Number Of DNA Single-strand Breaks Research Articles

DNA Repair Activation and Cell Death Suppression by Plant Polyphenols in Keratinocytes Exposed to Ultraviolet Irradiation.

This work investigated effects of plant polyphenolic compounds (PPs) on responses of cultured human HaCaT keratinocytes to ultraviolet radiation in the C range (UV-C). The experimental data obtained indicate a cytoprotective effect of PPs added immediately after UV-C exposure. The efficiency of PPs was lowered in the following order: acacetin ≥ silybin > quercetin. The influence of PPs on phosphorylation of histone H2AX and the number of single-strand DNA breaks in the nuclei of keratinocytes were also studied. Using the comet assay and γH2AX staining, followed by fluorescence microscopy, it has been established that PPs can reduce DNA damage in the nuclei of keratinocytes exposed to UV-C. It is concluded that PPs can diminish the destructive effect of UV radiation on skin cells, activating the process of repairing genetic damage.

Effect of ionizing radiation on the male reproductive system.

In the light of current data concerning the growing exposure to ionizing radiation (IR) originating from atrificial sources, especially from medical ones, and also related to occupational exposure, it is justifiable to systematize the state of knowledge concerning the effect of IR on the male reproductive system. There is no basis for the application of the hypothesis of hormesis in the area of male reproductive health. Regarding the impact of IR on spermatogenesis, spermatogonia are less susceptible to the occurrence of DNA damage after exposition to IR, but are characterized by slower DNA repair compared to somatic cells. Damage to the genes after exposure to IR is possible at each stage of spermatogenesis; however, haploidal spermatids show the highest radiosensitivity in this respect. The genetic risk of the cells differentiating during spermatogenesis is limited to one cycle of spermatogenesis, whereas the genetic instability may persist for the whole period of life, and DNA damage induced by IR may be transmitted to future generations. The minimum dose causing detectable DNA damage was 30 Gy. While exceeding this dose, the number of single-strand DNA breaks increases. Among males exposed to IR, a decrease was observed in sperm motility and in the percentage of morphologically normal spermatozoa as well as in an intensification of vacuolization. The genetic material in the sperm of these males showed higher fragmentation and methylation of genomic DNA. In the context of the epidemiological situation concerning the prevalence of infertility, while assessing the health effects of exposure to IR from artificial, including medical sources, the reproductive risk should be considered.

The effects of the HDAC inhibitor sodium butyrate on the expression of repair genes Rad51 and XRCC5 in fibroblast lines mEras-Waf1+/+ and mEras-Waf1–/–

Transformed mice fibroblasts with a knockout CDKN1A gene, which encodes р21/Waf1 protein (mEras-Waf1–/–cells), are characterized by a large number of single-strand DNA breaks and associated γ-H2A.X foci compared to the initial transformant line E1A+cHa-ras (mEras-Waf1+/+ cells). According to immunofluorescence and immunoblotting data, the nuclei of the cells from both studied lines mEras-Waf1+/+ and mEras-Waf1–/–contain significant amounts of Rad51 and Ku80 proteins, which participate in DNA repair, with a slight prevalence of Ku80 in CDKN1A knockout cells. Under a short-term effect of adriamycin, a DNA-damaging agent, on the cells of both lines, additional accumulation of Rad51 protein foci occurs in their nuclei. However, an HDAC inhibitor, sodium butyrate, notably decreases the contents of Rad51 and Ku80 proteins in both intact mEras-Waf1+/+ and mEras-Waf1–/–cells and cells treated with adriamycin. Results of RT-PCR and immunoblotting demonstrate that inhibiting effect of sodium butyrate (NaBut) becomes apparent at the level of Rad51 and XRCC5 gene transcription and at the level of translation of the respective repair proteins, Rad51 and Ku80. The observed suppressive effect of NaBut (HDAC inhibitor) on the components of DNA repair system can be partially explained by antiproliferative function of HDAC inhibitors. In addition, transcriptional activation of pluripotency genes Oct-4, Sox-2, and Klf4 was discovered in mEras-Waf1+/+ and mEras-Waf1–/–cells under the influence of NaBut, which implies that these genes are under negative control at the level of the chromatin structure.

