Fluorescent Analysis Of DNA Unwinding Research Articles

Induction of DNA-strand breaks after X-irradiation in murine bone cells of various differentiation capacities

During longterm space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. In addition to weightlessness, exposure to cosmic ionization radiation is another space related factor endangering health and productivity of astronauts. In order to elucidate changes in bone cell metabolism induced by ionizing radiation, ground-based bone cell models have been developed. The differentiation level of the bone cells may influence their radiation sensitivity. Therefore, our cell model comprises a collection of immortalized murine pre-osteoblast, osteoblast and osteocyte cell lines representing discrete stages of differentiation: the subclones 4 and 24 of the osteoblast cell line MC3T3-E1, the osteoblast cell line OCT-1 and the osteocyte cell line MLO-Y4 display varying potential to produce mineralized bone matrix upon incubation with ascorbic acid and β-glycerophosphate (osteogenic medium). The MLO-Y4 cells showed the highest and subclone 24 the lowest proliferation rate. The most intense von Kossa reaction after culture in osteogenic medium was observed in subclone 4, indicating mineralized bone matrix. The bone cell markers alkaline phosphatase and osteocalcin were determined to further characterize the differentiation stage. All cell lines expressed osteocalcin, as determined by reverse transcriptase polymerase chain reaction. The activity of alkaline phosphatase was highest in the cell line OCT-1 and very low in MLO-Y4 and S4. The peculiarity of the markers suggests a characterization of OCT-1 and S24 as preosteoblast, S4 as (mature) osteoblast, and MLO-Y4 as osteocyte. Survival after exposure to X-rays was determined using the colony forming ability test. The resulting dose–effect relationships revealed normal radiation sensitivity (compared to human fibroblasts). Cell clone specific variations (subclones 4 and 24) in the radiation sensitivity may be due to the differentiation level. The survival curve of MLO-Y4 shows a broad shoulder, suggesting a high repair capacity or a high DNA damage or misrepair tolerance. The quantitative acquisition of DNA-strand breaks was performed by fluorescent analysis of DNA unwinding and revealed a high level of DNA damage immediately after X-irradiation, which increases dose dependently. In conclusion, the cell line with the highest differentiation level (MLO-Y4) displays lower radiation sensitivity, regarding the shoulder width of the dose–effect curve, compared to the less differentiated osteoblast cell lines.

Peripheral blood lymphocytes DNA in patients with chronic liver diseases.

Viral replication in blood cells with nucleases may lead to the damage of lymphocytes genetic apparatus and the beginning of immunopathological reactions. Of this investigation is to reveal the damage to peripheral blood lymphocytes (PBL) DNA in the patients with chronic liver diseases. Sixteen-nine patients with chronic liver diseases (37 patients with chronic viral hepatitis, 2 patients with liver cirrhosis of mixed etiology (alcohol+virus G), 30 women with primary biliary cirrhosis-PBC) were examined. The condition of DNA structure of PBL was measured by the fluorescence analysis of DNA unwinding (FADU) technique with modification. Changes of fluorescence (in %) reflected the DNA distractions degree (the presence of DNA single-stranded breaks and alkalinelabile sights). The quantity of DNA single-stranded breaks and alkalinelabile sights in DNA in all patients with chronic viral hepatitis didn't differ from the control group, excluding the patients with chronic hepatitis (CH) C+G. Patients with HGV and TTV monoinfection had demonstrated the increase of the DNA single-stranded breaks PBL quantity. This fact may be connected with hypothesis about the viruses replication in white blood cells discussed in the literature. Tendency to increase quantity of DNA PBL damages in the patients with primary biliary cirrhosis (PBC) accordingly to the alkaline phosphatase activity increase was revealed. Significant decrease of the DNA single-stranded breaks and alkalinelabile sights in the PBC patients that were treated with prednisone was demonstrated. Probably, the tendency to increase the quantity of DNA single stranded breaks and alkalinelabile sights in lymphocytes of the PBC patients was depended on the surplus of the blood bile acid content.

