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At least 505 records · Page 28Linked to original sources
PubMed1988

Mitoxantrone-induced DNA damage in leukemia cells is enhanced by treatment with high-dose arabinosylcytosine.

In a phase II study, patients with chronic myelogenous leukemia in blast crisis (CML-BC) were treated with intravenous (IV) mitoxantrone (5 mg/m2 per day given over 30 min x 5 days and high-dose arabinosylcytosine (ara-C) (3 g/m2 IV q 12 h x 6). The effect of this treatment on DNA damage was studied in the leukemia cells of four patients using the alkaline elution technique modified to measure DNA in unlabeled human cells. A fluorescence assay using Hoechst 33258 dye was applied for the determination of eluted DNA. After a single infusion of mitoxantrone, neither frank nor protein-associated single-strand breaks (SSB) were observed. Even repeated treatment with mitoxantrone on 3 consecutive days did not induce significant SSB. However, after the combined sequential infusion of ara-C and mitoxantrone the DNA elution pattern changed, showing significant DNA damage. SSB remained apparent after 24 h and increased with subsequent doses of ara-C and mitoxantrone. Studies of other patients treated with ara-C alone did not reveal significant SSB (n = 5). Following mitoxantrone infusion the median peak concentrations of intracellular ara-CTP (the triphosphate of ara-C) exceeded 900 microM, a value greater than that observed in CML-BC patients receiving ara-C alone (230 microM, n = 15, P less than 0.02). The present study shows the applicability of the alkaline elution method for the assay of DNA damage in vivo. The enhanced DNA damage after combined treatment with mitoxantrone and high-dose ara-C suggests a synergistic drug effect.

Adult↗
PubMed1990

DNA damage and cytotoxicity of mitoxantrone and doxorubicin in doxorubicin-sensitive and -resistant human colon carcinoma cells.

The effects of mitoxantrone (Mx) and doxorubicin (Dx) on cytotoxicity and DNA damage as assayed by alkaline elution were studied in two human colon adenocarcinoma cell lines sensitive (LoVo) and resistant (LoVo/Dx) to doxorubicin. Mx was more cytotoxic than Dx to LoVo cells and was partially cross-resistant in LoVo/Dx. In LoVo cells, Mx produced about 5 times more DNA single-strand breaks (DNA-SSB) than Dx, but both drugs caused an equal number of DNA double-strand breaks (DNA-DSB). In LoVo/Dx cells, the number of DNA-DSB was very low for both Dx and Mx, but DNA-SSB were about 20 times higher for Mx. In LoVo cells, the number of DNA-DSB and protein-associated SSB were similar at equitoxic concentrations. For LoVo/Dx, the partial cross-resistance of Mx might be explained by the much higher number of DNA-SSB produced by this drug.

Colonic Neoplasms↗
PubMed1993

Cytotoxicity, DNA cross-linking, and DNA single-strand breaks induced by cyclophosphamide in a rat leukemia in vivo.

A study of cyclophosphamide (CP)-induced DNA damage and repair occurring in vivo was conducted in the brown Norway rat myelocytic leukemia (BNML) model. DNA single-strand breaks (SSB), DNA-DNA interstrand cross-links (DIC), DNA-protein cross-links (DPC), and DNA double-strand breaks (DSB) were measured by alkaline and neutral elution. After i.p. injection of 50 mg/kg CP, DIC were detectable at 1 h and peaked at 8 h. DPC were detectable at 2 h and peaked at 6 h. Both DIC and DPC persisted at a relatively high level until 28 h. Dose-response curves for both DIC and DPC were determined at 4 h after CP injection over the dose range of 25-150 mg/kg. These doses ranged from the minimally effective dose to doses curative for rats bearing this leukemia (1- to 9-log kill of leukemia cells). No SSB or DSB was observed at 4 h after CP injection over the dose range of 15-250 mg/kg, but a low level of SSB was observed at 18-28 h after CP treatment. These data suggest that the cytotoxic effect of CP in vivo is mediated mostly by DIC and DPC. SSB appearing late after CP injection in vivo may be a reflection of repair of DIC and DPC and an indication of the optimal timing for administration of DNA-repair inhibitors. This observation is of interest since our earlier work demonstrated that hydroxyurea can potentiate the therapeutic benefit of CP in this model when it is given over the 4-day period immediately after CP treatment.