Differential responses to high- and low-dose ultraviolet-B stress in tobacco Bright Yellow-2 cells.

Ultraviolet (UV)-B irradiation leads to DNA damage, cell cycle arrest, growth inhibition, and cell death. To evaluate the UV-B stress–induced changes in plant cells, we developed a model system based on tobacco Bright Yellow-2 (BY-2) cells. Both low-dose UV-B (low UV-B: 740 J m−2) and high-dose UV-B (high UV-B: 2960 J m−2) inhibited cell proliferation and induced cell death; these effects were more pronounced at high UV-B. Flow cytometry showed cell cycle arrest within 1 day after UV-B irradiation; neither low- nor high-UV-B–irradiated cells entered mitosis within 12 h. Cell cycle progression was gradually restored in low-UV-B–irradiated cells but not in high-UV-B–irradiated cells. UV-A irradiation, which activates cyclobutane pyrimidine dimer (CPD) photolyase, reduced inhibition of cell proliferation by low but not high UV-B and suppressed high-UV-B–induced cell death. UV-B induced CPD formation in a dose-dependent manner. The amounts of CPDs decreased gradually within 3 days in low-UV-B–irradiated cells, but remained elevated after 3 days in high-UV-B–irradiated cells. Low UV-B slightly increased the number of DNA single-strand breaks detected by the comet assay at 1 day after irradiation, and then decreased at 2 and 3 days after irradiation. High UV-B increased DNA fragmentation detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay 1 and 3 days after irradiation. Caffeine, an inhibitor of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) checkpoint kinases, reduced the rate of cell death in high-UV-B–irradiated cells. Our data suggest that low-UV-B–induced CPDs and/or DNA strand-breaks inhibit DNA replication and proliferation of BY-2 cells, whereas larger contents of high-UV-B–induced CPDs and/or DNA strand-breaks lead to cell death.

Calculation of DNA strand breaks due to direct and indirect effects of Auger electrons from incorporated 123I and 125I radionuclides using the Geant4 computer code

Purpose: In this work the number of DNA single-strand breaks (SSB) and double-strand breaks (DSB) due to direct and indirect effects of Auger electrons from incorporated 123I and 125I have been calculated by using the Geant4-DNA toolkit. We have performed and compared the calculations for several cases: 125I versus 123I, source positions and direct versus indirect breaks to study the capability of the Geant4-DNA in calculations of DNA damage yields.Materials and methods: Two different simple geometries of a 41 base pair of B-DNA have been simulated. The location of 123I has been considered to be in 123IdUrd and three different locations for 125I.Results: The results showed that the simpler geometry is sufficient for direct break calculations while indirect damage yield is more sensitive to the helical shape of DNA. For 123I Auger electrons, the average number of DSB due to the direct hits is almost twice the DSB due to the indirect hits. Furthermore, a comparison between the average number of SSB or DSB caused by Auger electrons of 125I and 123I in 125IdUrd and 123IdUrd shows that 125I is 1.5 times more effective than 123I per decay.Conclusions: The results are in reasonable agreement with previous experimental and theoretical results which shows the applicability of the Geant-DNA toolkit in nanodosimetry calculations which benefits from the open-source accessibility with the advantage that the DNA models used in this work enable us to save the computational time. Also, the results showed that the simpler geometry is suitable for direct break calculations, while for the indirect damage yield, the more precise model is preferred.

DNA damage after acute exposure of mice skin to physiological doses of UVB and UVA light

Solar ultraviolet (UV) radiation is an important risk factor in skin carcinogenesis. This has been attributed mainly to the UVB waveband because the high-energetic photons are capable of interacting with DNA and inducing DNA damage. Recently, UVA light has also gained increasing interest in relation to DNA alteration. Although UVA photons are less energetic than UVB, they comprise a major fraction of sunlight UV radiation and penetrate deep into the skin. The study was carried out to compare the acute effects of UVA and UVB light on SKH-1 mice in relation to DNA damage and associated parameters. Mice were exposed to UVA (10 and 20 J/cm(2)) or UVB (200 and 800 mJ/cm(2)) radiation. The number of DNA single-strand breaks (SSB) in lymphocytes, amount of phosphorylated histone H2AX (gamma-H2AX) and apoptosis or DNA fragmentation (TUNEL-positive cells) in skin sections and level of gamma-H2AX, activated caspase-3 and phosphorylated p53 in skin were evaluated after 4 and 24 h. SSB analyzed by alkaline comet assay were found to be 4 and 24 h following UVB and UVA treatment, respectively. TUNEL and gamma-H2AX-positive cell were observed only in UVB exposed animals at both time intervals. The level of activated caspase-3 and phospho-p53 was increased 24 h after UVA and UVB radiation and was more apparent in UVB treated mice. The results indicate that the mechanism of DNA damage caused by acute UVA exposure includes formation of SSB (oxidative damage), but not double-strand breaks.

