DNA Degradation In Cells Research Articles

Surface Modification by Media Organics Reduces the Bacterio-toxicity of Cupric Oxide Nanoparticle against Escherichia coli

Prevalence of antibiotic-resistant bacteria demands alternatives to antibiotics. Copper-based nanoparticles with a high antibacterial property may be a solution to the problem. It is, therefore, important to understand the mode of antibacterial action of the nanoparticles (NPs). Despite reports on induction of reactive oxygen species (ROS) in bacteria by copper and copper-oxide nanoparticles and involvement of such ROS in cell killing, it is still unclear (a) if surface modification of the nanoparticles by media organics has any role on their antibacterial potency and (b) whether the bactericidal effects of these NPs are ‘particle-specific’ or ‘ion-specific’ in nature. We address these issues for cupric oxide nanoparticle (CuO-NP) in this study. Instead of nutrient medium, when E. coli bacterial cells were suspended in saline (0.9% NaCl), CuO-NP had a more anti-bacterial effect, with MBC (minimum bactericidal concentration) value of 6 µg/mL, than in nutrient medium with MBC value of 160 µg/mL. Moreover, the lysine-modified CuO-NP in saline had MBC at 130 µg/mL. Thus, unmodified CuO-NP was more efficient killer than modified one. Our finding further revealed that in saline;CuO-NP had ‘particle-specific’ antibacterial effect through generation of ROS and consequent oxidative damage by lipid peroxidation, protein oxidation and DNA degradation in cells.

EFFECT OF ELEVATED TEMPERATURE, DARKNESS, AND HYDROGEN PEROXIDE TREATMENT ON OXIDATIVE STRESS AND CELL DEATH IN THE BLOOM-FORMING TOXIC CYANOBACTERIUM MICROCYSTIS AERUGINOSA(1).

This study assessed the implication of oxidative stress in the mortality of cells of Microcystis aeruginosa Kütz. Cultures grown at 25°C were exposed to 32°C, darkness, and hydrogen peroxide (0.5 mM) for 96 h. The cellular abundance, chl a concentration and content, maximum photochemical efficiency of PSII (Fv /Fm ratio), intracellular oxidative stress (determined with dihydrorhodamine 123 [DHR]), cell mortality (revealed by SYTOX-labeling of DNA), and activation of caspase 3-like proteins were assessed every 24 h. The presence of DNA degradation in cells of M. aeruginosa was also assessed using a terminal deoxynucletidyl transferase-mediated dUTP nick end labeling (TUNEL) assay at 96 h. Transferring cultures from 25°C to 32°C was generally beneficial to the cells. The cellular abundance and chl a concentration increased, and the mortality remained low (except for a transient burst at 72 h) as did the oxidative stress. In darkness, cells did not divide, and the Fv /Fm continuously decreased with time. The slow increase in intracellular oxidative stress coincided with the activation of caspase 3-like proteins and a 15% and 17% increase in mortality and TUNEL-positive cells, respectively. Exposure to hydrogen peroxide had the most detrimental effect on cells as growth ceased and the Fv /Fm declined to near zero in less than 24 h. The 2-fold increase in oxidative stress matched the activation of caspase 3-like proteins and a 40% and 37% increase in mortality and TUNEL-positive cells, respectively. These results demonstrate the implication of oxidative stress in the stress response and mortality of M. aeruginosa.

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: A putative mechanism for anticancer properties

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol–Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.

Bacteria recovered from dental pulp induce apoptosis of lymph node cells

Apoptosis is critical in the pathogenesis of several infectious diseases. The induction of apoptosis was assessed in mouse lymph node cells by four bacteria recovered from infected human dental pulp: Gemella morbillorum, Clostridium butyricum, Fusobacterium nucleatum and Bifidobacterium adolescentis. Smaller lymph nodes and smaller numbers of cells were observed after experimental dental pulp infection with C. butyricum, suggesting that this bacterium induces cell death. Apoptosis was evaluated by determination of cell ploidy and detection of DNA degradation in cells cultured with killed bacteria. Paraformaldehyde-killed C. butyricum and heat-killed G. morbillorum induced substantial cell death, while F. nucleatum and B. adolescentis induced cell death at lower levels. No bacterial preparations induced apoptosis in cells from mice genetically deficient for tumour necrosis factor receptor p55 (TNFRp55), implicating this receptor directly or indirectly as a mediator in the process. It was concluded that apoptosis may be induced during periapical lesions of pulpal origin.