Animals↗
PubMed2003

[A new irrigation-suction boring system facilities low-pressure intramedullary boring of isolated swine femurs].

AIM: Intramedullary nailing is the treatment of choice for the stabilization of fractures of long tubular bones. An important disadvantage of this method is the increase in intramedullary pressure and the resulting release of fat into the venous blood system during reaming of the medullary canal. We have developed a new type of rinsing-suction-reamer (SSB) in order to minimize these disadvantages. Trials were initiated to investigate whether it is possible to ream the medullary canal with the SSB without pressure increase in comparison with the standard AO-reamer (AOB). METHODS: Reamed intramedullary nailing was performed in 20 isolated pig femora. The intramedullary pressure was recorded continuously. RESULTS: While stepwise reaming was performed, the pressure only rose above the physiological level in AOB. During insertion of the guide wire and the nail, comparable values were measured for AOB and SSB. CONCLUSION: Our experiments show that reaming of the medullary canal is possible without a pressure increase using the SSB in comparison with AOB.

Animals↗
PubMed2005

Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana.

A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.

Amino Acid Sequence↗
PubMed1984

Two mechanisms of adriamycin-DNA interaction in L1210 cells.

Among the effects exerted by adriamycin (ADR), interaction with DNA is closely related to cytotoxicity. The interaction results in the formation of protein-associated DNA single-strand breaks (PA-SSB) and, at drug levels greater than or equal to 2.8 X 10(-6) M, also in "direct" (nonenzymatic) DNA single-strand breaks (D-SSB). To characterize the two types of DNA lesions, euoxic mouse leukemia L1210 cells were treated with various antioxidant agents in the presence of 2.8 X 10(-6), X 10(-5), or X 10(-4) M concentrations of ADR. The enzymes superoxide dismutase (200 micrograms/ml) or catalase (250 micrograms/ml), the OH' scavengers dimethyl sulfoxide (70 mM) or ethanol (70 mM), and an inhibitor of superoxide production, 2-deoxy-glucose (1 and 10 mM), reduced the frequency of D-SSB to 18.3 to 68.2% of its level in ADR-treated controls, while the frequency of PA-SSB remained unchanged. These observations seem to indicate that ADR-mediated free radicals cause discernible DNA damage in euoxic cells only at very high drug concentrations, greater than the peak plasma level achievable clinically following i.v. bolus. At lower ADR levels, relevant to clinical use, another type of interaction between the drug and DNA prevails, which apparently does not involve a free-radical mechanism.

Animals↗
PubMed1995

Human bioassays to assess environmental genotoxicity: development of a DNA break bioassay in HepG2 cells.

OBJECTIVES: Increasing interest in environmental health issues has created a demand for improved methods for the assessment of pollutant effects on humans. Our laboratory has developed an in vitro assay for the quantification of genotoxicity, monitored as DNA single strand breaks (SSB), in the HepG2 human hepatoma cell line. DESIGN AND METHODS: This assay procedure, which is based upon alkaline unwinding and hydroxylapatite DNA chromatography, is both rapid and simple to perform. RESULTS: HepG2 cells responded to the standard mutagen, 4-nitroquinoline N-oxide, demonstrating SSB formation at concentrations above 0.1 mumol/L. Phenanthrene-9,10-quinone, a component of diesel exhaust, mediated SSB formation at concentrations above 250 nmol/L. Finally, an extract of contaminated sediment from the Great Lakes Basin mediated SSB formation in a dose-dependent manner. CONCLUSIONS: These results illustrate the utility of this human genotoxicity assay for future use in screening of environmental pollutants.

4-Nitroquinoline-1-oxide↗
PubMed1990

DNA single-strand breaks in adult and embryonic avian erythrocytes.

We have investigated the possibility that the reactivation rate of adult avian erythrocytes, which is slower than that of embryonic erythrocytes, after fusion with metabolically active cells, is due to a greater number of single-strand breaks (ssb) in the DNA of the former. We have assayed ssb by measuring the template activity of the erythrocyte nuclei for added Escherichia coli DNA polymerase. We have found that differences in the numbers of ssb within polymerase-accessible regions between adult and embryonic cells are within experimental error. We conclude that, unless very localized clusters of damage exist within the DNA (which would not be detectable by this or other techniques), the difference in reactivation rate is not attributable to differences in ssb numbers.