Modulation of radiation-induced strand break repair by cisplatin in mammalian cells

Purpose: To investigate the repair of ionizing radiation-induced DNA lesions in human skin fibroblasts in the presence of cisplatin-DNA adducts and to determine the persistence of DNA repair inhibition by cisplatin. Materials and methods: Normal human fibroblasts (AG1522) treated with cisplatin were exposed to 4 Gy 60Co gamma-radiation and assayed for repair of radiation-induced damage under growth-permissive conditions. DNA damage was measured by the fluorescence analysis of DNA unwinding (FADU) and cytokinesis-blocked micronucleus assays. Results: Rejoining of strand breaks caused by 4 Gy radiation in cells without cisplatin pre-treatment appeared to be biphasic with an initial fast component (up to 15 min of repair time) followed by a slower component, and was completed by 90 min. Cisplatin treatment (10 mu g/ml, 30 min) immediately before irradiation had no effect on the fast rejoining component, but inhibited the slow component (p 0.01). The same cisplatin treatment 24 h prior to irradiation inhibited both slow and fast components (p 0.01). In contrast, decreasing the cisplatin exposure to 1.0 mu g/ml for 30 min, 24 h prior to irradiation, resulted in an increased amount of strand break repair at each time point measured compared with irradiated control cells. This mild cisplatin treatment (95% survival) also resulted in a reduction of radiation-generated micronuclei indicating an adaptive response. Conclusions: Cisplatin used in combination with ionizing radiation can produce differential cellular responses depending upon the severity of the cisplatin treatment and the time interval between cisplatin and radiation exposures.

Maximum tolerable doses of intravenous zidovudine in combination with 5-fluorouracil and leucovorin in metastatic colorectal cancer patients: Clinical evidence of significant antitumor activity and enhancement of zidovudine-induced DNA single strand breaks in peripheral nuclear blood cells

Experimental studies have demonstrated that 5-fluorouracil (5-FU) enhances zidovudine (AZT)-induced DNA strand breaks and cytotoxicity. Phase I studies have demonstrated that the maximum tolerable dose (MTD) of AZT is 8000 mg/sqm when administered i.v. over two hours after weekly 5-FU + l-leucovorin (LV), and that this combination has promising antitumor activity. The purpose of this study was therefore to evaluate the antitumor activity of weekly bolus 5-FU + LV + AZT, administered at its MTD, and to determine whether 5-FU enhances AZT-induced DNA strand breaks in blood nuclear cells. Twenty-nine chemotherapy-naïve metastatic colorectal cancer patients with measurable disease entered the study to evaluate the activity of a weekly 5-FU 500 mg/m2 i.v. bolus + LV 250 mg/m2 i.v. two-hour infusion + AZT 8000 mg/m2 i.v. two-hour infusion. In 10 different patients, who during three different weeks received 5-FU + LV, AZT and 5-FU + LV + AZT, DNA strand breaks in blood nuclear cells were determined by a fluorescent analysis of DNA unwinding. Treatment was generally well tolerated and WHO grades III-IV toxicities, consisting mostly of diarrhea (17%), were uncommon. One patient died of severe diarrhea with consequent hypokalemia and cardiac arrhythmia. All patients were considered evaluable for response, and 3 (10%) complete and 10 (35%) partial responses were observed, for an objective response rate of 45% (95% confidence limit interval 26%-64%). Both 5-FU + LV and AZT decreased the percentage of double stranded DNA in nuclear blood cells. The greatest effect was observed with 5-FU + LV + AZT, which reduced the percentage of double stranded DNA to 50% and 36% after 24 and 48 hours, respectively, and this interaction between 5-FU + LV and AZT was found to be cumulative. These studies demonstrate that the present dose and schedule of AZT in combination with 5-FU + LV has significant activity in metastatic colorectal cancer and that the combination of 5-FU + LV with AZT increases the amount of DNA damage. Therefore, AZT in combination with 5-FU + LV warrants further study in colorectal cancer.

