DNA SSBs Research Articles

Comparative studies of UV-induced DNA cleavage by structural isomers of an iodinated DNA ligand

Purpose : To evaluate the importance of the position of the halogen atom in iodinated DNA-binding bibenzimidazoles, with respect to sensitization of UV-A-induced DNA breakage. Methods and Materials : Three analogues of iodoHoechst 33258, denoted ortho-, meta- and paraiodoHoechst, according to the site of iodine substitution, were synthesized. Plasmid DNA (pBR322) was used to assay UV-A-induced DNA single-strand breaks (ssbs). The location of the sites of strand breakage was determined by DNA sequencing gel analysis, using a 32P-endlabelled oligo-DNA with a single binding site for the ligands. Results : A clear trend in decreasing activity of sensitization of UV-induced DNA ssbs was established: ortho- > meta-, para- > iodoHoechst 33258. The sequencing gel studies showed that orthoiodoHoechst was distinct from the other three compounds, with respect to the sites of DNA strand breakage and the chemistry of the cleavage reaction. Conclusion : The position of iodine substitution in iodinated bibenzimidazoles determines the location of the carbon-centered radical on the ligand in the minor groove of DNA. DNA strand cleavage is mediated by abstraction of a nearby deoxyribosyl H-atom. Hence, the position of the radical species determines: which deoxyribosyl group is attacked (i.e., site of cleavage relative to the ligand binding site); which H-atom is abstracted, more specifically which of the five deoxyribosyl carbons is involved (i.e., the chemistry of the cleavage reaction), and the stereochemistry of the transition state for the H-atom abstraction (and hence the efficiency or extent of strand breakage). The ortho-compound represents the best example to date of iodinated DNA ligands designed as potential radiation sensitizers, as an extension of the well-established sensitization by halogenated DNA precursors.

Yield of Strand Breaks as a Function of Scavenger Concentration and LET for SV40 Irradiated with 4 He Ions

We have measured by gel electrophoresis the yields of single- and double-strand breaks (SSBs and DSBs) induced in aqueous solutions of SV40 DNA and the SV40 minichromosome by 137Cs gamma rays (mean LET 0.3 keV micron-1) and 4He ions (mean LETs 85, 102, and 152 keV microns-1). DNA SSBs are caused mainly by the hydroxyl radicals under these conditions and are reduced in yield as either the hydroxyl radical scavenger concentration or the LET is increased (over the range studied). The G(SSB) for 4He ion irradiation is less by a factor of up to 10 than the G(SSB) for gamma irradiation, depending upon the scavenger concentration. The difference in the yields of SSBs agrees well with the difference in the yields of hydroxyl radicals for the radiations in question. In contrast, the yields of DSBs are similar for gamma and 4He ion irradiation over much of the range of scavenging capacity studied. However, at the highest scavenger concentrations the yields of DSBs are greater for 4He ion irradiation. In addition, the yields of DSBs remain almost constant with increasing LET (over the range studied). Therefore the relative yield of DSBs per SSB increases with increasing LET, supporting the hypothesis that increasing LET leads to an increased clustering of damage in DNA.

Hepatic DNA Damage in Mice Given Organochlorine Chemicals

The effects of organochlorine chemicals, mainly pesticides, on DNA single-strand breaks (DNA SSB) and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in DNA were examined in mouse liver. Male mice were treated with organochlorine chemicals per os for 5 days, and killed 4hrs after the last administration. The daily dose of each chemical was about one-fifth of the reported LD50. The chemicals examined were endrin, dieldrin, γ-BHC, chlorpyrifos, heptachlor, pentachloronitrobenzene, pp′-DDT, pentachlorophenol, chlordane, 2, 4-D, mirex, pentachlorobenzene, hexachlorobenzene, chlorobenzilate, methoxychlor and trichloroethylene. The administrations of dieldrin, heptachlor, chlordane, mirex, pentachlorobenzene, hexachlorobenzene, chlorobenzilate, and trichloroethylene significantly increased relative liver weight. The 8-OHdG level in hepatic DNA of vehicle-treated mice was 1.2per 105 dG and comparable values were found in all groups treated with the chemicals. Hepatic DNA SSBs in mice treated with trichloroethylene and pentachlorophenol were significantly enhanced, but the effect was slight.

Paracetamol inhibits UV-induced DNA repair in resting human mononuclear blood cells in vitro.

