Comet Assay to Assess the Non-target Effect of Neutronradiation in Human Peripheral Blood

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1 J. RADIAT. RES., 42, (2001) Comet Assay to Assess the Non-target Effect of Neutronradiation in Human Peripheral Blood NATARAJAN GAJENDIRAN 1,2 * KIMIO TANAKA 3 and NANAO KAMADA 1 1 Department of Cancer Cytogenetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima , Japan 2 Health and Safety Division, Indira Gandhi Center for Atomic Research, Kalpakkam , India 3 Department of Radiobiology, Institute for Environmental Sciences, Ienomae 1 7, Obuchi, Rokkasho Village, Aomori , Japan (Received on September 27, 2000) (Revision received on November 15, 2000) (2nd revision received on February 26, 2001) (Accepted on March 16, 2001) Comet assay/non-target effect/neutron/dna-damage The non-target effect of neutron-irradiation was assessed in unirradiated human peripheral blood lymphocytes using an alkaline comet assay. The isolated cells were incubated with an autologous plasma for 1 h at 37 C before performing the assay. The cells exhibited a significant increase in the tailmoment when the irradiated blood (2 Gy, 570 kev neutron) was the source of plasma. The genotoxic effect lasted for 96 h when stored at 20 C. It is believed to be mediated by the release of cytokines or other factors released by the irradiated cells. Plasma obtained from unirradiated blood or further irradiation of plasma did not enhance the tailmoment significantly. Thus, the adverse effect of neutron-exposure can be passed on to unirradiated neighbourhood through irradiated blood tissue without involving cell-cell contact. The non-targeted radiation effect assumes greater consequences in radiotherapy and biodosimetry. INTRODUCTION There is ample evidence in the literature to suggest that the medium from certain cell lines which have been exposed to radiation can reduce the survival of unirradiated cells or kill unirradiated cells in the vicinity 1 3). Similarly, the occurrence of a higher mutation frequency in CHO cells after the low-dose exposure of 238 Pu-α particle was attributed to the result of mutations arising in non-irradiated, bystander cells 4). Increases in the cytotoxic effect 5) and the chromosomal instability 6) have also been reported to occur in bystander cells. However, the nature of the signaling molecule(s) that mediates the response remains to be established, even *Corresponding author: Phone; , Fax; , E. mail; ngj@igcar.ernet.in

2 158 N. GAJENDIRAN ET AL. though reactive oxygen species, extra-nuclear originating signaling pathways, and secreted diffusable factors were thought to be involved 7). A variety of endpoints, such as cell-survival 3), sister chromatid exchange 8), and HPRT mutation 4), were used to assess the non-targeted radiation effect. Single-cell gel electrophoresis (comet assay) is considered to be a sensitive and rapid technique to visualize individual cells for their various levels of DNA damage 9). In this study, we used this assay to assess the non-targeted effect of neutron irradiation in human lymphocytes. MATERIALS AND METHODS Radiation source A neutron irradiating system, the Hiroshima University Radiobiological Accelerator (HIRRAC), was used to deliver 570 kev neutrons. HIRRAC consists of a Schenkel-type 3 MV ion accelerator (HN-3000 BL; manufactured by Nisshin High Voltage Co. Ltd.) and a target assembly for neutron generation. The details of the irradiation system have been described elsewhere 10,11). In brief, irradiation was carried out by a vertical beam (V-Line) using a Li target. The contamination of the γ -rays was less than 3% when using a 10 µm-thick 7 Li target. The dose rate at 10-cm distance was cgy/min. Whole blood or plasma in 1 ml was put in 35 mm-diameter plastic dishes and irradiated to 2 Gy at room temperature. Sham-irradiated samples were used for a comparison. Sample preparation Human peripheral blood collected from a healthy volunteer in heparinized tubes was used. The blood was layered onto Lymphoprep TM (Nycomed, Oslo, Norway) and subjected to gradient centrifugation. A clear top layer was collected as plasma and a puffy layer contained mononuclear lymphocytes. A portion of the plasma from irradiated- or unirradiated- blood was stored at 20 C to study the genotoxicity over a period of time. The cells were washed twice in a RPMI 1640 medium at room temperature. Finally, the washed cells were suspended to a titer of cells/ml in the autologous plasma and further incubated for 1 h at 37 C before performing a comet assay. The isolated plasma was separately subjected to a comet assay to check for any cross-cellular contamination. The schematic representation of samples prepared for comet assay is provided in Fig. 1. Single-cell gel electrophoresis (comet assay) The basic alkaline technique described by Singh et al 12) was followed with some modifications. About 10 4 cells in 5 µl were mixed with 80 µl of 0.7% low-melting agarose (Gibco BRL, FRG) at 37 C in a microfuge and spread on a fully frosted microscopic slide pre-coated with 200 µl of 0.1% agarose. An Oncor Chromosome in situ System plastic cover slip (Oncor Science Inc.) was laid over the gel mixture to produce a uniform surface for microscopic observations. After gelling at 0 C for one minute, the cover slip was gently pealed off from the agarose layer. The cells were lysed by dipping the slides in a lysing solution (100