Characterization of tumour necrosis factor α release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines—CCRF-HSB2 and WIL-2NS—were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0–50 μM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 °, respectively. Using 50 μM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 μM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 μM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10–15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40–60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor α (TNFα) was present in the > 30,000 Da fraction. Evidence that TNFα was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNFα antibody. Treatment of granulocytederived supernatants with this antibody led to the specific removal of a significant portion of both the cyto- and genotoxicity (> 50% in both cases). Our data suggest a highly effective mechanism for the promotion of drug-induced lupus by PA. The specific induction of TNFα represents a highly effective mechanism for the induction of the inflammatory response commonly associated with this syndrome and provides a potential route for the specific induction of severe adverse drug reactions. Furthermore, the biochemical mechanisms of action of lupus-inducing drugs may provide insight into the molecular mechanisms leading to development of idiopathic lupus.

Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes.

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3'-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose gamma-radiation (0.1 Gy) or mild hyperthermia (40 degrees C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.

Effects of pulsed ultrasound and ultraviolet radiation on Vitamin E and olive oil

SynopsisThe effects of ultrasound and ultraviolet radiation on Vitamin E and its pharmacological excipient, olive oil, were tested by fluorescent analysis of DNA unwinding (FADU) on DNA extracted from human lymphocytes of healthy donors. The results show that Vitamin E may be inactivated and behave as a radical species, while olive oil appears unaffected by treatment either with ultrasound or with ultraviolet radiation.

Detection by fluorescence analysis of DNA unwinding and unscheduled DNA synthesis, of DNA damage and repair induced in vitro by direct-acting mutagens on human lymphocytes

The sensitivity and reliability of UDS and FADU in detecting mutagenic effects were compared by measuring DNA damage and repair in PBL treated in vitro with UV light. MMS and BPDE. The results indicate that FADU is more sensitive than UDS, as it is able to detect DNA damage at doses 3–4-fold lower. We also determined the DNA damage and repair induced by the above agents on lymphocyte samples from different donors by FADU and UDS, confirming that the DNA repair process in humans is characterized by interindividually variable efficiency.

DNA Strand Break Formation Following Exposure of Bovine Pulmonary Artery and Aortic Endothelial Cells to Reactive Oxygen Products

Experiments were performed to investigate the hypothesis that exposure of vascular endothelial cells to low levels of reduced oxygen products results in DNA strand breakage as an early event and to determine if endothelial cells derived from bovine pulmonary artery demonstrate a susceptibility to oxidant injury that is different from that of cells derived from bovine aorta. Endothelial cells grown in culture were exposed to H2O2 (either added directly or generated from glucose oxidase) or superoxide radical (generated from xanthine oxidase), and DNA strand breakage was determined using fluorescent analysis of DNA unwinding. Cell injury was also assessed by measuring the release of lactate dehydrogenase (LDH) or the release of 51Cr from prelabeled cells. Whereas LDH or 51Cr release detected injury resulting from exposure of endothelial cells to greater than or equal to 100 microM H2O2 and was apparent only 2 or more h after exposure, DNA strand breakage was detectable after 15 min of exposure of endothelial cells to 50 microM H2O2. Approximately equivalent DNA strand breakage resulted from exposure to 50 microM H2O2, to 25 mU glucose oxidase, or to 10 mU xanthine oxidase; this injury is similar to that seen following exposure to 10 gray X-radiation. DNA strand breakage following exposure of cells to xanthine oxidase was preventable by catalase but not by superoxide dismutase or hydroxyl radical scavengers, suggesting that H2O2 is the active extracellular oxidant mediating DNA strand breaks. No differences were seen in the susceptibility of pulmonary artery or aortic endothelial cells to oxidant injury.

Fuel smoke condensate induced DNA damage in human lymphocytes and protection by turmeric (Curcuma longa)