Aging↗
PubMed1985

Intermediates in homologous pairing promoted by recA protein. Isolation and characterization of active presynaptic complexes.

recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA. This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized. At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA. Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein. Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.

Adenosine Diphosphate↗
PubMed1989

Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells.

The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH-) and non-depleted (GSH+) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH- and GSH+ cells. The aberrations were then repaired in GSH+ cells but those in GSH- cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.

Animals↗
PubMed1994

Dietary butylated hydroxytoluene counteracts with paraquat to reduce the rate of hepatic DNA single strand breaks in senescence-accelerated mice.

The present study was designed to assess the age-related changes of DNA single strand breaks (SSB) in the liver of senescence-resistant and senescence-prone mice, SAMR1/Fky and SAMP1@Fky. In the first series of experiments, the animals were fed a commercial diet for 12-18 months. The mice were killed and the livers were excised at 3- or 6-month intervals for the analysis of the rate of DNA SSB by the ethidium bromide fluorescence method. With advancing age, the rate of DNA SSB was increased in both strains of mice but the increase was significantly higher in SAMP1@Fky than in SAMR1/Fky. In the second series of experiments, the mice were fed one of the following diets for 12 weeks: 20% casein diet (basal diet), basal diet with 300 ppm butylated hydroxytoluene (BHT) added, basal diet with 200 ppm paraquat (PQ) added, and basal diet with 200 ppm PQ plus 300 ppm BHT added. Added dietary PQ increased the rate of DNA SSB in both SAMP1@Fky and SAMR1/Fky. The increases were offset by co-administration of BHT. Dietary BHT, therefore, may suppress the oxidative stress developed by paraquat administration.

Aging↗
PubMed1993

Caffeine-derived N-nitroso compounds. III: Mutagenicity in S. typhimurium and in vitro induction of DNA single-strand breaks in rat hepatocytes by mononitrosocaffeidine and dinitrosocaffeidine.

Mutagenesis in S. typhimurium and in vitro induction of DNA single-strand breaks in primary rat hepatocytes (DNA-SSB) have been investigated for two new N-nitroso compounds, mononitrosocaffeidine (MNC) and dinitrosocaffeidine (DNC). Mononitrosamidocaffeidine (MNAC) and tert.-(butyloxy)carbonyl-mononitrosamidocaffeidine (t-BOC-MNAC), both nitrosated derivatives of caffeidine with nitrosation at methylcarboxamide-N only, were also similarly studied. MNC, an asymmetric nitrosamine, failed to show mutagenicity in any of the tester strains used, and also did not induce DNA-SSB in rat hepatocytes. DNC, having both N-nitrosamide and N-nitrosamine groups in the molecule, showed direct mutagenicity in TA100, TA1535 and TA102. The mutagenic potential of the compound was found to increase on S9 activation. However, it was non-mutagenic in TA98 and TA1537. DNC also exhibited a high potential for inducing alkali-labile DNA-SSB in rat hepatocytes (70-78% C-T value) and was cytotoxic at concentrations over 0.1 mumole/ml. Both MNC and DNC were found to produce formaldehyde on S9 activation. MNAC was not mutagenic directly but showed weak mutagenicity on metabolic activation, whereas t-BOC-MNAC was mutagenic both with and without S9 activation in TA100, TA1535 and TA102. t-BOC-MNAC was more cytotoxic to hepatocytes than MNAC, though both caused DNA-SSB to the same extent (62% C-T value). On the basis of the presented data it is inferred that while DNC is a direct-acting mutagen in TA100, TA1535 and TA102 due to the presence of a reactive N-methylnitrosamido group, its mutagenic potential is greatly enhanced in the presence of S9 possibly due to the synergistic influence of an activated N-methylnitrosamino group in the molecule. Additionally, the study shows a qualitative consistency between Salmonella mutagenicity, genotoxicity in hepatocytes and the reactivity of the methyl group at the nitrosamido-N in nitrosated caffeidine compounds.