DNA damage in thymocytes of mice after exposure of the whole body to combined action of cadmium ions and γ radiation

The effect of combined action of cadmium chloride at a dose of 0.5 mg/kg of body weight and γ-radiation of 1 Gy on induction of DNA damage in thymocytes and the total number of cells in the thymus of mice was studied. We found that injection of CdCl2 0.5 h prior to irradiation decreased the number of single-strand DNA breaks and the number of alkali-labile sites in thymocytes 48 h after irradiation as compared to the γ-radiation effect only. This effect was associated with a strong decrease in the total number of thymocytes in this organ as compared to the action of cadmium ions and γ radiation separately. This masked the general genotoxic effect of combined treatment and created an illusion of a radioprotective effect of cadmium ions. Injection of cadmium chloride into mice 24 h prior to irradiation was followed by an additive increase in the number of the single-strand DNA breaks and the number of alkali-labile sites in thymocytes as compared to the respective controls such as the separate effects of cadmium ions and irradiation. We revealed a simultaneous decrease in the part of DNA tightly bound to proteins, i.e., DNA-protein cross-links as compared to the effect of γ-radiation only. We did not observe any statistically significant changes in the total number of thymocytes as compared to the separate effects of cadmium ions and irradiation. Thus, our data show that exposure of murine thymocytes to combined action of cadmium ions and γ-radiation at the doses and with the methods of treatment used induced additive effects but not antagonistic effects or protection against radiation.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Disparity in the induction of glutathione depletion, ROS formation, poly(ADP-ribose) polymerase-1 activation, and apoptosis by quinonoid derivatives of naphthalene in human cultured cells

The purpose of this study is to examine the differences in the induction of cytotoxic effects and poly(ADP-ribose) polymerase-1 activation in human MCF-7 breast cancer cells by quinonoid derivatives of naphthalene, including 1,2-naphthalenediol (NCAT), 1,4-naphthalenediol (NHQ), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1,4-NQ). Results from the cytotoxic response analyses in cells indicated that all naphthalene quinonoids induced cell death in MCF-7 cells at concentrations ranging from 0.1 to 100microM where NHQ and 1,4-NQ were more efficient than NCAT and 1,2-NQ in the induction of cell death. Results from Western blot analyses confirmed that treatment of cells with NCAT and NHQ resulted in up-regulation of p53 protein expression and a significant shift in bax/bcl2 ratio, suggesting the induction of p53-dependent apoptosis in MCF-7 cells. Additionally, we observed that all naphthalene quinonoids induced increases in reactive oxygen species (ROS) formation and glutathione (GSH) depletion in MCF-7 cells. The induction of ROS formation and GSH depletion in cells by naphthalene quinonoids decreases in the rank order 1,4-NQ>NHQ>1,2-NQ approximately equal to NCAT. Further investigation indicated that least-squares estimates of the overall rates of elimination (k(e)) of naphthalene quinonoids in MCF-7 cells decreased in the rank order 1,4-NQ>1,2-NQ>NHQ>NCAT. Values of k(e) were estimated to be between 0.280h(-1)(T(1/2)=151min) and 13.8h(-1)(T(1/2)=3.05min). These results provide evidence that the para-isomeric form of naphthalene quinonoids tend to induce acute production of ROS and alterations in intracellular redox status in cells, leading to the subsequent cell death. Further, all naphthalene quinonoids induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells at non-cytotoxic concentrations. The reduction of intracellular NAD(P)H in cells exposed to NCAT and 1,2-NQ was blocked by two types of poly(ADP-ribose) polymerase (PARP) inhibitors whereas PARP inhibitors did not prevent the reduction of NAD(P)H in cells exposed to NHQ and 1,4-NQ. Further investigation confirmed that increases in the number of DNA single-strand breaks were detected in MCF-7 cells exposed to NCAT and 1,2-NQ as measured by the single-cell gel electrophoresis (Comet) assay whereas NHQ and 1,4-NQ did not induce increases in the number of single-strand breaks in MCF-7 cells. Overall, results from our investigation suggest that while NHQ and 1,4-NQ are more efficient in the induction of cell death, NCAT and 1,2-NQ are prone to induce depletion of NAD(P)H and NAD(+) mediated by PARP-1 activation through formation of DNA single-strand breaks in human cultured cells.

