Production Of DNA Strand Breaks Research Articles

Ferritin enhances production of DNA strand breaks by 6-hydroxydopamine, ascorbic acid and H2O2 in CCC PM2 bacteriophage DNA

Damage of CCC PM2 DNA by 6-hydroxydopamine (6-OHDA) and ascorbic acid (AA), compounds that are both able to release iron from ferritin, was significantly enhanced in the presence of ferritin. H2O2, a product of 6-OHDA autoxidation, did not induce DNA strand breaks in the absence of ferritin and only to a minor extent in the presence of ferritin. DNA damage by 6-OHDA and AA could be reduced by the hydroxyl radical scavenger mannitol, the iron chelator desferrioxamine, and, partly, by a combination of superoxide dismutase and catalase. These inhibitory effects were clearly less pronounced in the presence of ferritin. Ferritin obviously played an important role as a source of iron in the pro-oxidative processes of 6-OHDA and AA. These features might be of importance in cancer therapy since many tumor cells contain elevated ferritin levels.

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

Scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair.

C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.

DNA strand breaks induced in cultured human and rodent cells by chlorohydroxyfuranones—mutagens isolated from drinking water

Chlorohydroxyfuranones, by-products of chlorine disinfection and drinking water contaminants, are shown to produce DNA strand breaks in human and rodent cells. One chlorohydroxyfuranone, 3-chloro-4-dichloromethyl-5-hydroxy-2[5H]-furanone (MX), a potent bacterial mutagen, induces 232 +/- 89 DNA strand breaks.(cell-microM)-1 in human CCRF-CEM cells over a concentration range of 4.4 to 220 microM. This constitutes a DNA damage potency comparable to dimethylsulfate (DMS). By comparison, 3,4-dichloro-5-hydroxy-2[5H]-furanone (MA), another chlorohydroxyfuranone which is approximately four orders of magnitude less mutagenic than MX in Salmonella typhimurium strain TA100, is only about tenfold less potent as an inducer of DNA strand breaks in these cells, i.e., 18.2 +/- 3.1 strand breaks.(cell-microM)-1. The DNA strand-breaking potential of MX is inactivated by prior incubation with a rat liver S9 homogenate. In addition, both chlorohydroxyfuranones are ineffective at producing DNA strand breaks in primary rate hepatocytes (PRH) at concentrations below those which produce cytotoxicity as assessed by release of the cellular enzyme lactate dehydrogenase (LDH). Prior treatment of the PRH with 750 microM diethyl maleate, a glutathione-depleting agent, did not enhance the cytotoxicity nor the DNA strand-breaking potential of either chlorohydroxyfuranone. This could indicate that glutathione-glutathione-S-transferase is not an important mechanism for the detoxification of these compounds in PRH.

A General Theory of DNA Strand Break Production by Direct and Indirect Effects

A General Theory of DNA Strand Break Production by Direct and Indirect Effects

A General Theory of DNA Strand Break Production by Direct and Indirect Effects

Abstract A general theory of the production of strand breaks by energetic electrons and heavy charged particles has been developed. This theory is based on chemical and biochemical mechanisms and does not require adjustable parameters. In order to avoid the complexities of a cellular system, we have referenced our calculations to systems consisting of aqueous solutions of SV40 DNA containing different amounts of tris-HCl buffer. It appears from the calculations that at 500 mM tris, the strand break yields are similar to those observed in a cellular complex when normalised to account for DNA length. Furthermore, the results indicate that the indirect action is not negligible in creating strand breaks. The difference between DNA breaks with and without histones has also been analysed.

Production of DNA Strand Breaks by Direct Effects of Heavy Charged Particles

A theoretical model has been developed to calculate the yields of single- and double-strand breaks in DNA induced by direct effects of ionizing radiation. In this model, which involves no fitted parameters, elements of track structure and stopping power theory are combined with a detailed geometrical description of DNA to calculate the energy deposited by fast charged particles to DNA molecules. The average energy per interaction with a DNA molecule is estimated to be 30 eV from the available data on oscillator strength measurements. These ideas have been incorporated in a Monte Carlo computer program using Poisson statistics to treat the stochastic nature of the energy deposition processes and thereby determine the excitation and ionization states of the molecule. Each ionization reaction on the DNA backbone is assumed to lead to a DNA strand break. In our model double-strand breaks result from nearby independent breaks on opposite strands. Our calculated single- and double-strand break yields compare well with measured cellular data under conditions such that direct effects are thought to dominate strand break production.