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation.

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.

Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): the embryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in Medaka (Orizias latipes).

Vertebrate embryos are extremely sensitive to environmental contaminants known as planar halogenated hydrocarbons (PHHs). The physiological targets that mediate PHH-induced embryotoxicity are not known. We have characterized embryotoxicity in medaka (Orizias latipes) caused by 2,3,7,8-tetrachlorodibezo-p-dioxin (TCDD), the prototypic PHH. DNA degradation in cells of the embryonic vasculature and loss of functional integrity of the medial yolk vein were demonstrated in TCDD-exposed embryos. Pharmacological intervention with piperonyl butoxide inhibited TCDD-induced DNA degradation, restored the functional integrity of the medial yolk vein, and protected against the embryotoxicity of TCDD. Treatment of TCDD-exposed embryos with the antioxidant N-acetylcysteine also provided significant protection against the embryotoxicity of TCDD. These results demonstrate that DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity.

Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): Theembryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in medaka (Orizias latipes)

Vertebrate embryos are extremely sensitive to environmental contaminants known as planar halogenated hydrocarbons (PHHs). The physiological targets that mediate PHH-induced embryotoxicity are not known. We have characterized embryotoxicity in medaka (Orizias latipes) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypic PHH. DNA degradation in cells of the embryonic vasculature and loss of functional integrity of the medial yolk vein were demonstrated in TCDD-exposed embryos. Pharmacological intervention with piperonyl butoxide inhibited TCDD-induced DNA degradation, restored the functional integrity of the medial yolk vein, and protected against the embryotoxicity of TCDD. Treatment of TCDD-exposed embryos with the antioxidant N-acetylcysteine also provided significant protection against the embryotoxicity of TCDD. These results demonstrate that DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity.

Apoptosis of monocytes and prolonged survival of granulocytes as a result of phagocytosis of bacteria.

Monocytes and granulocytes were incubated with suspensions of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or Salmonella enteritidis and, after being washed free of bacteria, cultured for up to 48 h. Every few hours, samples of cultured cells were taken for DNA isolation. Monocytes which phagocytosed bacteria showed features of apoptotic cells, as determined by light microscopy and DNA fragmentation detected by gel electrophoresis. The phenomenon was observed 2 to 4 h after phagocytosis, in contrast, control monocytes did not show signs of apoptosis until 48 h of culture. Intact control granulocytes spontaneously became apoptotic after 12 h of culture. In contrast, degradation of DNA in cells exposed to bacteria was delayed by 12 to 24 h. In conclusion, our observation suggests that granulocytes and monocytes react differently to phagocytosis of bacteria.

Teratogen-induced apoptosis may be affected by immunopotentiation

Intra-uterine immunization of mice with paternal allogeneic or xenogeneic (rat) splenocytes was found to increase embryo tolerance to cyclophosphamide (CP)-induced teratogenesis. As the CP-induced teratogenic effect was shown to be associated with apoptosis, the present study was designed to investigate whether the protective effect of immunopotentiation may be realized via an alteration of CP-induced apoptosis. Various doses of CP were injected intraperitoneally into ICR mice on day 12 of pregnancy. Intra-uterine immunization with xenogeneic rat splenocytes was carried out 3 weeks before mating. Implantation sites, resorptions, live and dead fetuses, as well as soft tissue anomalies and external malformations, were recorded to evaluate the CP-induced embryotoxic effect. In parallel, flow cytometric analysis and DNA fragmentation assay were used for evaluation of CP-induced apoptosis in limbs, tail and whole embryos. The treatment of mothers with a high dose of CP induced the death of almost all embryos and striking fetal growth retardation in survivors. This strong embryotoxic effect was accompanied by very prominent DNA degradation in cells collected from whole embryos. Immunostimulation caused a dramatic decrease of embryonal loss (by ~ 50%) and a significant (about 30%) increase in fetal weight. Such an increase in fetal survival and in fetal weight was found to be accompanied by a clear decrease in apoptosis level in embryo cell populations as judged by DNA gel electrophoresis with subsequent quantitation of DNA fragmentation in negatives by an image analysis technique. After treatment with a low dose of CP, a decrease in the proportion of fetuses with limb and tail anomalies in immunized females was accompanied by a decrease in the proportion of apoptotic nuclei in cells taken from limbs and tails. The results of this study suggest that the teratogen-induced apoptosis may, at least partly, be dependent on fetomaternal immune interactions.