Activation of Ataxia Telangiectasia Mutated by DNA Strand Break-inducing Agents Correlates Closely with the Number of DNA Double Strand Breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.

OZONE-INDUCED DNA SINGLE STRAND-BREAKS IN GUINEA PIG TRACHEOBRONCHIAL EPITHELIAL CELLS IN VIVO

Two-month-old male guinea pigs (Dunkin-Hartley strain, specific pathogen free), 4 in each group, were exposed to 0.00, 0.45, and 1.00 ppm O 3 for 72 h. The trachea with two main bronchi was removed from each animal after O 3 exposure. The trachea lavage fluid was used to measure the protein content as an index of altered tracheobronchial epithelial (TE) cell membrane permeability after O 3 exposure. The TE cells were isolated and employed for the determination of DNA single-strand breaks (SSBs) by fluorometric analysis of DNA unwinding (FADU). The statistical significance level was set atα = .05. The results show that neither the yield nor the viability of the TE cell from various O 3 treatment groups was different from that of controls. Compared to controls, the protein content was elevated significantly after 0.45 ppm O 3 exposure; however, the amount of DNA SSBs was not. The number of DNA SSBs increased significantly in the 1.00 ppm O 3 exposure group when compared to controls. Regardless of the alkali incubation time at 15°C, the double-stranded DNA left in the alkali TE cell lysate was a linear function of O 3 exposure concentrations.

Activation of genotoxins to DNA-damaging species in exfoliated breast milk cells

Exfoliated cells, isolated from breast milk samples donated by UK-resident women ( n=15), were incubated, either immediately or after culture for 7 days, with one of a series of genotoxins, either in the presence or absence of the DNA-repair inhibitors, hydroxyurea (HU), and cytosine arabinoside (ara-C). The numbers of DNA single-strand breaks induced were then assessed as comet tail length (CTL) (μm) using the alkaline single cell–gel electrophoresis (‘Comet’) assay; cell viability was measured by trypan blue exclusion. The heterocyclic aromatic amines (HAAs) (2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) (0.4 mM), 3-amino-1,4-dimethyl-5H-pyrido[4,3- b]indole (Trp-P-1) (1.67 mM), 3-amino-1-methyl-5H-pyrido[4,3- b]indole (Trp-P-2) (1.77 mM)), a polycyclic aromatic hydrocarbon (benzo[ a]pyrene (B[ a]P) (0.36 mM)), a nitro-polycyclic aromatic hydrocarbon (1-nitropyrene (1-NP) (1.84 mM)) and aromatic amines ( o-toluidine (0.85 mM), p-chloroaniline (0.71 mM)) each induced statistically significant ( P<0.0001, Mann–Whitney test) increases in median CTLs in breast milk cells from all the donors examined when incubated (30 min, 37°C) in the presence of HU/ara-C. In some cases, these compounds were also active in the absence of the repair inhibitors. There were marked variations in comet formation between donors and between genotoxins. Cell culture appeared to increase the epithelial cell proportion and cultured cells retained their ability to activate genotoxins. The results suggest that breast milk is a valuable source of human mammary cells for the study of the metabolic activation of possible carcinogens.

Tungsten Particle-Induced Nicking of Supercoiled Plasmid DNA

Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes. In such applications, tungsten was observed to affect the integrity of plasmid DNA. Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks. As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected. The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA. No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers. Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged. Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts.

