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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.

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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.

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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.