Potentiation of sodium chromate(VI)-induced chromosomal aberrations and mutation by vitamin B2 in Chinese hamster V79 cells.

The effect of vitamin B2, which is capable of reducing chromium(VI) to chromium(V), on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 200 microM vitamin B2 (riboflavin) for 24 h prior to exposure to Na2CrO4 (2.5-5 microM) resulted in an increase of metal-induced chromosomal aberrations and mutation at the HGPRT locus. These and other previous studies suggest that vitamin B2 enhances the clastogenic and mutagenic action of chromate compounds, through its ability to directly reduce chromium(VI) in cells.     

Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide

Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose-phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose-phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed.     

Lethal heat induces single strand breaks in the DNA of bacterial spores

Lethal heating induces DNA single strand breakage in bacterial endospores as detected by the alkaline sucrose gradient centrifugation technique. Heating of spores of $\text{Bacillus}\text{subtilis}$ 168 at 90°C for 10, 30, and 60 min induced 6, 15, and 15 single strand breaks, respectively and inactivated 6%, 98.2%, and 99.974% of the spores. This is the first report to our knowledge identifying specifically single strand DNA breakage with lethal heat injury of bacterial spores.     

Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA

Poly(ADP-ribose)polymerase is a chromatin-associated enzyme of eukaryotic cell nuclei that catalyses the covalent attachment of ADP-ribose units from NAD+ to various nuclear acceptor proteins. This post-translational modification has been postulated to influence several chromatin functions, particularly those where nicking and rejoining of DNA occur. Poly(ADP-ribosyl)ation reactions are strictly dependent upon the presence of interruptions on DNA. We have recently demonstrated that the DNA-binding domain of the protein containing two putative "zinc-fingers" binds DNA in a zinc-dependent manner. The basis for the recognition of the DNA strand breaks by this enzyme, and more precisely, its 29,000 Mr N-terminal part, which contains the metal binding sites, needed to be clarified. DNA probes harbouring a single strand interruption at a defined position were constructed from synthetic oligonucleotides. DNase I protection studies show that poly(ADP-ribose)polymerase specifically binds to a DNA single-strand break by its metal-binding domain depending upon the presence of Zn(II). These results support the idea that the enzyme participates to the maintenance of DNA integrity in eukaryotes.     

Photodynamic action of chlorpromazine on adenovirus 5: Repairable damage and single strand breaks

Chlorpromazine, a substituted phenothiazine, commonly used as a sedative, has been found to photosensitize the inactivation of human adenovirus 5 to wavelengths of light between 330 and 390 nm. The slope of the inactivation curve is three fold greater when fibroblasts from people having xeroderma pigmentosum (XP) were used as viral hosts than when normal fibroblasts were used, showing that at least two-thirds of the damage produced in the virions is repairable by normal human fibroblasts. The phototreatment of chlorpromazine sensitized virions also results in the production of DNA strand breaks, which correlate fairly well with the production of lethal viral damage as measured in XP fibroblasts. These findings suggest that the photosensitization of the skin observed in patients treated with chlorpromazine might be due to DNA damage.     

Interspecies differences in DNA single strand breaks caused by benzo(a)pyrene and marine environment

The presence of DNA single strand breaks in untreated specimens of selected species, mosquito fish Gambusia affinis, painted comber Serranus scriba, blue mussel Mytilus galloprovincialis, spiny crab Maja crispata and sea cucumber Holothuria tubulosa as well as in 10 microg/g benzo(a)pyrene (BaP) treated mosquito fish, blue mussel and spiny crab was measured, using alkaline filter elution. Interspecies differences in alkaline elution profiles were observed and attributed to different lengths of DNA from different sources and to differences in the number of strand breaks present during normal cellular events in different phyla. Spiny crab hemocytes are more sensitive to action of BaP then blue mussel hemocytes and mosquito fish hepatocytes that could be explained by differences in the rates of distinct metabolic reactions and DNA repair among the investigated species. In field study, DNA single strand breaks were measured in hepatocytes of painted comber and in hemocytes of blue mussel and spiny crab from natural population specimens collected at eight sampling sites along Istrian coast, Croatia. Spatial variations in DNA integrity for each species were detected and revealed for the first time that spiny crab is responsive to different environmental conditions. Interspecies variations in the DNA integrity due to environmental conditions, confirmed species specific susceptibility to genotoxicity of certain environment that in long-term may modify the structure of marine communities. The multi-species approach in designing biomonitoring studies was suggested.     

DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions

Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these ‘complex’ lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIα and DNA polymerase β. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB and identified it as DNA polymerase δ (Pol δ). Using recombinant protein we confirmed that the 3′-5′-exonuclease activity of Pol δ can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol δ are partially deficient in the repair of these ‘complex’ lesions, demonstrating the importance of Pol δ during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.     

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.     