3 NON-TARGET EFFECT OF NEUTRON 159 Fig. 1. Schematic representation of the sample-preparation for a comet assay. The number in a circle represents the sample types for study. mm Na-EDTA, 10 mm Tris, 2.5 M NaCl, 1% Triton X-100 and 10% DMSO, ph 10) for 1 h at 4 C to remove the membrane and proteins. The slides were rinsed free of salt and detergent in a buffer (1 mm Na-EDTA, 300 mm NaOH, ph>13) and subsequently submersed in a horizontal gel-electrophoresis apparatus by adding fresh buffer, and remained in the buffer for 20 min to allow the unwinding of the DNA and the expression of alkali labile damage. Then, a weak electric field was applied (300 ma; 0.8 V/cm for 20 min at 24 C, under dim yellow light) to draw negatively charged DNA towards the anode. After electrophoresis, the slides were washed twice for 5 min in a neutralizing buffer (0.4 M Tris, ph 7.5) and stained with 75 µl of propidium iodide (20 µl/ml). The slides were analysed within 3 h. Under this condition, each cell could be identified with a landmark of migrating DNA. The amount and length of migration greatly depends on the DNA fragment size. Comet image analysis The fluorescent signals of the stained comets were examined using a fluorescence microscope with excitation at nm, detection >580 nm, coupled with an intensified target camera with a self-designated image-analysis system (Olympus, Tokyo). The comet images were stored using a CCD camera and analyzed using software in BAS 1500 (Fuji Co., Tokyo). In the first step of the measurement, the head of each comet was marked by a circle, followed by a measurement of the DNA content within this circle. Finally, the total fluorescence of the

4 160 N. GAJENDIRAN ET AL. head and tail was calculated by subtracting the head fluorescence from the total fluorescence. The genetic integrity of the cells was understood as being the tailmoment. The tailmoment was calculated by multiplying the tail length by the amount of DNA in the tail. The comet tail was set to be the area from the edge of the head to the end of the tail. Relative units were used in the graphic presentations. For each sample, about images were randomly analyzed. Repeat experiments were conducted within two days of the initial assessment. Each data point of the DNA damage in the graphic presentation represented the mean ± SD of at least 3 individual experiments. Statistical analyses were carried out using the INSTAT GRAPH-PAD program. The tailmoment data using the Shapiro Wilks test indicated that the data fit to a Gaussian bell-shaped distribution. Therefore, the mean values were calculated. Probability values close to significance were obtained for data groups by employing the two-tailed ANOVA test. Significance was assumed if P<0.05. RESULTS AND DISCUSSION Human peripheral blood lymphocytes, collected from the irradiated (monoenergetic neutrons 570 kev; 2 Gy) whole blood (sample 2) or unirradiated lymphocytes when suspended (1 h) in the plasma of irradiated whole blood (sample 4), exhibited extensive DNA damage (Fig. 2) (Table 1). The plasma isolated from irradiated whole blood (sample 4) evoked a significantly higher tailmoment than the response obtained either with the plasma from unirradiated blood (sample 3) (P=0.0296) or with the plasma that received radiation after isolation (sample 5) (P=0.0362). The lymphocyte-isolation technique probably marks for the differences in the tailmoment between two unexposed lymphocyte groups (sample 1 and 3). It is understood that Fig. 2. Non-target effect of neutron-irradiation as assessed by a comet assay. The columns are the mean and the error bars represent ±SD. The data are based on 100 comet cells per sample. The values of sample 4 are significantly different from those samples 3 and 5 at P< and P<0.0362, respectively.