Protection from radiation-induced DNA single-strand breaks by induction of nuclear metallothionein

Purpose : To examine the extent to which nuclear metallothionein protects from radiation-induced DNA damage under aerobic and hypoxic conditions. Materials and methods : A semiquantitative fluorescence image analysis method measured the nuclear content of metallothionein (MT) in ME180 and SiHa human squamous cervical carcinoma cell lines under normal growth conditions, and following MT induction by zinc. The extent of initial DNA damage following 60 Co irradiation under aerobic and hypoxic conditions was assessed using the alkaline comet assay. Results : Provided that cells were maintained at 37°C, most of the cellular content of MT was in the nucleus. Incubation at 4°C caused the rapid translocation of MT from the nucleus into the cytoplasm in both cell lines, with no net loss of cellular MT. Baseline nuclear MT levels were about four times greater in ME180 cells, and were much more readily induced by treatment with 100 μ m zinc acetate, compared with SiHa cells. Under aerobic conditions, MT induction by zinc resulted in no protection in either of the cell lines. Under hypoxic conditions, however, the number of DNA single-strand breaks in zinc-treated cells was reduced by ~40% in ME180, but not in SiHa cells, when compared with non-induced controls. Conclusions : Nuclear MT can exert a significant level of protection from radiation by a mechanism that involves competition with oxygen for DNA radical sites and/or scavenging of free radicals. Because increased MT levels have been reported in hypoxic micro-regions of some solid tumours, this protective mechanism might have clinical relevance.

Induction of DNA damage and expression of heat shock protein HSP70 by polychlorinated biphenyls in the marine sponge Suberites domuncula Olivi

The effects of different polychlorinated biphenyls (PCBs) including toxic coplanar non- ortho (PCB77) and non-planar mono- ortho (PCB118) and di- ortho (PCB153) congeners on the extent of DNA damage as well as on the expression of heat shock protein 70 (HSP70) were investigated in the marine sponge Suberites domuncula Olivi (Porifera; Demospongiae). A time-dependent increase in the number of DNA single-strand breaks, expressed as strand scission factor (SSF), was found after injection of a single dose of 25 μg of PCB118 or PCB153 per gram wet mass of S. domuncula, using Fast Micromethod assay, which is based on the unwinding of DNA under alkaline conditions. The number of strand breaks induced by PCB153 was about two-fold higher than that observed for PCB118 after an incubation period of six days. Unexpectedly, a strong reduction in the rate of DNA unwinding compared to untreated control, that could be indicative for the formation of DNA crosslinks, was found following treatment of S. domuncula with PCB77, which is, in vertebrate systems, the most toxic among the PCB congeners studied. The three selected PCB congeners are able to induce the expression of both “constitutive” (HSP73; M r 73 kDa) and “inducible” (HSP75; M r 75 kDa) HSP70 proteins. The expression of HSP75 was markedly lower than that observed for HSP73, and varied considerably among different sponge individuals exposed to PCB77. The results in this work demonstrate that: (1) marine sponges ( S. domuncula) respond to distinct PCB congeners by induction of different degrees of DNA damage, and (2) expression of HSP is a potential (but not specific) biomarker for PCB exposure in sponges.

Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.

Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents. Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli. We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks. dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide. In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles. In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps. The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.

Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated by copper and iron

Oxidative DNA damage is involved in diverse biological phenomena and consists of several kinds of lesions, mainly, strand breaks, base modifications, and DNA-protein crosslinking. However, little is known about the existence of a chemical relationship among them or the ratio by which these different types of lesions are produced. In the present study we investigated whether a relationship exists between DNA strand breakage and base modification. We selected cupric [Cu(II)] and ferric [Fe(III)] ions for this study because these transition metals are active catalysts of DNA damage in vivo. Supercoiled plasmid DNA pZ189 was treated with Cu(II) or Fe(III) in the presence of different reducing agents. We measured in each sample both the number of DNA single-strand breaks (SSB) by quantitative electrophoresis and the amount of a modified DNA base, 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC with simultaneous electrochemical (EC) and spectrophotometric detection. The number of DNA SSBs produced was linearly related to the number of 8-OHdG present. The regression of the number of SSBs as a function of the number of 8-OHdG is expressed by the equation [SSBs] = b × [8-OHdG], where b = 1.7, 2.0, 2,7, 1.7, and 9.4, for Cu(II) in the presence of H 2O 2, l-cysteine and l-ascorbate, and for Fe(III) in the presence of H 2O 2 and l-ascorbate, respectively. The linear correlation we observed between the production of SSB and 8-OHdG mediated by Fe(III) and by Cu(II) suggests that these products may arise via a common chemical mechanism and could allow an easier and more precise estimation of DNA breakage.

