Radiation Protection Dosimetry (2014), Vol. 159, No. 1–4, pp. 111 –117 Advance Access publication 28 May 2014

doi:10.1093/rpd/ncu172

GENOTOXICITY IN EARTHWORM AFTER COMBINED TREATMENT OF IONISING RADIATION AND MERCURY Tae Ho Ryu1, Kwang-Guk An2 and Jin Kyu Kim1,* 1 Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea 2 College of Biological Science and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea *Corresponding author: [email protected]

INTRODUCTION All living organisms are being exposed to harmful agents present in environments such as air, water and soil. A risk assessment of hazardous substances is usually performed based on the simplest assumption that any harmful factor acts independently of others. However, the combined exposure to multiple harmful factors might result in a higher effect than expected from the addition of the effect produced by each factor. Recent studies show that the combined and cumulated exposures are an issue of global concern(1, 2). Ionising radiation is one of the most common risk factors for damage to cells. It has been well known that ionising radiation can cause macromolecule damage, including lipids, proteins and DNA, either directly or indirectly through interaction with water(3). Singleand double-strand breaks on a DNA helix, in particular, were induced by generating oxygen reactive species in the cells(4). The radioactive fallout from Chernobyl and other nuclear accidents combined with chronic contamination has resulted in morphologic, physiologic and genetic variations in all animal species in the area including mammals, birds, fish and invertebrates(5). If such soil contamination occurs, radioactive materials can accumulate in the human body through natural food chains, although humans are not directly exposed to radiation. Radiological protection has generally focused on human beings, but the International Commission on Radiological Protection (ICRP) requires the data of ionising radiation effects on nonhuman biota(6, 7). Earthworms, as the most important key indicator species in soil environments, are easily exposed to natural and non-natural radiation. In addition, earthworms have been recommended for the

testing of acute and sub-acute toxicity of soil by the OECD and in previous studies(8 – 12). Mercuric compounds are also related to many reports indicating its genotoxic potential in a variety of organisms including humans and aquatic species(13). The mercury exists in numerous forms, metallic mercury, mercuric sulphide (HgS), mercuric chloride (HgCl2) and methyl mercury, which are commonly found in the environment. HgCl2 adversely affects the central and peripheral nervous system, the kidney, the eye, and reproductive fitness(14). Mercury and its compounds participate in four main mechanisms that can cause genotoxicity in the cells: direct action on DNA, the generation of free radicals and oxidative stress, the inhibition of mitotic spindle formation, and an influence on the DNA repair mechanisms(15). This study was performed to investigate the acute genotoxic effects of ionising radiation and mercury on the earthworm, Eisenia fetida. These overall researches provide the fundamental information on the combined effects of radiation and mercury on the organisms. In addition, experiments were done to identify the levels of DNA damage and repair kinetics in the coelomocytes of E. fetida treated with ionising radiation and HgCl2 by means of the alkaline comet assay. MATERIALS AND METHODS Chemicals HgCl2 (.99.5 % purity) was obtained from Samchun Pure Chemical, Co., Ltd., Korea. All other chemicals were of reagent grade and purchased from Sigma Aldrich, Co.

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This study was performed to investigate the acute genotoxic effects of mercury and radiation on earthworms (Eisenia fetida). The levels of DNA damage and the repair kinetics in the coelomocytes of E. fetida treated with mercuric chloride (HgCl2) and ionising radiation (gamma rays) were analysed by means of the comet assay. For detection of DNA damage and repair, E. fetida was exposed to HgCl2 (0– 160 mg kg21) and irradiated with gamma rays (0– 50 Gy) in vivo. The increase in DNA damage depended on the concentration of mercury or dose of radiation. The results showed that the more the oxidative stress induced by mercury and radiation the longer the repair time that was required. When a combination of HgCl2 and gamma rays was applied, the cell damage was much higher than those treated with HgCl2 or radiation alone, which indicated that the genotoxic effects were increased after the combined treatment of mercury and radiation.