Animals↗
PubMed1989

Hydrogen peroxide insult in cultured mammalian cells: relationships between DNA single-strand breakage, poly(ADP-ribose) metabolism and cell killing.

We examined the effect of exposure to H2O2 at 37 degrees C on Chinese hamster ovary cell survival, DNA single-strand break (SSB) induction and rejoining, and activation of poly(ADP-ribose) (ADPR) polymerase. The effect of the ADPR polymerase inhibitor 3-aminobenzamide on each of these processes was also determined. SSB induction increased progressively with increasing H2O2 concentration. SSB levels were maximal after approx. 5 min of exposure to H2O2 (100 microM) and then decreased at longer times. This decrease, which paralleled the time-dependent depletion of H2O2, was due to the rejoining of SSBs. 3-Aminobenzamide enhanced the level of SSBs at each time point. H2O2 increased the level of both ADPR synthesis and NAD+ depletion (both measures of ADPR polymerase activity) in a concentration-dependent fashion, with the maximum effect being reached after approx. 20 min. After 100 microM H2O2, the effects on both ADPR and NAD+ were reversible. 3-Aminobenzamide completely blocked the effects of the oxidant on both NAD+ and ADPR levels. Thus, SSB induction by H2O2 at 37 degrees C was accompanied by a marked but reversible stimulation of ADPR polymerase. However, cell killing by H2O2 was only slightly enhanced in the presence of 3-aminobenzamide (5 mM), so the above-mentioned effects do not appear to be relevant to the cytotoxic effect of H2O2 under these conditions. Comparing these results with data obtained previously for cells treated with H2O2 at 4 degrees C suggests that the mechanisms of DNA strand breakage and cell killing may be quite different at the two temperatures, and that DNA damage at 37 degrees C may be indirectly mediated by temperature-dependent metabolic events.

Animals↗
PubMed1985

Repair of DNA single-strand breaks in X-irradiated yeast. II. Kinetics of repair as measured by the DNA-unwinding method.

The kinetics of disappearance of single-strand breaks (SSB) from the DNA of X-irradiated stationary yeast cells under liquid-holding conditions was found to proceed in a dose-independent manner up to a dose of at least 2400 Gy, and was found to be complete after incubation of cells for 1 h. This was deduced from data for a yeast wild-type (WT) haploid and diploid strain as well as for rad52 haploid cells defective in DNA double-strand break (DSB) repair. In all cases an initial fast repair component assumed to correspond to SSB repair was observed whereby about 80% of the induced 'unwinding points' disappeared from the DNA with a time constant of about 3 min. Following this fast component, a slower component of removal of 'unwinding points' occurred with a time constant estimated to be 20 min. The molecular nature of these two components of repair is not known. We could find no evidence for the induction of secondary (enzymatic) breaks in the DNA during post-irradiation incubation. Incubation of cells in growth medium after irradiation resulted in similar kinetics as those under liquid-holding conditions. In the absence of an energy source in the medium (i.e. when cells were incubated in buffer or distilled water after irradiation) only 60-80% of the SSB were removed from yeast DNA. Residual SSB disappeared from the DNA only when cells were transferred to a medium containing glucose. The relative mass of DNA unwound per induced strand break (i.e. represented by the slope of the dose-effect curve immediately after irradiation) was found to change slowly with the age of the cell culture under liquid-holding conditions. This effect had to be corrected for in the measurements of strand break repair under these conditions.

Culture Media↗
PubMed1989

DNA-lesion and cell death by alpha-particles and nitrogen ions.

