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1

Treatment with silver nanoparticles delays repair of X-ray induced DNA damage in HepG2 cells

Wojewódzka M., Lankoff A., Dusińska M., Brunborg G., Czerwińska J., Iwaneńko T., Stępkowski T., Szumiel I., Kruszewski M.

Nukleonika

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2011

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Vol. 56, No. 1

29-33

EN

Nanoparticles (NPs) defined as particles having at least one dimension below 100 nm have been applied in the last decade in industry and medicine. Recently, there is an increased concern about the biohazard aspect of the presence of NP in consumer goods and in the environment. Silver NP (Ag NP) cause oxidative stress in mammalian cells in result of generation of reactive oxygen species (ROS). This results in genotoxicity and mutagenicity, disturbed mitochondrial respiration, slowed proliferation and cell death. Using the alkaline comet assay, we examined the effect of combined treatment with Ag NP 20 nm or 200 nm and X-rays (2 Gy) in HepG2 cells. In addition, combined treatment with X-rays and titanium dioxide NP (TiO2 NP) 21 nm was also studied. No effect of NP pre-treatment on X-ray induced initial deoxyribonucleic acid (DNA) damage levels was observed for all three NP. In contrast, Ag NP treatment preceding exposure to X-rays caused a marked decrease in the rate of single strand break rejoining. The effect was particularly strong for Ag NP 20 nm. TiO2 NP pre-treatment had no effect on DNA repair.

2

DNA damage in subpopulations of human lymphocytes irradiated with doses in the range of 0-1 Gy of X-radiation

Wojewódzka M., Machaj E. K., Goździk A., Iwaneńko T., Ołdak T., Kruszewski M., Pojda Z.

Nukleonika

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2008

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Vol. 53, No. 4

145-149

EN

We compared three methods usually applied in biological dosimetry for estimation of radiation-induced DNA damage in human T and B lymphocytes: alkaline comet assay, micronucleus (MN) test and formation of histone gamma-H2AX foci. Human peripheral blood lymphocytes were fractionated using T cells and B cells isolation kits. Cells were irradiated with doses in the range of 0-1 Gy of X-rays. Induction of DNA damage was assessed by the standard alkaline comet assay, MN test and histone gammaH2AX foci immunofluorescence assay. Notwithstanding different end-points measured by the applied methods, all tests revealed a similar induction of DNA damage in B lymphocytes as compared with T lymphocytes. The results indicated that all three tests detect DNA damage with similar sensitivity, the lowest dose being approximately 0.3 Gy. The difference between irradiated and control cells was expressed as the ratio of the value obtained for irradiated cells (1 Gy) to that for control cells. The highest ratio was obtained for formation of gammaH2AX foci and was 6.2 for T and 13.8 for B lymphocytes, whereas those for comet assay and micronucleus test were 3.5; 3.6 and 5.6; 4.8, respectively.

3

Ogniska histonu [gamma]-H2AX marker pęknięć podwójnoniciowych DNA

Wojewódzka M., Szumiel I.

Postępy Techniki Jądrowej

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2006

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z. 3

15-18

4

Effects of an antimutagen of 1,4-dihydropyridine series on cell survival and DNA damage in L5178Y murine sublines

Dalivelya O., Savina N., Kuzhir T., Buraczewska I., Wojewódzka M., Szumiel I.

Nukleonika

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2006

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Vol. 51, nr 3

141-146

EN

In a series of studies it was shown that 1,4-dihydropyridine derivatives (1,4-DHP) show antimutagenic and anticlastogenic properties and accelerate repair of oxidant and ionising radiation generated DNA damage. Here, effects of one of 1,4-DHP compounds (sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate denoted as DHP) in X-irradiated L5178Y cells (murine lymphoma sublines, LY-R and LY-S) are reported. DHP treatment 1 h before, during and after X-irradiation gave a radioprotective effect in double strand break (DSB) repair competent LY-R cells: there was an increase in post-irradiation proliferation and cell viability as well as a slight acceleration of break rejoining as measured by the neutral comet assay. In the radiosensitive LY-S cells with impaired non-homologous end-joining system, the radioprotective effect was seen as enhanced growth and viability. There was, however, no effect on the DSB repair rate. Notably, there was no dependence of the biological effects on DHP concentration in the range of concentrations studied (1 nM - 100 mM), suggesting an all-or-none effect, as in cellular signaling induction observed in radioadaptation or bystander effect. We assume that DHP acts by decreasing fixation of radiation inflicted DNA damage, among others, by increasing the rate of DNA repair and enhancing the efficiency of checkpoint control. Direct confirmation of this assumption is necessary.

5

DNA double-strand break rejoining in radioadapted human lymphocytes: evaluation by neutral comet assay and pulse-field gel electrophoresis

Wojewódzka M., Buraczewska I., Szumiel I., Grądzka I.