5 NON-TARGET EFFECT OF NEUTRON 161 Table 1. Neutron-induced DNA damage in non-targeted human blood lymphocytes, as assessed by a comet assay. Sample type* Tailmoment Mean ± SD * Refer Fig. 1 The mean value represents the average of 3 individual experiments; ± SD represents the variation among the mean values. blood cells, in response to radiation excrete a signal, which can bring about an adverse effect of radiation in unexposed lymphocytes. The effect lasted for 96 h when stored at 20 C (Fig. 3). Goh and Sumner 13) have shown that irradiated plasma can induce chromosomal breaks in the cultures of normal leukocytes. The effectiveness of the chromosome-breaking factor in the plasma remains unchanged with increasing the dose and quality of radiation. Such a factor was also found to be present in the plasma of the survivors of atomic bombing 14). Narayanan et al 15) have presented evidence that the effect was mediated by the release of cytokines or other factors, which ultimately increased the production of reactive oxygen species in non-target cells. The biological effect of radiation not restricted to the irradiated cells has been made evident in low-fluence α-particle studies 4,7). Our experimental results serve as a simple and direct measurement of the bystander effect in neutron irradiation. Also, it is evident that Fig. 3. Genotoxicity of the plasma from irradiated blood over a period of storage ( 20 C). The error bars represent ±SD. 1 & 2 refer to sample types, described in Fig. 1.

6 162 N. GAJENDIRAN ET AL. physical contact between irradiated- and unirradiated cells may not be necessary to bring about the bystander effect, at least in blood tissue. Radiation-induced lesions are severe at high LET 16). The scope of using neutron-irradiation in the form of boron neutron capture therapy (BNCT) 17) becomes high for the treatment of malignant tumours. Hence, the non-targeted radiation effect assumes greater consequences in radiotherapy, the radioresponse of tumours and biodosimetry. Along the same line, the possibility of irradiated feeder cells in tissue culture to modify the experimental outcome cannot be ignored. Also, it is relevant to note that the initiation in radiation carcinogenesis may not be a targeted mutational event, but rather a process that involves a significant fraction of the irradiated cell population 18). ACKNOWLEDGEMENTS We are grateful to Dr.H.Tauchi of the Department of Radiation Biology, and to Messrs. S. Takeoka, K. Kitagawa and S. Suga of the Radiation Research Center for Frontier Sciences, Hiroshima University for experimental help. NG was supported by HICARE, Japan and Indira Gandhi Center for Atomic Research, India. REFERENCES 1. Bertho, J.-M. and Gourmelon, P. (1998) Human thymic stromal cell irradiation reduces intra-thymic T cell precursor proliferation: evidence for a soluble mediator. Int. J. Radiat. Biol. 74: Lloyd, D. C. and Moquet, J. E. (1985) The clastogenic effect of irradiated plasma. Int. J. Radiat. Biol. 47: Mothersill, C. and Seymour, C. (1997) Medium from irradiated human epithelial cells but not human fibroblasts reduces the clastogenic survival of unirradiated cells. Int. J. Rad. Biol. 71: Nagasawa, H. and Little, J. B. (1999) Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect. Radiat. Res. 152: Mothersill, C. and Seymour, C. (1998) Cell-cell contact during gamma irradiation is required to induce a bystander effect in the normal human keratinocytes: evidence for release during irradiation of a signal controlling survival into the medium. Radiat. Res. 149: Lorimore, S. A., Kadhim, M. A., Pocock, D. A., Papworth, D., Stevens, D. L., Goodhead, D. T. and Wright, E. G. (1998) Chromosomal instability in the descendants of unirradiated surviving cells after alpha-particle irradiation. Proc. Nat. Acad. 94: Azzam, E. I., de Toledo, S. M., Gooding, T. and Little, J. B. (1998) Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of alpha particles. Radiat. Res. 150: Deshpande, A., Goodwin, E. H., Bailey, S. M., Marrone, B. and Lehnert, B. (1996) Alpha-particle-induced sister chromatid exchanges in normal human lung fibroblasts: evidence for an extranuclear target. Radiat. Res. 145: Olive, P. L. (1999) DNA damage and repair in individual cells: applications of the comet assay in radiobiology. Int. J. Radiat. Biol. 75: Endo, S., Hoshi, M., Tauchi, H., Takeoda, S., Kitagawa, K., Suga, S., Maeda, N., Komatsu, K., Sawada, S., Iwamoto, E., Sakamoto, S., Takeyama, K. and Omura, M. (1995) Neutron generator at Hiroshima University for

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