Halina Lisowska

A cross-platform public domain PC image-analysis program for the comet assay.

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact.

FociCounter: a freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.

Gamma-H2AX foci are sensitive and specific indicator for the induction of DNA double-strand breaks (DSBs) and an immunocytochemical assay with antibodies recognizing gamma-H2AX has become the gold standard for the detection of this type of DNA lesion. Quantification of gamma-H2AX foci can be achieved by various methods such as Western blotting, flow cytometry, visual analysis and computational analysis with a fluorescence microscope. The best sensitivity is achieved by computer analysis. Since no freeware programme for the analysis of gamma-H2AX foci exists for a PC platform, the aim of our study was to develop a simple and user-friendly public-domain software. The algorithm applied in our programme allows determination of the number of foci in a single cell, a focus intensity per cell, as well as a cell intensity. Its graphical user interface is based on a GTK+ library and the whole application can be run under a variety of operating systems, including MS Windows and Linux. The programme called FociCounter is publicly available at http://focicounter.sourceforge.net. Application of the programme was tested by analysing gamma-H2AX foci in CHO and MO59K cells irradiated in vitro with X-rays and validated by comparing the results obtained with the outcome of automated image analysis and flow cytometry.

Effect of surface modification of silica nanoparticles on toxicity and cellular uptake by human peripheral blood lymphocytes in vitro

Abstract Silica nanoparticles have an interesting potential in drug delivery, gene therapy and molecular imaging due to the possibility of tailoring their surface reactivity that can be obtained by surface modification. Despite these potential benefits, there is concern that exposure of humans to certain types of silica nanomaterials may lead to significant adverse health effects. The motivation of this study was to determine the kinetics of cellular binding/uptake of the vinyl- and the aminopropyl/vinyl-modified silica nanoparticles into peripheral blood lymphocytes in vitro, to explore their genotoxic and cytotoxic properties and to compare the biological properties of modified silica nanoparticles with those of the unmodified ones. Size of nanoparticles determined by SEM varied from 10 to 50 nm. The average hydrodynamic diameter and zeta potential also varied from 176.7 nm (+18.16 mV) [aminopropyl/vinyl-modified] and 235.4 nm (−9.49 mV) [vinyl-modified] to 266.3 (−13.32 mV) [unmodified]. Surface-modified silica particles were internalized by lymphocytes with varying efficiency and expressed no cytotoxic nor genotoxic effects, as determined by various methods (cell viability, apoptosis/necrosis, oxidative DNA damage, chromosome aberrations). However, they affected the proliferation of the lymphocytes as indicated by a decrease in mitotic index value and cell cycle progression. In contrast, unmodified silica nanoparticles exhibited cytotoxic and genotoxic properties at high doses as well as interfered with cell cycle.

Radioprotective effect of hypothermia on cells – a multiparametric approach to delineate the mechanisms

Purpose: Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection. Materials and methods: Human lymphoblastoid TK6 cells were incubated for 20 min at 0.8 or 37°C and subsequently exposed to 1 Gy of γ- or X-rays. The influence of ataxia telangiectasia mutated (ATM)-mediated double-strand break signalling and histone deacetylase-dependent chromatin condensation was investigated using the micronucleus assay. Furthermore, the effect of hypothermia was investigated at the level of phosphorylated histone 2AX (γH2AX) foci, clonogenic cell survival and micronuclei in sequentially-harvested cells. Results: The radioprotective effect of hypothermia (called the temperature effect [TE]) was evident only at the level of micronuclei at a single fixation time, was not influenced by the inhibition of ATM kinase activity and completely abolished by the histone deacetylase inhibition. No TE was seen at the level of γH2AX foci and cell survival. Conclusions: We suggest that low temperature during irradiation can induce a temporary cell cycle shift, which could lead to a reduced micronucleus frequency. Future experiments focused on cell cycle progression are needed to confirm this hypothesis.

Biological effectiveness of 12 C and 20 Ne ions with very high LET

Purpose: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of 12C and 20Ne ions. Materials and methods: Chinese hamster ovary (CHO-K1) cells were exposed to high LET 12C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and 20Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up. Results: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440–830 keV/μm for 12C and for 1020–1600 keV/μm for 20Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET. Conclusions: The RBE decreases with increasing LET in the range between 440 and 1600 keV/μm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.