Twigs-dry leaves smoke condensate (TDS) was investigated for its DNA damaging activity in human peripheral lymphocytes, by using a sensitive method, fluorescence analysis of DNA unwinding (FADU). An aqueous turmeric component (Aq.T) was studied as a protective agent. TDS at one to 100 folds dilution induced 55% DNA damage at 20 min, while 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml induced only 25% damage. Aq.T at 300 ng/microliter afforded 90% protection to DNA against TDS and 65% against TPA. The mechanism of Aq.T protection was investigated by using (i) inhibitors of arachidonate cascade, viz., indomethacin (28 microM), NDGA (10 microM), DBAP (36 microM), (ii) antioxidant enzymes viz., CAT (0.2 U/microliter), SOD (0.6 U/microliter), (iii) antioxidants--BHA, curcumin (40 microM), mixed gangliosides (20 nM) and protease inhibitor TLCK (100 microM). These compounds offered the following extents of protection to DNA against TDS: indomethacin--40%, NDGA--83%, DBAP--70%, SOD--38%, CAT--40%, BHA--38%, curcumin--60%, mixed gangliosides--88%, TLCK--85%. Against TPA as clastogenic agent, the extents of protection were: indomethacin--73%, NDGA--32%, DBAP--72%, SOD--60%, CAT, BHA-negligible, curcumin--23%, mixed gangliosides--60%, TLCK--59%. These results indicate that (i) TDS and TPA induce DNA damage possibly by different mechanisms, (ii) Aq.T is a more effective protectant against TDS whereas it is on par with other inhibitors against TPA.

Reactive oxygen-mediated damage to murine mammary tumor cells

We have shown, in a preliminary report, that macrophages can induce strand breaks in the DNA of co-cultured tumor cells (Chong et al., 1988). The present study is designed to determine if oxygen-centered species generated by the cell-free enzyme-substrate combination of hypoxanthine and xanthine oxidase can induce similar lesions and to identify the specific mediator(s). We report that co-incubation of murine mammary tumor cell lines with hypoxanthine and xanthine oxidase leads to the induction of DNA-strand breaks as determined by fluorescence analysis of DNA unwinding (FADU) assay or alkaline elution techniques. This damage is preventable by catalase which removes hydrogen peroxide but no protection is provided by agents to remove or prevent the formation of superoxide anion (superoxide dismutase), or hydroxyl radical (mannitol or the iron chelator o-phenanthroline). Likewise, cyclooxygenase or lipoxygenase inhibitors of arachidonate metabolism (indomethacin, nordihydroguaiaretic acid, caffeic acid) or bromophenacyl bromide do not alter the degree of DNA scission. Treatment with higher doses of oxygen species leads to significant toxicity as determined by evaluation of cell growth potential or colony-forming ability. Again, toxicity is prevented only by the presence of catalase. Tumor cells are able to rejoin strand breaks at lower, less toxic doses. When comparing different tumor cell subpopulations at various stages of progression, i.e., metastatic vs. nonmetastatic, for sensitivity to hydrogen peroxide-induced strand breakage, we found that at lower concentrations (less than 5μM) metastatic populations are sensitive whereas nonmetastatic populations exhibit no significant breakage. At higher concentrations of hydrogen peroxide, all lines were sensitive, suggesting that a lower threshold of sensitivity may exist for more progressed tumour cell lines.

The interplay between mixed function oxygenases and DNA alteration under PAH pollution

Our experience in DNA alteration assessment tells us that aquatic vertebrates, in general, and also some of the invertebrates, by living in polluted areas acquire altered DNA; DNA alterations being defined either as single-strand breaks, and/or alkali sensitive or single-strand specific nuclease-cleavable sites (sse). This can be shown by several methods: (i) Alkaline filter elution; 1 (ii) fluorescence analysis of DNA unwinding; 2 (iii) alkaline sucrose density gradient centrifugation; 3 (iv) alkaline unwinding analysis with hydroxyapatite differentiation; 4 (v) nucleoid sedimentation analysis; 5 (vi) supercoiled DNA relaxation detection by electrophoresis; 6 and (vii) measurement of DNA molecular weight distributions with the electron microscope. 7 The amount of sse per DNA mass can be a function of either an increased activity of DNA damaging agents or agents that inhibit DNA repair. Among DNA damaging agents are polycyclic aromatic hydrocarbons (PAH), that can cause sse by the ‘fast effect’ 8 or after induction upon uptake, through the action of mixed function oxygenase (MFO). Since MFO induction within certain limits is a function of PAH concentration measurement of this enzyme activity enables at least partial interpretation of the DNA alteration status, which can be a two-phase process.