Radiosensitization of Chinese Hamster V79 Cells by Bromodeoxyuridine Substitution of Thymidine: Enhancement of Radiation-Induced Toxicity and DNA Strand Break Production by Monofilar and Bifilar Substitution

Bromodeoxyuridine (BrdU) competes with thymidine (TdR) for incorporation into DNA of exponentially growing V79-171 cells. Such cells show an enhancement of the radiation response as determined by clonogenic survival and DNA damage measured by filter elution techniques after doses up to 15 Gy. The degree of radiosensitization for both survival and rates of alkaline and neutral elution are dependent on percentage BrdU substitution and independent of whether BrdU is in one strand only (monofilar) or both strands (bifilar) of the DNA duplex: e.g., for 16% BrdU substitution distributed either monofilarly or partially bifilarly, there is an enhancement factor for Do of 1.55. At this percentage substitution, the enhancement factor for the rate of alkaline elution is 1.75 and that for the rate of neutral elution is 1.54. The greater the percentage BrdU substitution, the larger was the enhancement ratio for survival and radiation-induced strand breaks in both monofilarly and bifilarly substituted cells. The increase in cell radiosensitivity caused by BrdU substitution shows a better correlation with the increase in radiation-induced double-strand breaks than with the increase in radiation-induced single-strand breaks.

Radiobiological effects of high-let particles: DNA strand breaks

Fundamental studies in radiation biology with high-LET charged particles involve a systematic study of the physical, chemical, molecular and cellular processes. Water molecules and DNA present inside a cell constitute the important targets for energy deposition which eventually lead to either cell death or mutation or transformation. High-LET charged particles are very efficient in causing these types of damages. One of the primary lesions for causing injury to a cell is the production of DNA strand breaks. A good understanding of these breaks is essential before ultimate biological effects of heavy particles can be predicted. Based on known molecular mechanisms of the formation of strand breaks, a theoretical model is presented along with a comparison between the predictions of the model and experimental data. The studies have shown that LET is not a convenient physical parameter to relate the extent of strand breaks and one needs to know the microscopic distribution of energy (track structure). A discussion has also been presented to provide a background on various radiobiological characteristics of high-LET charged particles from the point of view of their uniqueness in damaging cancer cells.

O-phenanthroline protects mammalian cells from hydrogen peroxide-induced gene mutation and morphological transformation.

The radiomimetic agent hydrogen peroxide is known to produce DNA strand breaks, chromosome damage and cell death. It has also been identified as one of the cytotoxic agents formed during certain drug metabolism and by phagocytic cells in the respiratory burst. Our laboratory recently identified the ultimate reactive species responsible for the DNA-damaging and cytotoxic effect of H2O2 as being hydroxyl radical. This was achieved by the use of the specific iron chelator o-phenanthroline, which prevents the occurrence of a Fenton reaction between H2O2 and chromatin bound ferrous ions. In this paper we show that H2O2 is able to induce mutation at the HGPRT locus in V79 cells and morphological transformation of C3H/10T1/2 cells. o-Phenanthroline abolishes both effects, indicating that hydroxyl radical is directly involved in mutation and carcinogenesis.

Inhibition of bleomycin-induced DNA strand breaks in V 79 Chinese hamster cells by the antioxidant propylgallate

Bleomycin induced DNA single-strand breaks in Chinese hamster V 79 cells which were detected by the alkaline filter elution assay. In the presence of propylgallate, an antioxidant, the amount of DNA single-strand breaks was significantly reduced. The production of DNA strand breaks by methylnitronitrosoguanidine used as a positiv control was not influenced by propylgallate. It is suggested that propylgallate inhibits the generation of DNA single-strand breaks by trapping reactive oxygen species produced by the bleomycin-iron(II) complex.

The role of catalytic iron in asbestos induced lipid peroxidation and DNA-strand breakage in C3H10T1/2 cells.

The involvement of catalytic iron in the vitro activities of crocidolite asbestos has been investigated. Exposure of C3H10T1/2 cells to either the UICC crocidolite standard reference sample or a non fibrous (milled) derivative resulted in an increase of thiobarbituric acid reactive substances. This catalytic activity was inhibited by pretreatment with the iron chelator desferrioxamine. The effect of this activity on cellular DNA was measured in an assay based on the production of DNA-strand breaks. Increased levels of DNA-strand breaks were detected in cultures treated with both the milled and UICC crocidolite. Inclusion of desferrioxamine with the asbestos inhibited DNA-strand breakage. It is concluded the catalytic iron present on the dust is capable of damaging both lipid and DNA and that this could be an important mechanism in asbestos pathogenicity.

