Effects of hyperthermia and nicotinamide on DNA repair synthesis, ADP-ribosyl transferase activity, NAD+ and ATP pools, and cytotoxicity in gamma-irradiated human mononuclear leukocytes

Effects of hyperthermia and nicotinamide on ADP-ribosyl transferase activity (ADPRT), unscheduled DNA synthesis (UDS), NAD+- and ATP-pools and cytotoxicity were investigated in gamma-irradiated human mononuclear leukocytes. A significant decrease in radiation-induced UDS after heat treatment for 45 min was found. Nicotinamide increased the UDS levels in irradiated cells, but no effect of hyperthermia on these increased UDS values was observed. In the presence of 2 mM nicotinamide radiation-induced ADPRT activity was reduced to about 50 per cent. However, hyperthermia for 45 min was found to have no effect on the enzyme activity for temperatures below 46 degrees C. Nicotinamide increased the NAD+ pool in unirradiated cells. Damaging the cells with gamma-radiation leads to a severe depletion of the NAD+ pool. The NAD+ pool is restored, however, if the cells repair for 5 h at 37 degrees C. When radiation-damaged cells were treated with hyperthermia, exogenously supplied nicotinamide could not be converted to NAD+ in sufficient amounts to prevent NAD+ depletion. These data indicate that the radiosensitizing effect of heat and nicotinamide could both be explained by effects on the enzyme ADPRT, i.e. nicotinamide by directly blocking the enzyme and hyperthermia by limiting the co-substrate (NAD+).     

Influence of hyperthermia and gamma radiation on ADP-ribosyl transferase, NAD+, and ATP pools in human mononuclear leukocytes

Effects of hyperthermia (42.5 degrees C) and gamma radiation (30 Gy) on ADP-ribosyl transferase, NAD+, and ATP pools in human mononuclear leukocytes have been investigated. It was found that the gamma-ray activation level of the enzyme was not influenced by this hyperthermia for 45 min. Following deprivation of ATP synthesis by 2,4-dinitrophenol, an uncoupler of the oxidative phosphorylation, and omitting glucose from the culture medium, the NAD+ pool was reduced to about 60% of control value. The potentiation of ATP production by exogenously supplied adenosine was reduced after a combined treatment of the cells with hyperthermia and gamma radiation. Mitochondrial and endoplasmic changes within the mononuclear leukocytes were also observed. Based on these findings a model for the hyperthermia effect is proposed.     

alpha-Interferon increases immunoglobulin production in cultured human mononuclear leukocytes

Interferons (IFN) are known to modulate immune responses in either an inhibitory or a stimulating manner. The present study was initiated to investigate the mechanisms by which alpha-IFN modulates Ig production of human peripheral blood mononuclear cells (PBMC). IgG and IgM production was measured in pokeweed mitogen- (PWM) stimulated 7-day cultures of PBMC. Significant enhancement of IgM and IgG production was observed when alpha-IFN was added. Overnight preincubation followed by washing also produced significant enhancement. The effect of alpha-IFN was not obtained in the absence of PWM or T cells. The effect of alpha-IFN on cultures of B and T cells was not altered by irradiation of T cells (2000 rad). alpha-IFN was not shown to enhance the production of helper factor but did increase the responsiveness of B cells to helper factor if the B cells were preincubated with alpha-IFN. Finally, alpha-IFN did not increase the Ig production of PBMC induced by Epstein Barr virus (EBV), and the outgrowth of EBV-infected PBMC was not affected. Overall, these results show for the first time that the effect of alpha-IFN on PBMC is due to an enhanced responsiveness of B cells to helper factors produced by radioresistant T cells.     

Correlation between DNA synthesis and intracellular NAD in cultured human leukemic lymphocytes.

The NAD⁺ level in lymphocytes obtained from an individual with acute monocytic leukemia increased five-fold and then remained constant when the cells were adapted to growth in suspension culture. When the NAD⁺ level of established cells was lowered by means of a nicotinamide-poor medium or by the action of 1-methyl-1-nitrosourea, there was a concomitant decrease in the rate of DNA synthesis. These results indicate that there is a direct correlation between intracellular NAD⁺ and the synthesis of DNA in cultured leukemic lymphocytes. However, the exact nature of the relationship remains speculative.     

Quantification of unscheduled DNA synthesis in mononuclear leukocytes of the horse

This study compares the relationship between N-acetoxy-2-acetylaminofluorene (NA-AAF) and u.v. induced unscheduled DNA synthesis (UDS) and their respective relationships to age and blood pressure in horse mononuclear leukocytes with earlier, similar investigations on human leukocytes. U.v. induced UDS was found to proceed more rapidly than NA-AAF induced UDS. A pronounced lag period associated with the rapid demand for 3H-dThd into DNA after u.v. damage was observed. NA-AAF induced UDS correlated significantly with NA-AAF binding, age and the blood pressure of male horses. UDS values, induced by either method, were about half the level calculated for human leukocytes.     