Effect of hypothermia on radiation-induced micronuclei and delay of cell cycle progression in TK6 cells

Abstract Purpose: Low temperature (hypothermia) during irradiation leads to a reduced frequency of micronuclei in TK6 cells and it has been suggested that perturbation of cell cycle progression is responsible for this effect. The aim of the study was to test this hypothesis. Materials and methods: Human lymphoblastoid TK6 cells were treated by a combination of hypothermia (0.8°C) and ionizing radiation in varying order (hypothermia before, during or after irradiation) and micronuclei were scored. Growth assay and two-dimensional flow cytometry was used to analyze cell cycle kinetics following irradiated of cells at 0.8°C or 37.0°C. Results: The temperature effect was observed at the level of micronuclei regardless of whether cells were cooled during or immediately before or after the radiation exposure. No indication of cell cycle perturbation by combined exposure to hypothermia and radiation could be detected. Conclusions: The protective effect of hypothermia observed at the level of cytogenetic damage was not due to a modulation of cell cycle progression. A possible alternative mechanism and experiments to test it are discussed.

Individual variations in the micronucleus assay for biological dosimetry after high dose exposure.

The micronucleus assay is widely used as a biological dosimeter. Due to an inhibitory effect of radiation on cell proliferation the assay yields satisfactory results only when the absorbed dose is below about 5Gy. In 2002 Müller and Rode suggested that a modified version of the test, based on the analysis of the ratio of trinucleated to tetranucleated cells and the frequency of micronuclei (Mn) in binucleated cells containing at least one Mn, can be applied to detect a dose reaching 15Gy (Mutat. Res. 502 (2002) 47-51). Their conclusion was based on the results of experiments with lymphocytes from one donor and nothing is known about the possible influence of individual variability on the applicability of the Mn test to detect high doses of radiation. The aim of the present study was to validate the modified micronucleus assay with lymphocytes of 5 donors. Their blood was exposed to 0, 5, 10, 15 and 20Gy of (60)Co gamma rays. The levels of Mn and of cell proliferation were assessed using various approaches. A strong inter-individual variability was observed for all endpoints. The results clearly show that the assessment of cell proliferation is essential for the interpretation of results. Unfortunately, it was not possible to identify one single proliferation marker that gives all necessary information.

Chromosomal aberrations in peripheral blood lymphocytes exposed to a mixed beam of low energy neutrons and gamma radiation

Cells exposed to thermal neutrons are simultaneously damaged by radiations with high and low linear energy transfer (LET). A question relevant for the assessment of risk of exposure to a mixed beam is whether the biological effect of both radiation types is additive or synergistic. The aim of the present investigation was to calculate whether the high and low LET components of a thermal neutron field interact when damaging cells. Human peripheral blood lymphocytes were exposed to neutrons from the HB11 beam at the Institute for Energy and Transport, Petten, Netherlands, in a 37 °C water phantom at varying depths, where the mix of high and low LET beam components differs. Chromosomal aberrations were analysed and the relative biological effectiveness (RBE) values as well as the expected contributions of protons and photons to the aberration yield were calculated based on a dose response of aberrations in lymphocytes exposed to (60)Co gamma radiation. The RBE for 10 dicentrics per 100 cells was 3 for mixed beam and 7.2 for protons. For 20 dicentrics per 100 cells the respective values were 2.4 and 5.8. Within the limitations of the experimental setup the results indicate that for this endpoint there is no synergism between the high and low LET radiations.

The dose-response relationship for dicentric chromosomes and γ-H2AX foci in human peripheral blood lymphocytes: influence of temperature during exposure and intra- and inter-individual variability of donors.

Abstract Purpose: Hypothermia during in vitro irradiation of human peripheral blood lymphocytes (PBL) affects the level of chromosome aberrations. The molecular mechanisms of this phenomenon are not fully understood. The aim of our study was to examine the effect of hypothermia on the dose-response relationship for dicentric chromosomes and the level of γ-H2AX (phosphorylated histone H2AX) foci. In addition, the inter- and intra-individual variability was assessed in relation to temperature. Materials and methods: PBL were kept at 0.8, 20 and 37°C and then exposed to gamma-rays (from 0–3 Gy). Dicentric chromosomes were scored in first post-treatment mitoses. γ-H2AX foci were scored 15, 30, 60, 120 min and 24 h post irradiation. Results: Our results revealed that the frequency of dicentric chromosomes in cells exposed at 37°C to gamma-rays was higher than after exposure at 0.8 and 20°C. No effect of temperature was observed on the number of γ-H2AX foci as well as on the intra- and inter-individual variations of the dicentric yield and the number of γ-H2AX foci. Conclusions: Temperature at exposure to ionizing radiation has a pronounced effect on the level of cytogenetic damage but not γ-H2AX foci.