Characterisation of a human small-cell lung cancer cell line resistant to the DNA topoisomerase I-directed drug topotecan.

Camptothecins are DNA topoisomerase I-directed anti-tumour drugs with a novel mechanism of action. Topotecan (TPT), a hydrophilic derivative of camptothecin, is currently undergoing phase II clinical trials in small-cell lung cancer (SCLC). Human SCLC OC-NYH cells were made more than 6-fold resistant to topotecan by stepwise drug exposure and resistance was stable for 70 passages without drug. NYH/TPT cells had half the topoisomerase I level and activity of wild-type cells. However, no difference in camptothecin or topotecan inhibition of topoisomerase I-mediated DNA relaxation was found, indicating that the enzyme itself was unchanged in the resistant cell. In NYH/TPT cells, topoisomerase II alpha and beta levels were increased approximately 2-fold. Accordingly, the topoisomerase II-directed drug etoposide (VP-16) induced an increased number of DNA single-strand breaks in NYH/TPT cells. However, sensitivity to different topoisomerase II-targeting agents in NYH/TPT cells varied from increased to decreased, indicating a role for as yet unidentified factors acting on the pathway to cell death after topoisomerase II-induced DNA damage has occurred. Of 20 anti-cancer agents tested, only hydroxyurea showed marked collateral hypersensitivity in NYH/TPT cells.

Final Report on the Safety Assessment of Sodium Iodate

Sodium Iodate is an inorganic salt that is intended for use as an oxidizing agent in cosmetics, but no current uses have been reported. It is approved by the European Union for use as a preservative in rinse-off cosmetic products at concentrations no greater than 0.1%. Pure Sodium Iodate is a sufficiently strong oxidizing agent that it presents a fire risk near organic material, and it can react violently with aluminum, arsenic, carbon, copper, hydrogen peroxide, phosphorous, potassium, sulfur, and metal sulfides. Sodium Iodate is toxic to the retina; injection of 10-4 M Sodium Iodate into the vitreous of rabbit eyes inactivated the electroretinogram in 1 day. Acute toxicity studies in mice show that concentrations of 505 mg/kg delivered orally is expected to cause convulsions and death in half the animals. No mutagenic activity was seen in Ames tests. Exposure of repair proficient strains of Escherichia coli to ionizing radiation and Sodium Iodate increased the number of DNA single-strand breaks over those seen with exposure to ionizing radiation alone. Sodium Iodate combined with aflatoxin B1 showed fewer mutations in the Ames test than did aflatoxin alone. These available data were not sufficient to support the safety of Sodium Iodate for use in cosmetic formulations. Additional data were considered necessary to evaluate the safety of this ingredient, including the purpose of use and likely concentration of use in cosmetics; 28-day dermal toxicity data; and animal irritation data as a function of dose. It cannot be concluded that this ingredient is safe for use in cosmetic products until the listed safety data have been obtained and evaluated.

A Mathematical Formalism Describing the Yield of Radiation-Induced Single- and Double-Strand DNA Breaks, and Its Dependence on Radiation Quality

The purpose of this paper is to describe mathematically the observed dependence of the number of DNA single-strand breaks (SSBs) per unit dose on radiation quality (e.g., LET) by invoking the concept of matrix saturation (i.e., the fact that a nucleotide can be the site of one SSB only and therefore further local energy deposition is wasted), and to show that, based on this, one obtains a characteristic variation of the yield of DNA double-strand breaks with variation of LET, in qualitative agreement with certain experimental determinations. The formalism developed here makes use of methodology from the field of integral geometry, introduced by Kellerer [Radiat. Prot. Dosim. 31, 9-16, 1990] in microdosimetry.