The repair of DNA single strand breaks in human cells with genetical disorders

The aim of this work was to make a preliminary examination of some hereditary diseases, related to precocious aging in order to determine whether the defects involved resulted from an impairment of the DNA repair capacity. To test for an impairment in DNA single strand capacity, in cells from patients with different diseases, we have followed the rate of the DNA after X-irradiation by sedimentation on alkaline sucrose gradients. It was found that all cells observed from lesch nyhan, cystic fibrosis, trisomy 15 et 21, retinoblastoma, were able to repair the single strand breaks in their DNA to the same extent and at the same initial rate.     

Inhibition of B(a)P induced strand breaks in presence of curcumin

Incidence of cancer at different sites may be related to oxidative damage to host genome by genotoxicants. These oxidative actions may be modified by phytochemicals present in foods. The non-nutritive dietary constituents which possess antimutagenic property appear to be promising chemopreventive agents. This study reports the protective effect of curcumin on B(a)P induced DNA damage in human peripheral blood lymphocyte cells. The study group consisted of 10 male smokers, 10 non-smokers and 10 non-smoking females aged between 25 and 45. The DNA damage was assessed using comet assay. In all the groups curcumin showed a dose-dependent inhibitory effect. The effect appeared to be sex dependent. There was no correlation between DNA damage and GST-Mu levels and levels of micronutrients namely Vitamins A, E and beta carotene. The results of this study are in line with our earlier observations on turmeric/curcumin as a potential chemopreventer.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

ESR characterization and metallokinetic analysis of Cr(V) in the blood of rats given carcinogen chromate(VI) compounds.

It has been shown that bio-trace metal elements are related to many diseases and the aging process. For many years, carcinogen hexavalent chromium (VI) has been known to be toxic to animals, but its dynamic toxicological mechanism is not sufficiently elucidated. Bioinorganic chemistry in terms of metallokinetic analysis of beneficial or toxic metal ions and their complexes is an important investigation for understanding their biochemical and physiological roles. We have tried to examine the real-time behavior of paramagnetic metal ions and complexes in animals, in which electron spin resonance (ESR) was capable of measuring paramagnetic species in chemical and biological systems. On the basis of our previous results on stable nitroxide spin probes, we have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze time-dependent ESR signal changes due to paramagnetic metal ions and their complexes in real time. When K2Cr2O7 or Na2Cr2O7 in saline was intravenously administered to rats, two ESR signals due to pentavalent chromium(V) were detectable in the circulating blood of rats. Cr(V) detected in the blood was indicated to be in the CrO(O4) and CrO(S2O2) coordination modes after the study on model complexes. From the changes of ESR signal intensities due to Cr(V) in the blood, the metallokinetic parameters were obtained using the pharmacokinetic analysis and the curve-fitting methods. The obtained results are important for understanding carcinogen chromate in terms of the formation of Cr(V) in animals. In addition, we propose the BCM-ESR method, which is useful to analyze the disposition of paramagnetic metal species in the blood of living animals.     

Isolation and characterization of mAMSA-hypersensitive mutants. Cytotoxicity of Top2 covalent complexes containing DNA single strand breaks

Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro(473) to Leu and Gly(737) to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly(737) --> Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro(473) --> Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro(473) --> Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro(473) --> Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs.     

Asp-Ala-His-Lys (DAHK) inhibits copper-induced oxidative DNA double strand breaks and telomere shortening

Both DNA and the telomeric sequence are susceptible to copper-mediated reactive oxygen species (ROS) damage, particularly damage attributed to hydroxyl radicals. In this study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. Asp-Ala-His-Lys (DAHK), a specific copper chelating tetrapeptide d-analog of the N-terminus of human albumin, attenuated DNA strand breaks in a dose dependent manner. d-DAHK, at a ratio of 4:1 (d-DAHKCu), provided complete protection of isolated DNA from double strand breaks and, at a ratio of 2:1 (d-DAHKCu), completely protected DNA in Raji cells exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres and Raji cell treated samples showed some conservation of telomere sequences. d-DAHK provided complete telomere length protection at a ratio of 2:1 (d-DAHKCu). The human albumin N-terminus analog, d-DAHK, protects DNA and telomeres against copper-mediated ROS damage and may be a useful therapeutic adjunct in ROS disease processes.     

The effect of temperature shock on the yield of gamma-induced single strand breaks in bacterial DNA

The existence of the super fast repairing system for single-strand breaks in DNA, induced in cells exposed to gamma-irradiation at 0°C in a phosphate buffer under aerobic conditions, has been shown in three strains of Escherichia coli K-12 (AB 1157 $\text{rec}{}^{\mathrm{+}}$, AB 2463 $\text{rec A}\text{13}$ and JC 4457 $\text{rec B}$). Super fast repairing system rejoins up to 90–95 % of initial single-strand breaks during irradiation. An estimation of the lower limit of the rate of repairing the breaks by this system has been made.