Enhancement of X ray induced DNA damage by pre-treatment with halogenated pyrimidine analogs

The use of the halogenated pyrimidine analogs, including bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd), as potential clinical radiosensitizers is an area of renewed clinical research. Cellular radiation effects of these analogs using clinically achievable steady state plasma levels (10 −7−10 −5 M) were measured in vitro using exponentially growing Chinese hamster V79 cells. Radiation response was determined by clonogenic survival and by the production of DNA single strand (SSB) and double strand breaks (DSB) using filter elution techniques. Replacement of thymidine in DNA by BrdUrd or IdUrd was also determined. Drug exposure of BrdUrd or IdUrd for two population doublins (17 hr) prior to irradiation resulted in a progressive reduction of n̄ and D 0 compared to untreated controls over the drug dose range of 10 −7-10 −5 M. The percent thymidine replacement increased from 1% at 10 −7 M to 5% at 10 −6 M to 23% at 10−5 M. Using a 17 hr exposure at 10 −5 of BrdUrd of IdUrd, the production of SSB and DSB was increased by ≥2× and ≥1.5× respectively. No differences in the kinetics of repair of SSB and DSB were found following 3 hr of post-irradiation repair. We conclude that in this in vitro model, there appears to be a direct relationship between percent thymidine replacement, reduction of radiation survival parameters (n, D 0), and the production of DNA strand breaks in the clinically achievable dose range of these halogenated pyrimidine analogs. These filter elution assays may be adapted to in vivo studies and possibly may allow for monitoring of radiation-induced DNA damage and repair in clinical tumor specimens treated with these radiosensitizers.

Haloacetonitriles: metabolism, genotoxicity, and tumor-initiating activity.

Haloacetonitriles (HAN) are drinking water contaminants produced during chlorine disinfection. This paper evaluates metabolism, genotoxicity, and tumor-initiating activity of these chemicals. The alkylating potential of the HAN to react with the electrophile-trapping agent, 4-(p-nitrobenzyl)pyridine, followed the order dibromoacetonitrile (DBAN) greater than bromochloroacetonitrile (BCAN) greater than chloroacetonitrile (CAN) greater than dichloroacetonitrile (DCAN) greater than trichloroacetonitrile (TCAN). When administered orally to rats, the HAN were metabolized to cyanide and excreted in the urine as thiocyanate. The extent of thiocyanate excretion was CAN greater than BCAN greater than DCAN greater than DBAN much greater than TCAN. Haloacetonitriles inhibited in vitro microsomal dimethylnitrosamine demethylase (DMN-DM) activity. The most potent inhibitors were DBAN and BCAN, with Ki = 3-4 X 10(-5) M; the next potent were DCAN and TCAN, with Ki = 2 X 10(-4) M; and the least potent inhibitor was CAN, with Ki = 9 X 10(-2) M. When administered orally, TCAN, but not DBAN, inhibited hepatic DMN-DM activity. The HAN produced DNA strand breaks in cultured human lymphoblastic (CCRF-CEM) cells. TCAN was the most potent DNA strand breaker, and BCAN greater than DBAN greater than DCAN greater than CAN, which was only marginally active. DCAN reacted with polyadenylic acid and DNA to form adducts in a cell-free system; however, the oral administration of DBAN or DCAN to rats did not result in detectable adduct formation in liver DNA. None of the HAN initiated gamma-glutamyltranspeptidase (GGT) foci when assayed for tumor-initiating activity in rat liver foci bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxic properties of haloacetonitriles: Drinking water by-products of chlorine disinfection

Chlorinated and brominated haloacetonitriles (HAN), known drinking water contaminants which form during chlorine disinfection, were investigated for genotoxic activity. The HAN produced DNA strand breaks in cultured human lymphoblastic (CCRF-CEM) cells, bound to the nucleophilic trapping agent 4-( p-nitrobenzyl)pyridine and formed a covalent bond to polyadenylic acid in a cell-free reaction system. Thus, we have demonstrated that these chemicals are genotoxic, which would indicate a potential for carcinogenic activity and for human health hazard.