Degradation of DNA in cells and extracts of the obligately anaerobic bacterium Roseburia cecicola upon exposure to air.

High-molecular-weight chromosomal DNA from Roseburia cecicola, an oxygen-intolerant anaerobe, could be isolated only when the bacterial cells were kept under anaerobic conditions up to the time of cell lysis. When the cells were exposed to oxygen before lysis, the chromosomal DNA degraded. Likewise, linear but not covalently closed circular DNAs degraded in cell extracts of the organism that were exposed to atmospheres containing O2 but not in extracts that were maintained in a reduced state. Covalently closed circular DNAs were nicked but not degraded in the oxidized extracts.

Independent mutations in Ad2ts111 cause degradation of cellular DNA and defective viral DNA replication.

An adenovirus mutant, Ad2ts111, has previously been shown to be temperature sensitive for viral DNA replication in vivo and also to induce degradation of cellular DNA. Soluble nuclear extracts prepared from Ad2ts111-infected HeLa cells grown at either the permissive (32 degrees C) or the nonpermissive (39.5 degrees C) temperature are thermolabile for elongation but not for initiation of DNA replication in vitro. Adenovirus single-stranded-DNA-binding protein purified from wild-type-infected cells can complement these extracts at the restrictive temperature in vitro. The DNA-binding protein synthesized in Ad2ts111-infected cells is stable at the nonpermissive temperature and is phosphorylated, as is the wild-type protein. In contrast, the mutant DNA-binding protein synthesized in Ad5ts125-infected cells is unstable. Ad2ts111 and Ad5ts125 do not complement each other for virus growth in vivo. These results suggest that Ad2ts111 contains a mutation in the DNA-binding protein that affects viral DNA synthesis. Finally, we demonstrated that, unlike viral DNA synthesis, the induction of cellular DNA degradation in Ad2ts111-infected cells is not temperature sensitive and that this phenotype is a result of a mutation in early region 1 on the virus genome. Thus, the two phenotypes displayed in Ad2ts111-infected cells, namely, the temperature-sensitive replication of viral DNA and the degradation of cell DNA, are the result of two separate mutations.

Possible participation of Ca 2+, Mg 2+-dependent endonuclease in liver DNA fragmentation after N-methyl- N-nitrosourea treatment

We examined the fragmentation of DNA treated with N-methyl- N-nitrosourea under conditions in which Ca 2+, Mg 2+-dependent endonuclease is active. The molecular mass of DNA found in mouse liver slices treated with methylnitrosurea in the presence of Ca 2+ plus Mg 2+ was 4 · 10 5 Da. Similar results were obtained with a reconstituted system containing partially purified Ca 2+, Mg 2+-dependent endonuclease and methylnitrosurea-treated DNA. The enzyme extensively cleaved methylnitrosurea-treated DNA, compared with non-treated DNA. The methylnitrosurea-treated nuclear proteins obtained from mouse liver nuclei had no effect on the DNA fragmentation by the enzyme. Using closed-circular DNA treated with methylnitrosurea, the enzyme produced single-strand cuts in the DNA, as was seen in non-treated, closed-circular DNA, however, the rate of hydrolysis was increased. Ca 2+, Mg 2+-dependent endonuclease thus warrants further investigation, with regard to the precise mechanism of extensive degradation of DNA in cells treated with carcinogenic alkylating agents.