When the natural logarithm of the surviving fraction is plotted against the dose of radiation, curves with shoulders at relatively high survival levels are obtained after gamma-rays. The curves were practically linear in case of HMV-I and HA-1 cells irradiated by charged particle beams. These cells were derived from human malignant melanoma and Chinese hamster cells, respectively. The amount of DNA single strand breaks (ssb) by gamma-rays or nitrogen-ions (LET=530KeV/micrometers) in HMV-I cells increases linearly with increment in dose, when the ssb is detected using the alkaline elution technique. There is no close relationship between the dose-response curve of the ssb and the dose-survival curves after gamma-rays or N-ions. The amount of DNA double strand breaks (dsb) by gamma-rays increases quadratically with increment of dose, in both HMV-I cells and HA-1 cells, when the dsb is detected using the neutral elution technique. The survival fraction for HA-1 cells is slightly higher than that for HMV-I cells, at the same dose, and the amount of dsb for HA-1 cells is considerably greater than that for HMV-I cells. These results suggest that the radiosensitivities to gamma-rays in different cell lines do not correspond to the number of DNA strand breaks. The amount of both non-repairable ssb and dsb also increases quadratically with increment of dose for gamma-rays and almost linearly with increment of dose for N-ions and alpha-particles (LET=36keV/micrometers for HA-1 cells and LET=77keV/micrometers for HMV-I cells). The dose-response curves for non-repairable dsb in case of these radiations seemed to mirror image the dose-survival curves for these radiations, in both cell lines. The number of non-repairable DNA strand breaks in the two cell lines, at the same level of survival was much the same. These results show the close relationship between the induction of non-repairable DNA strand breaks and cell killing.

Alpha Particles↗
PubMed1992

DNA damage induced by alachlor after in vitro activation by rat hepatocytes.

We investigated the ability of alachlor to cause DNA damage by measuring single-strand breaks (SSB) in DNA, after metabolic activation by freshly isolated rat hepatocytes. Incubation of different concentrations of alachlor with rat hepatocytes led to numerous metabolites. The majority, isolated and identified by GC-MS analysis, were products arising from reactions catalyzed by the P-450 monooxygenase system, arylamidase and flavin mixed-function oxidase/cytochrome P-450 monooxygenase. The results, using freshly isolated rat hepatocytes, showed that in these conditions several potentially DNA damaging metabolites were produced; this experimental condition was used to assess DNA damage induced by the mixture of alachlor and its metabolites. The alkaline elution technique showed that at 200 microM and more clearly at 400 microM there were some small fragments that eluted in the first fraction. This fragmentation was probably due to alachlor cytotoxicity. In addition to the small DNA fragments eluting in the first fraction there were other larger DNA fragments. These DNA-SSB were most evident at the alachlor concentration of 400 microM, but also at 200 microM and 100 microM, whereas at 10 microM the DNA elution rate appeared comparable to that of controls. The results suggest that some unstable and DNA-reactive metabolites might interact with DNA causing SSB and such interaction might be important in relation to the mechanism of alachlor-induced DNA damage. However, it may not be possible to clarify whether SSB are the result of direct DNA interaction of the compound or of secondary cellular processes after chemical treatment.

Acetamides↗
PubMed1991

A possible interaction of single-strand binding protein and RecA protein during post-ultraviolet DNA synthesis.

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.

DNA Replication↗
PubMed1991

Ultrasonic cavitation indirectly induces single strand breaks in DNA of viable cells in vitro by the action of residual hydrogen peroxide.

Direct exposure of cells to vigorous ultrasonic cavitation results predominantly in mechanical cell lysis, but latent effects due to production of toxic sonochemicals can also be present. Phosphate buffered saline (PBS) was exposed to 1.61 MHz ultrasonic cavitation at 20 degrees C in a rotating tube exposure system to build up sonochemical products. Single strand DNA breaks (SSBs) were then induced by treating Chinese hamster ovary (CHO) cells with the cavitated PBS for 30 min on ice. The SSBs resided in viable cells, as evidenced by their ability to repair the breaks when warmed. This indirect effect could be explained by the action of cavitation-generated hydrogen peroxide that had built up (e.g., to 16 microM after 30 min exposure) in the PBS. Dissolution of argon gas in the PBS before exposure enhanced the SSB effect and the H2O2 production. Addition of catalase to the cavitated PBS before cell treatment eliminated the H2O2 and the SSB gamma effect. Tests with hydrogen peroxide showed that 16 microM H2O2 treatment for 30 on ice was as effective as 1 Gy dose of 60Co gamma rays in producing single strand breaks. The SSB effect of H2O2 and gamma rays was reduced by addition of the radical scavenger cysteamine to the cells before treatment, but cysteamine did not reduce the SSB effect of direct exposure to ultrasonic cavitation. These results help to clarify the potential for genetic effects from ultrasonic cavitation. These effects help to clarify the potential for genetic effects from ultrasonic cavitation.

Animals↗