Mental-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells

Cloning efficiency and DNA strand breaks induction were compared in human diploid fibroblasts (HSBP) and chinese hamster ovary (CHO) cells treated with various metal salts. Cadmium (Cd2+), nickel (Ni2+) and chromate (Cr2O7) reduced the cloning efficiency of HSBP cells more than that of CHO cells whereas the reverse was true after treatment with mercury (Hg2+), manganese (Mn2+) and cobalt (Co2+). The effects on cloning efficiency did not consistently correlate with DNA strand breaking activity as all metals except Cr(VI) were more effective at producing DNA strand breaks in CHO cells than in human cells. The differential responses of the two cell types was shown to be only partially due to differences in cellular uptake of metals. DNA breaks induced in human cells by Hg2+ and Cr2O7 were shown most likely to be alkaline labile sites rather than true strand breaks since no damage was detected in a nick translation assay which measures the amount of free 3'-OH terminals. Damage induced by Mn2+ and Co2+, however, appeared to be comprised at least in part by true DNA strand breaks. DNA damage was also induced in HSBP cells following treatment with selenium but only in the presence of reduced glutathione. These studies indicate that DNA damage is not as major a consequence following some metal treatments in human cells as it appears to be in rodent cells. This suggests that rodent models for risk estimation of metal-induced tumorigenesis may not always be appropriate for extrapolation to humans.

Genotoxic Properties of Haloacetonitriles: Drinking Water By-Products of Chlorine Disinfection

Genotoxic Properties of Haloacetonitriles: Drinking Water By-Products of Chlorine Disinfection. DANIEL, F. B., SCHENCK, K. M., MATTOX, J. K., LIN, E. L. C., HAAS, D. L., AND PEREIRA, M. A. (1986). Fundam. Appl. Toxicol. 6,447–453. Chlorinated and brominated haloacetonitriles (HAN), known drinking water contaminants which form during chlorine disinfection, were investigated for genotoxic activity. The HAN produced DNA strand breaks in cultured human lymphoblastic (CCRF-CEM) cells, bound to the nucleophilic trapping agent 4-(p-nitrobenzyl)pyridine and formed a covalent bond to polyadenylic acid in a cell-free reaction system. Thus, we have demonstrated that these chemicals are genotoxic, which would indicate a potential for carcinogenic activity and for human health hazard.

The production of DNA strand breaks in human leukocytes by superoxide anion may involve a metabolic process.

H2O2 is known to be capable of inducing strand-break damage in intracellular DNA, but whether O2- also can do so in the absence of H2O2 is uncertain. The difficulty in distinguishing the effects of the two is that, under physiological conditions, dismutation of O2- to H2O2 can readily occur. When human leukocytes are stimulated with phorbol 12-myristate 13-acetate (PMA), they release O2-, and within a few minutes strand breakage in intracellular DNA can be observed. We have attempted to determine whether the O2- produced is itself capable of causing DNA damage or whether H2O2 alone, or in combination with O2-, is responsible for the observed damage. Addition of catalase (up to 250 micrograms/ml) to remove H2O2 prevented no more than about 50% of the DNA damage. The majority of the remaining damage could be blocked, in a dose-dependent manner, by superoxide dismutase (SOD) or a SOD-mimetic copper complex, identifying a fraction of damage to intracellular DNA dependent upon extracellular O2-. We studied this O2(-)-specific fraction through the use of three metabolic poisons (fluoride, 2-deoxyglucose, and A23187). These agents largely blocked DNA damage, while affecting extracellular O2- levels only slightly. For comparison, H2O2-induced DNA damage was studied with glucose oxidase to generate a flux of H2O2. The first two metabolic poisons had little effect, whereas A23187 did inhibit H2O2-induced DNA damage. We conclude that O2(-)-induced damage occurs through a mechanism that differs, at least in part, from the H2O2 damage pathway and that the former may involve one or more metabolic steps.

Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species

Active oxygen species are suspected as being a cause of the cellular damage that occurs at the site of inflammation. Phagocytic cells accumulate at these sites and produce superoxide ion, hydrogen peroxide and hydroxyl radical. The ultimate killing species, the cellular target and the mechanism whereby the lethal injury is produced are unknown. We exposed mouse fibroblasts to xanthine oxidase and acetaldehyde, a system which mimics the membrane of phagocytic cells in terms of production of oxygen species. We observed that the generation of these species produced DNA strand breaks and cellular death. The metal chelator o-phenanthroline completely abolished the former effect, and at the same time it effectively protected the cells from lethal injuries. Because complexing iron o-phenanthroline prevents the formation of hydroxyl radical by the Fendon reaction (Fe(II) + H 2O 2 → Fe(III) + OH − + OH ·), it is proposed that most of the death and DNA damage are brought about by OH radical, produced from other species by iron-mediated reactions.