Nukleonika

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2006

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Vol. 51, nr 4

185-191

EN

Adaptive response (AR), an enhanced resistance to a high dose of ionising radiation acquired after pretreatment with a very low dose, was estimated in normal human lymphocytes. The question posed was whether the extent of radioadaptation, assessed by micronucleus test, would be related to the rate of DNA double-strand break (DSB) rejoining. Phytohemagglutinin-stimulated G1-lymphocytes from 5 healthy male volunteers were pre-treated (or not) with an adaptive (5 cGy) dose of X-rays, followed by a higher (5 or 10 Gy) challenge dose after 20-22 h. DSB rejoining after the challenge dose was monitored with the use of two methods: neutral comet assay, modified to reduce the contribution of single-strand breaks (SSBs) and thermolabile sites, and pulse-field gel electrophoresis (PFGE), specific for DSBs. At the level of micronuclei, an AR was observed in lymphocytes of 3 of 5 donors. Up to 60 min, comet assay showed no statistically significant differences in DNA break rejoining between adapted and non-adapted lymphocytes, independently of AR appearance. PFGE gave similar results, although in three donors it revealed secondary increases in DSBs levels at 30 min and/or 60 min post-irradiation in the adapted vs. the non-adapted samples. Failure to demonstrate changes in DSBs rejoining rate in the adapted lymphocytes could be due to diversity of AR intensity/timing at the level of DNA repair in not fully homogenous cell populations. Also, “rare” DNA cuts characteristic of early apoptosis/necrosis could overlap the process of DNA break rejoining.

6

Modified neutral comet assay for human lymphocytes

Wojewódzka M., Grądzka I., Buraczewska I.

Nukleonika

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2002

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Vol. 47, nr 1

1-5

EN

Comet assay under neutral conditions allows the detection of DNA double-strand breaks, considered to be the biologically relevant radiation-induced lesion. In this report we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA double strand breaks in human lymphocytes irradiated with doses of 60Co gamma-rays (from 10 to 100 Gy). The analysis carried out according to this protocol takes less time than those published so far. Also, the use of lysis at 50°C is avoided; this is important in view of the presence of heat-labile sites in the DNA of irradiated cells, recently reported by Rydberg [12]. The comets have well defined, sharp limits, are suitable for computer image analysis and chromatin of the control cells remains condensed.

7

Application of the DNA comet assay for detection of irradiated meat

Kruszewski M., Malec-Czechowska K., Dancewicz A. M., Iwaneńko T., Szot Z., Wojewódzka M.

Nukleonika

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1998

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Vol. 43, nr 2

147-160

EN

Radiation induces damage to the DNA. This damage (fragmentation) can be assessed in the irradiated food using Single Cell Gel Electrophoresis (SCGE), known as DNA comet assay. Fragmentation of DNA may also be caused by improper storage of meat and repeated freezing and thawing. This makes identification of irradiated meat by this assay not reliable enough. In order to know the scale of the processes imitating irradiation effects in DNA of the comets, their shape and lenghts were examined in both unirradiated and irradiated fresh meat (D = 1.5 or 3.0 kGy) stored at 4°C or frozen (-21°) up to 5 months. Comets formed upon SCGE were stained with DAPI or silver and examined in fluorescent or light microscope. They were divided arbitrarily into 4 classes. Comets of class IV were found quite often in fresh meat stored at 4°C. In meat samples that were irradiated and stored frozen, comets of class, I, II and III were observed. The negative comet test is univocal. Positive comet test, however, needs confirmation. The meat should be subjected to further analysis with other validated methods.

PL

Promieniowanie powoduje uszkodzenia DNA. Te uszkodzenia (fragmentację) można ocenić w napromieniowanej żywności stosując elektroforezę w żelu pojedynczej komórki, zwaną także testem kometowym. Fragmentację DNA w mięsie mogą także wywoływać: nieprawidłowe przechowywanie mięsa oraz powtarzane zamrażanie i rozmnażanie. Czyni to identyfikację napromieniowania mięsa mniej wiarygodną. W celu poznania procesów imitujących napromieniowanie, tj. powodujących powstawanie kometek DNA, oceniano ich kształt i długość w mięsie nie napromieniowanym i napromieniowanym dawką 1.5 lub 3.0 kGy przechowywanym w 40C lub w stanie zamrożenia do 5 miesięcy. Otrzymane kometki DNA barwiono barwnikiem fluorescencyjnym DAPI lub srebrem i badano w mikroskopie fluorescencyjnym lub zwykłym. Kometki podzielono na 4 klasy. Kometki IV klasy znajdowano często w mięsie przechowywanym w 40C. W próbkach mięsa napromieniowanego i przechowywanego w stanie zamrożenia obserwowano kometki klasy l, II i III. Negatywny test kometowy jest jednoznaczny. Test dodatni wymaga potwierdzenia przez zastosowanie innych metod dających miarodajne wyniki.