Interferon-mediated inhibition of prostaglandin synthesis in human mononuclear leukocytes

In this study, the question of whether human leukocyte-derived and fibroblast-derived interferon had an effect on prostaglandin metabolism in human peripheral blood mononuclear cells has been considered. Both leukocyte- and fibroblast-derived interferon were potent inhibitors of mononuclear cell prostaglandin synthesis at low physiological concentrations. Inhibition required a minimum incubation of 1 hr. Interferon had no effect on release of arachidonic acid; synthesis of hydroxy fatty acids was slightly increased.     

Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes

The effects of prostaglandin (PG) E1, PGE2, the stable prostacyclin analogue Iloprost, and PGF2 alpha on low density lipoprotein (LDL) receptor activity and cholesterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 45 hr in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of 125I-labeled LDL. Addition of PGE1 in increasing concentrations to the incubation medium inhibited cholesterol synthesis and the specific accumulation and degradation of 125I-labeled LDL; at a concentration of 10 microM, the inhibitions were 61%, 70%, and 67%, respectively, after an incubation of 20 hr. The effects of PGE2 and Iloprost were similar. The action of the prostaglandins on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. The prostaglandins yielded sigmoidal log concentration-effect curves. In contrast, PGF2 alpha had no influence on cholesterol synthesis or LDL receptor activity up to a concentration of 10 microM. PGE1, PGE2, and Iloprost, but not PGF2 alpha, led to an increase in the concentration of intracellular cyclic AMP. Dibutyryl cyclic AMP mimicked the effects of the E-prostaglandins and Iloprost on the LDL receptor activity. The results suggest that PGE1, PGE2, and prostacyclin affect LDL receptor activity and cholesterol synthesis and, therefore, may play a role in the regulation of cholesterol homeostasis and in the development of atherosclerosis.     

Effects of homogeneous and inhomogeneous static magnetic fields combined with gamma radiation on DNA and DNA repair

The aim of this study was to reveal whether static magnetic fields (SMFs) influence the repair of radiation‐damaged DNA on leukocytes or has any effect on DNA. After 4 Gy of ⁶⁰Co‐γ irradiation, some of the samples were exposed to inhomogeneous SMFs with a lateral magnetic flux density gradient of 47.7, 1.2, or 0.3 T/m by 10 mm lateral periodicity, while other samples were exposed to homogeneous SMF of 159.2 ± 13.4 mT magnetic flux density for a time period of 0.5 min, 1, 2, 4, 6, 18, 20, or 24 h. Another set of samples was exposed to the aforementioned SMFs before gamma irradiation. The following three groups were examined: (i) exposed to SMF only, (ii) exposed to SMF following irradiation by ⁶⁰Co‐γ, and (iii) exposed to SMF before ⁶⁰Co‐γ irradiation. The analysis of the DNA damage was made by single‐cell gel electrophoresis technique (comet assay). Statistically significant differences were found at 1 h (iSMF), 4 h (hSMF), and 18 h (hSMF) if samples were exposed to only SMF, compared to control. When the SMF exposure followed the ⁶⁰Co‐γ irradiation, statistically significant differences were found at 1 h (iSMF) and 4 h (hSMF). If exposure to SMF preceded ⁶⁰Co‐γ irradiation, no statistically significant difference was found compared to 4 Gy gamma‐irradiated group. Bioelectromagnetics 31:488–494, 2010. © 2010 Wiley‐Liss, Inc.     

Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes

The effects of microwave radiation (2450 MHz, continuous wave, mean specific absorption rate of 103.5 +/- 4.2 W/kg) and convection heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 degrees C were investigated. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photometer. A significant stimulation after microwave exposure (p less than 0.005) over total CL of matched 37 degrees C incubator controls was observed. A similar degree of stimulation compared to incubator controls was also detected after sham treatment. There was no significant difference between changes in total CL or stimulation indices of the microwave and sham exposed groups. It appears that exposure to microwave radiation, under normothermic (37 +/- 0.03 degrees C) conditions, has no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between microwave or sham exposed cells and their respective incubator controls occurred because the temperature of the incubator controls did not exceed 35.9 degrees C and this temperature required 39 minutes to reach from 22 degrees C. Slow heating of incubator controls must be accounted for in thermal and radiofrequency radiation studies in vitro.     

NAD+: The convergence of DNA repair and mitophagy

ATM is a 350 kDa serine/threonine kinase best known for its role in DNA repair and multiple cellular homeostasis pathways. Mutation in ATM causes the disease ataxia telangiectasia (A-T) with clinical features including ataxia, severe cerebellar atrophy and Purkinje cell loss. In a cross-species study, using primary rat neurons, the roundworm C. elegans, and a mouse model of A-T, we showed that loss of ATM induces mitochondrial dysfunction and compromised mitophagy due to NAD⁺ insufficiency. Remarkably, NAD⁺ repletion mitigates both the DNA repair defect and mitochondrial dysfunction in ATM-deficient neurons. In C. elegans, NAD⁺ repletion can clear accumulated dysfunctional mitochondria through restoration of compromised mitophagy via upregulation of DCT-1. Thus, NAD⁺ ties together DNA repair and mitophagy in neuroprotection and intimates immediate translational applications for A-T and related neurodegenerative DNA repair-deficient diseases.     

Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts

The effect of Lys-bradykinin on net Na+ influx in serum-deprived cultured human fibroblasts (HSWP cells) was measured. It was found that Lys-bradykinin stimulates net Na+ influx with a K1/2 of 1 nM. When Lys-bradykinin was combined with epidermal growth factor, vasopressin and insulin, the net Na+ influx stimulated was comparable with that measured in response to 10% serum. The above combination of growth factors also was found to stimulate DNA synthesis to 92% of the serum-stimulated levels in HSWP cells and to support cell growth over a period of 6 days. In addition, it was observed that the Na+ influx stimulated by Lys-bradykinin or by the combination of four growth factors was completely inhibited by the amiloride analog benzamil. Thus Lys-bradykinin presumably stimulates the same Na+ transport system as is stimulated by serum. Finally, the present results suggest that an increase in Na+ influx always accompanies DNA synthesis in HSWP cells. On the basis of these observations, it can be hypothesized that Na+ influx is a necessary event to stimulate DNA synthesis.     

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.     

Dependence of cell survival on DNA repair in human mononuclear phagocytes.

Mononuclear phagocytes play a central role in the pathogenesis of chronic inflammatory diseases. It is therefore important to define chemotherapeutically exploitable metabolic pathways that distinguish monocytes from other cell types. Blood monocytes do not synthesize deoxynucleotides de novo, and their transformation to macrophages occurs without cell division. Whether or not monocytes can repair DNA damage, and whether or not DNA repair is necessary for their survival, is unknown. The present experiments demonstrate that normal human monocytes, unlike neutrophils, rapidly repair DNA strand breaks induced by gamma-irradiation. Monocyte extracts contain functional immunoreactive DNA polymerase-alpha. DNA repair synthesis in normal monocytes is blocked by aphidicolin, an inhibitor of DNA polymerase-alpha with respect to dCTP. Aphidicolin is also directly toxic to normal monocytes, but has no effect on nondividing lymphocytes or fibroblasts. Compared to most other cell types, monocytes and macrophages have very low dCTP pools, but abundant deoxycytidine kinase activity. This suggests that dCTP derived from salvage pathways is important for DNA repair in these cells. Consistent with this notion, exogenous deoxycytidine could partially protect monocytes from aphidicolin killing. The unexpected toxicity of aphidicolin toward normal human monocytes may be attributable to their high rate of spontaneous DNA strand break formation, to the importance of DNA polymerase-alpha for DNA repair in these cells, and to their minute dCTP pools.     

Influence of iron-overload on DNA damage and its repair in human leukocytes in vitro

Iron is an important element that modulates the production of reactive oxygen species, which are thought to play a causative role in biological processes such as mutagenesis and carcinogenesis. The potential genotoxicity of dietary iron has been seldom studied in human leukocyte and only few reports have investigated in human colon tumor cells. Therefore, DNA damage and repair capacity of human leukocytes were examined using comet assay for screening the potential toxicity of various iron-overloads such as ferric-nitrilotriacetate (Fe-NTA), FeSO(4), hemoglobin and myoglobin, and compared with 200μM of H(2)O(2) and HNE. The iron-overloads tested were not cytotoxic in the range of 10-1000 microM by trypan blue exclusion assay. The exposure of leukocytes to Fe-NTA (500 and 1000 microM), FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) for 30 min induced significantly higher DNA damage than NC. Treatment with 500 and 1000 microM of Fe-NTA showed a similar genotoxic effect to H(2)O(2), and a significant higher genotoxic effect than HNE. The genotoxicity of FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) was not significantly different from that of H(2)O(2) and HNE. Iron-overloads generated DNA strand break were rejoined from the first 1h. Their genotoxic effect was not observed at 24h. These data from this study provide additional information on the genotoxicity of iron-overloads and self-repair capacity in human leukocytes.     

DNA repair in cultured mouse cells of increasing population doubling level

Cultures of mouse cells of various population doubling levels (PDL) were examined for DNA-repair capabilities as estimated by (i) the excision of pyrimidine dimers; (ii) unscheduled DNA synthesis (UDS) in response to UV-irradiation or N-methyl-N'-nitrosoguanidine (MNNG) treatment; (iii) the levels of two DNA-repair enzyme activities, uracil DNA glycosylase and AP endonuclease. The responses to ultraviolet light and MNNG decreased rapidly within the first two PDL and more slowly thereafter until essentially no repair was detected by PDL 12. A continuous cell line which emerged from the cultured cells after a crises period had some restoration of repair capability. The amount of uracil DNA glycosylase activity decreased by approximately 40% before the crises period then decreased by 90% in the continuous cell line. In contrast, the amount of AP endonuclease activity present in the precrises cells showed no significant change until PDL 12, then increased 6-7-fold in the continuous cell line.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.