The effects of paracetamol on the repair of DNA damage in resting human peripheral mononuclear blood cells (MNC) in vitro were investigated by means of the alkaline elution technique. Low doses of UV light (254 nm, 3 J/m2) caused a transient increase in the amount of DNA single-strand breaks and alkali-labile sites (SSBs). Paracetamol (0.1-1.0 mM) present during post-irradiation incubation approximately doubled the maximum level of UV-induced (1-3 J/m2) SSBs and delayed the completion of repair. Although there were considerable variations between cells prepared from different donors, the level of UV-induced DNA SSBs was always higher with paracetamol. Hydroxyurea (0.3 mM), an inhibitor of ribonucleotide reductase, caused a similar increased accumulation and slow removal of SSBs, whereas cytosine-1-beta-D-arabinofuranoside (Ara C) (10 microM), an inhibitor of DNA polymerases, led to a steady accumulation of DNA SSBs. The increased levels of SSBs caused by paracetamol or hydroxyurea were both completely suppressed by concomitant addition of deoxyribonucleosides; this supports the notion that paracetamol as well as hydroxyurea inhibits ribonucleotide reductase. About the same rates of formation and removal of UV-induced SSBs were observed in T lymphocytes, B lymphocytes and monocytes. In both isolated T lymphocytes and B lymphocytes, paracetamol (0.3 mM) markedly increased the level of DNA SSBs induced by UV, whereas monocytes seemed to be less sensitive to the effect of paracetamol. It is concluded that the inhibition of DNA repair may contribute to the clastogenic effects of paracetamol.

Mechanism of Radiosensitization by Halogenated Pyrimidines: Effect of BrdU on Repair of DNA Breaks, Interphase Chromatin Breaks, and Potentially Lethal Damage in Plateau-Phase CHO Cells

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Elaboration of cellular DNA breaks by hydroperoxides

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl ( tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01–1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1–6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37°C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37° C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cells lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10 −4 of greater, but prevent strand breaks and stimulate cell growth at 10 −5M.

Increase in topoisomerase‐II‐mediated dna breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate

Pre-treatment with low, non-toxic concentrations (0.04 microM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)-induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre-treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre-treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug-induced DNA breaks as assessed by the alkaline elution method after proteinase K digestion. The amount of DNA single-strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre-treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre-treatment were similar to those seen in cells without pretreatment. The amount of DNA double-strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VP16-induced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar to those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G1-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre-treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non-toxic MTX pre-treatment is due to a larger fraction of S-phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.

Characterization of resistance to VP-16 in human leukemic cell line.

Resistance mechanism was studied in the VP-16-resistant human leukemic cell line (THP-1/E) which was developed by continuous drug exposure. The drug uptake and efflux studies revealed no decrease in net cellular drug accumulation. VP-16-induced DNA single- and double-strand breaks in the whole THP-1/E cells decreased significantly compared to the sensitive counterpart as assessed by alkaline elution methods. Decrease in DNA SSBs was also observable in the isolated nuclei from the THP-1/E cells. The resistance to VP-16 in THP-1/E appeared to be independent of altered membrane permeability, and more likely to be associated with decreased VP-16-mediated DNA cleavage.

Induction of DNA strand breaks by RSU-1069, a nitroimidazole-aziridine radiosensitizer: Role of binding of both unreduced and radiation-reduced forms to DNA, in vitro

[2- 14C]-RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol], either as a parent (unreduced) or following radiation reduction, binds to calf thymus DNA in vivo. Radiation-reduced RSU-1069 binds to a greater extent and more rapidly than the parent compound. RSU-1137, a nonaziridino analogue of RSU-1069, binds following radiation reduction. Radiation-reduced misonidazole (1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol) exhibits binding ratios a thousand-fold less than those of reduced RSU-1069. There is no evidence for binding of parent misonidazole. Both parent and reduced RSU-1069 cause single strand breaks (ssbs) in pSV2 gpt plasmid DNA with thereduced compound causing a greater number of breaks. Parent and reduced RSU-1137 and misonidazole do not cause ssbs. It is inferred that the aziridine moiety present in both parent and reducedRSU-1069 is required for ssb production. RSU-1069 reacts with inorganic phosphate probably via nucleophilic ring-opening of the aziridine fragment. Incubation of plasmid DNA with reduced RSU-1069 in the presence of either phosphate or deoxyribose-5-phosphate at concentrations greater than 0.35 mol dm −3 prevents strand breakage, whereas 1.2 mol dm −3 deoxyribose does not protect against strand breakage formation. From these findings it is proposed that the observed binding to DNA occurs via the aziridine and the reduced nitro group of RSU-1069 and that these two have different target sites. Binding to DNA via the reduced nitro group may serve to increase aziridine attack due to localization at or near its target.

Radioprotection of euoxic bacteria by phenothiazine drugs.

Survival studies on irradiated euoxic E. coli B/r cells in presence of various concentrations of four radioprotecting phenothiazine drugs have been carried out. Maximum radioprotection was obtained at a optimal concentration for each drug and it decreased on either side of it. The DNA strand break studies at the maximum protective and non-protective concentrations of chlorpromazine and promethazine revealed that the number of ssbs in DNA were less at the protective concentration which were efficiently repaired by the type-III repair process. On the other hand, at the non-protective concentrations, inhibition of DNA repair was noticed and a higher number of DNA ssbs were detected. We suggest that the membrane is fluidized to a greater extent at the protective concentrations allowing the chemical restitution of damaged sites by NPSH compounds. At the non-protective high concentrations of the drugs, the membrane may be too grossly disorganised to allow any repair and at the same time high concentrations of the drugs or their radicals may also react with radioprotective intracellular sulphhydryls.