Antagonistic effect of magnesium chloride on the nickel chloride–induced inhibition of DNA replication in chinese hamster ovary cells

The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl₂) was analyzed by radiolabeled-thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni²⁺-induced inhibition was time- and concentration- dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl₂, which was the highest noncytotoxic concentration utilized. After six hours, 500 μM and 1 mM as well as 2.5 mM NiCl₂ all produced the same 50% to 60% reduction in [³H]-thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 μM or 1 mM NiCl₂ treatment began to increase after washout of nickel, but a six-hour exposure of cells to 2.5 mM NiCl₂ produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl₂ used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [³H]-thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl₂ for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl₂ on DNA replication was prevented by the addition of magnesium chloride (MgCl₂) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg²⁺, since the maximally effective dose of Mg²⁺ reduced ⁶³Ni²⁺ uptake by no more than 25% while the inhibition of replication was completely reversed.     

DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193

The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Sodium selenite enhances glutathione peroxidase activity and DNA strand breaks in hepatoma induced by N-nitrosodiethylamine and promoted by phenobarbital

An element/compound that acts as an antioxidant as well as, can increase the oxidative stress offers a new approach in differentiation therapy. Experiments were carried out to determine the effect of selenite on DNA damage and glutathione peroxidase (GPx) activity in N-nitrosodiethylamine (DEN) induced, phenobarbital promoted rat hepatoma. Supra-nutritional level of selenite (4 ppm) was supplemented at either, before-initiation/after-initiation and/or during entire period of the study. At the end of experiment period (20 weeks), extent of DNA damage (alkaline comet assay), selenium concentration, and GPx activity were assessed on nodular tissue (NL) cells, surrounding liver (SL) cells, and whole liver tissue (control) cells. Hepatic selenium level and GPx activity were decreased in DEN and PB-administered animals, whereas the DNA damage was found to be increased in both NL and SL cells compared with control group. However, the DNA damage is more in SL cells than in NL cells. Pre-supplementation of selenite did not show any difference in DNA (strand breaks) damage, selenium, and GPx activity. Increased hepatic selenium concentration and GPx activity were observed in both NL and SL cells in post-supplementation and entire period of selenite supplemented animals compared to DEN + PB treated animals. However, DNA damage was increased in NL but decreased in SL cells. Supplementation of selenite alone for 16 or 20 weeks had shown increased DNA damage, selenium concentration, and GPx activity compared to normal control animals. In summary, cancer bearing animals increased DNA damage and decreased Se level and GPx activity in NL and SL cells and other organs in cancer bearing animals, supplementation of Se further provoked DNA damage (no change in pretreatment) in NL cells, however it decreased DNA damage SL cells and other organs (kidney, lungs, and spleen). On the other hand Se levels and GPx activity were increased in NL and SL cells and other organs of Se-supplemented rats (no difference in group 3 animals). These results demonstrate that, in addition to chemopreventive and chemotherapeutic role of selenite, it also prevents cellular DNA damage induced in cancerous condition.     

Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 °C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.     

Increased lymphocyte DNA strand breaks in rubber workers

Objective: To study the effect of occupational exposure to rubber processing, smoking, and alcohol drinking on lymphocyte DNA damage. Subjects and Methods: Of 371 employees (197 men and 174 women) from a rubber factory in Guangzhou, 281 were rubber processing workers from five production sections and 90 were managerial workers. Information on occupational exposure, smoking, and drinking was collected by interviews. Blood samples were taken in the morning by venipuncture. DNA damages were measured by the Comet assay. Possible DNA-protein crosslinks were broken down by proteinase K. Tail moment, measured by Komet 4.0 image analysis software, was the measure of DNA damage. Results: The rubber processing workers had larger tail moment than the managerial workers (Geometric mean, 95%CI) [1.77 μm (1.64–1.90) versus 1.52 μm (1.36–1.71), P=0.04]. Both smoking [1.93 μm (1.74–2.13) versus 1.59 μm (1.47–1.71), P=0.003] and alcohol drinking [2.21 μm (1.87–2.62) versus 1.63 μm (1.53–1.74), P<0.001] increased tail moment. Tail moment differed significantly among job categories (F=3.21, P=0.008), the largest was observed in mixers. In the non-smoking and non-drinking workers, rubber processing workers had larger tail moment than managerial workers after adjusting for age (P=0.033). General linear model analysis showed that after adjusting for each other, occupational exposure (P=0.027), smoking (P=0.012), and alcohol drinking (P=0.013) was associated with larger tail moment, whereas age and gender had no effect. Conclusions: Occupational exposure to rubber processing, smoking, and alcohol drinking can cause DNA damage.     

