNA repair and Pi production. Inhibition of calmodulin by trifluoperazine and W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide] reduced DNA repair in part. We investigated the role of the Ca(2+)-independent phosphatases PP1 and PP2A using specific inhibitors. Calyculin A, which inhibits both phosphatases, reduced DNA repair. Endothall, a PP2A inhibitor, had no effect on DNA repair. Okadaic acid, which is mostly a PP2A inhibitor but also a weak inhibitor of PP1, reduced DNA repair only slightly. We suggest that DNA repair is mediated by way of Ca(2+)-dependent and Ca(2+)-independent pathways, with calcineurin and PP1 being the respective phosphatases involved in each pathway.

The contribution of an arteriovenous access for hemodialysis to left ventricular hypertrophy.

BACKGROUND: The long-term isolated contribution of hemodialysis arteriovenous access (AVA) to cardiac hemodynamics has not been previously investigated in a prospective manner. METHODS: Twelve predialysis patients were studied before and 1 and 3 months after creation of a primary AVA. Evaluation included relevant clinical parameters, echocardiographic studies, and hemodynamic hormones. RESULTS: After creation of an AVA, there was no change in patient weight, blood pressure or hemoglobin level. Cardiac index increased and systemic vascular resistance decreased. Left ventricular mass (LVM) corrected to height increased from 63.8 +/- 5.5 to 68.9 +/- 4.9 g/m(2.7) at 1 month (P = 0.05) and 72.5 +/- 8.9 g/m(2.7) at 3 months (P < 0.05). This increase in LVM was accounted for mostly by an increase in interventricular septal thickness, whereas left ventricular end-diastolic diameter and posterior wall thickness did not change. The incidence of left ventricular hypertrophy (LVH) increased from 67% at baseline to 83% and 90% at 1 and 3 months, respectively. Left atrial area increased from 17.6 +/- 1.0 cm(2) at baseline to 19.7 +/- 1.3 cm(2) at 1 month (P < 0.01) and 20.2 +/- 1.2 cm(2) at 3 months (P < 0.05). Early diastolic transmitral flow increased from 68.0 +/- 4.2 cm/s at baseline to 85.6 +/- 7.3 and 89.2 +/- 6.5 cm/s at 1 and 3 months, respectively (P < 0.01). Inferior vena cava diameter increased at 1 month and did not change at 3 months. Plasma atrial natriuretic polypeptide levels increased from 268 +/- 35 pg/mL (87 +/- 11 pmol/L) at baseline to 461 +/- 63 pg/mL (150 +/- 20 pmol/L) at 1 month (P < 0.01) and 610 +/- 96 pg/mL (198 +/- 31 pmol/L) at 3 months (P < 0.01). Plasma renin activity and serum aldosterone levels decreased. Plasma angiotensin II, angiotensin-converting enzyme, and endothelin levels did not change. CONCLUSION: Creation of a hemodialysis AVA is independently associated with further progression of already existing LVH.