镁离子对谷氨酸诱发的海马神经元损伤的保护作用

目的 :探讨镁离子 (Mg2 )在 0 .1mmol/L谷氨酸诱发的海马神经元损伤中的作用。方法 :将神经元从新生SD大鼠海马中分离后培养 6～ 9d ,即随机分成三组 :A .单纯培养基 ;B .培养基 谷氨酸 ;C .培养基 Mg2 ,30min后再加入谷氨酸。结果 :①B组海马神经元存活率与A组相比显著地呈剂量依赖性降低。②与B组对照 ,C组加用低浓度的Mg2 可提高海马神经元的存活率。结论 :低浓度的Mg2 能保护谷氨酸诱发损伤的海马神经元     

Effects of essential divalent metals on carcinogenicity and metabolism of nickel and cadmium

Interactions between the physiologically essential metals calcium, magnesium, and zinc and the carcinogenic metals nickel and cadmium were investigated to help elucidate the mechanisms of action of the carcinogenic metals. Bioassay studies revealed several significant findings, including: (1) the ability of magnesium and calcium to inhibit nickel-induced elevation of pulmonary adenoma incidence in strain A mice; (2) the ability of magnesium, but not of calcium, to prevent cadmium-induced subcutaneous sarcoma formation; and (3) the ability of magnesium, but not of calcium, to inhibit nickel-induced muscle tumor formation. Biochemical studies indicated a direct relationship between the antitumorigenic potential of magnesium and the capacity of this metal to: (1) inhibit nickel and cadmium uptake by the target tissues in vivo; (2) inhibit nickel-induced disturbances in DNA synthesis in vivo; (3) inhibit nuclear and cytosolic uptake of nickel by the target tissue cells in vivo; and (4) inhibit nickel and cadmium binding to DNA in vitro. Calcium, which in most cases did not prevent carcinogenesis, had no consistent influence on the uptake of carcinogenic metals or their biochemical effects in the target tissues. Magnesium and zinc, but not calcium, were also found to attenuate the acute toxic effects of nickel, indicating a possible correlation between prevention of acute effects and reduction in tumorigenicity. Zinc, which antagonizes cadmium tumorigenicity in the rat testis, was found to reduce markedly cadmium uptake into isolated testicular interstitial cells. Also, zinc was found to inhibit strongly cadmium binding to DNA in vitro.     

The oxidative DNA base damage in testes of rats after intraperitoneal cadmium injection

Cadmium is known to be a carcinogenic metal that especially its compounds have sufficient evidence in both humans and experimental animals beneath its environmental effects. Testis tissue is highly sensitive to the effects of cadmium. It is proposed that cadmium also increases oxygen derived free radicals and lipid peroxidation. As indicators of oxidative DNA damage, 6 oxidative DNA bases were determined by using Gas Chromatography/Mass Spectrometry-Selected Ion Monitoring technique. 45 Sprague-Dawley rats (225-300 g) were used as experimental animals and were divided into 3 groups of 15 rats. A single 2 mg NaCl/kg body wt, 0,5 and 1,25 mg CdCl2/kg body wt were injected intraperitoneally to control, low and high dose groups, respectively. 5-OH Cytosine, 8-OH Adenine and Fapy Guanine lesions were elevated significantly in high dose group in the first day. A clear dose-response relationship was seen between dose groups and 8-OH Adenine levels related with time in all periods. There was a significant dose-response relationship in 2-OH Adenine, Fapy Guanine and 8-OH Guanine, especially in the second week suggesting the inhibition of XPA protein by cadmium after first week. In contrast, the observation of a significant decrease of 5-OH Cytosine levels after first week showed that cadmium could not affect the enzymes repairing the cytosine base lesions.     

Protection of DNA from gamma-radiation induced strand breaks by Epicatechin

Epicatechin (EC), a polyphenolic antioxidant compound found in tea, apples and chocolate offered protection to DNA against ionizing radiation induced damages. Under in vitro conditions of radiation exposure, plasmid pBR322 DNA was protected by EC in a concentration dependent manner. The dose modifying factor for 0.2 mM EC for 50% protection of the plasmid DNA was found to be 6.0. EC when administered to mice 1 h prior to exposure to 4 Gy gamma-radiation protected cellular DNA against radiation-induced strand breaks in peripheral blood leukocytes, as revealed in alkaline comet assay studies. Thus, EC was found to protect DNA from gamma-radiation indiced strand breaks under in vitro as well as in vivo conditions of radiation exposure.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a “constant ^·OH radical scavenging environment”, k of 1.5 × 10⁷ s^-1 by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0 ± 0.2 at [F] < 100 μM by a mechanism other than through direct scavenging of ^·OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H^· atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from ^·OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.     

Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays

Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while xrs-5 cells showed the lowest survival, and irs-20 cells had an intermediate survival. These results are very similar to the cell survival curves induced by X-rays in these three cell lines. Our data also show the dose dependent induction of DNA-DSBs in these cell lines exposed to arsenite. However, in order to obtain a similar cell survival in wild type cells, twice as many DNA-DSBs are necessary with arsenite exposure when compared with X-rays, suggesting that the types of DNA lesions leading to DSB induced by arsenite are different from those by X-rays. Based on these data, further mechanistic investigations including the involvement of DNA-DSB repair proteins are warranted in the recovery process from arsenic (As) exposure.     

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O₃) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta™Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene.These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta™Mice.     

Three classes of DNA strand breaks induced by X-irradiation and internal beta-rays

Repair kinetics of DNA strand breaks were investigated after exposing exponentially growing CHO cells to X-radiation or to internal beta-rays from incorporated tritium, respectively. DNA strand breaks were analysed by the alkaline unwinding technique followed by chromatography on hydroxyapatite. For either type of radiation, the repair kinetics are statistically best described by a sum of three exponential components. The half-times determined are tau I approximately 2 min, tau II approximately 20 min and tau III approximately 170 min; they are identical for both types of radiation. But the initial fractions of the components are different for X- and internal beta-rays; X-rays; fI = 0.70, fII = 0.25, fIII = 0.05; internal beta-rays: fI = 0.40, fII = 0.40, fIII = 0.20. Components I and II are considered to represent the repair of two different classes of single-strand breaks and component III the repair of double-strand breaks. Two alternative interpretations for the occurrence of the two classes of single-strand breaks are discussed.     

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.     

Influence of cadmium and nickel on growth,net photosynthesis and carbohydrate distribution in rice plants

Six day old rice seedlings (Oryza sativa L. cv. Bahía) were grown for 5 or 10 days in a nutrient solution with either Cd (0.01, 0.1 mmol/l) or Ni (0.1, 0.5 mmol/l). Both Cd and Ni reduced the length of shoots and roots depending on the concentration and type of ion tested. On the other hand, the dry weight to fresh weight ratio was increased by heavy metal treatments, especially in the aerial part of 0.5 mmol/l Ni treated plants. The application of 0.1 mmol/l Cd and 0.5 mmol/l Ni to the seedlings produced an inhibition of the transport of carbohydrate reserves from the seeds from which plants were developing, to the rest of the plant. Net photosynthesis was also inhibited in treated plants. However, the total carbohydrate content in the shoots of these plants was higher than in controls. Thus, the starch, soluble sugars and sucrose content in the shoots of 0.5 mmol/l Ni treated plants was respectively up to 2.6, 2.8 and 4 times greater compared to controls. The distribution of assimilates between organs was also affected by the treatment: the carbohydrate content increased in the stem and second leaf but it was not affected or decreased in the root and third leaf. Although less evident, the effect of Cd on carbohydrate distribution and content was similar to that of Ni. The possible mechanisms involved in the abnormal carbohydrate accumulation and distribution are discussed.Abbreviations 0 DT plants at day zero of treatment - 5 DT five days treated plants - 10 DT ten days treated plants - DW dry weight - FW fresh weight     

Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.