T. H. RYU ET AL.

Test animals

Combined treatment of mercury and radiation

The earthworm, E. fetida, was chosen for this study because it is an internationally accepted model species for toxicity testing with a cosmopolitan distribution(16). The species of E. fetida belongs to the taxa of phylum annelida and class clitellata, is hermaphrodite and fertilises its eggs inside a cocoon secreted by the clitellum(17). These worms are live in the upper layer of the soils containing rotting vegetation, compost and manure. E. fetida is native to Europe and found on every continent, except for Antarctica. These are most adaptive to various moisture (range of moisture content: 43–90 %) and temperature extremes (ideal temperature range: 20–25 8C; Min and Max range: 33–35 8C) than other species. Adult E. fetida with sexually matured and welldeveloped clitellum (average weight, 350 mg) was used for this experiment. Earthworms were maintained in the dark in a 6:3:1 mixture of artificial soil (gardening soil, Sanglim Co., Ltd., Korea), rice bran and cattle manure at 23+2 8C. The moisture content was adjusted to 65+5 % of the final weight with dechlorinated water.

Experiments were conducted to identify the combined effects of radiation and mercury in the coelomocytes of E. fetida. The animals were exposed to HgCl2 (0– 160 mg kg21) for a period of 48 h, and gamma rays (0 –50 Gy) simultaneously.

Mercury treatment For in vivo exposures, artificial soil were prepared by dissolving the amount of HgCl2 in distilled water and mixing it thoroughly into 120 g of dried artificial soil in clear plastic containers (105`  82`  68 mm) with ventilated lids to attain a moisture content of 70 %. HgCl2 was mixed to artificial soil for the treatment concentrations of 10, 20, 40, 80 and 160 mg of HgCl2 per soil weight (kg21), respectively. These exposure concentrations were based on the range-finding test limiting the exposure range to concentrations below the point without causing lethality. The worms were exposed to these soils for periods of 24 and 48 h in an ambient condition-controlled room. Sets of twenty worms were exposed to each concentration. No feed was added during the exposure period. At the end of the exposure test, all worms were transferred to clean soil. Irradiation A group of ten worms was transferred to a plastic Petri dish with moist filter paper and then acutely irradiated with 0, 2.5, 5, 10, 20 and 50 Gy (1 h21), respectively. External gamma radiation was provided by a 60Co source (7.4 PBq of capacity; AECL, Canada at the Korea Atomic Energy Research Institute). After irradiation, the samples were covered in black cloth and on ice while carrying the worms to minimise the effects by light or temperature. At the end of the exposure tests, all worms were transferred to clean soil.

Eisenia fetida coelomocytes were obtained using a simple non-invasive technique described by Eyambe et al. with a slight modification(18). The comet assay was performed under alkaline conditions following the modified procedure of Singh et al.(19). Avolume of 20 ml of a cell suspension mixed in 200 ml of a 0.5 % low melting-point agarose (LMA) dissolved in PBS were spread onto fully frosted microscope slides precoated with 1 % normal melting-point agarose. The third layer of 0.5 % LMA was added and solidified. The cells were then lysed for 2 h at 4 8C in a lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1 % Triton X-100 and 10 % DMSO, pH 10.0). After lysis, slides were placed in an electrophoresis tank and the DNA was allowed to unwind for 20 min in an electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH . 13.0). Electrophoresis (0.7 V cm21, 300 mA, 25 min) was performed in the same buffer. The slides were washed twice for 5 min in a neutralisation buffer (0.4 M Tris, pH 7.5) before dehydration in absolute ethanol for 10 min. The slides were dried in darkness and stained with 100 ml of ethidium bromide (20 g ml21). The comets were analysed using an image analysis system (Komet 4.0 from Kinetic Imaging Ltd., Liverpool, UK). Fifty cells per slide were scored out of a total of 100 cells per dose. Statistical Analysis Each treatment was performed in triplicate, and the standard deviation (SD) was calculated. The statistical program, OriginPro 7.5, was used for the data analyses. The data were subjected to one-way analysis of variance (ANOVA test) followed by the least significance difference test at p-value levels of ,0.005 and ,0.001, respectively. RESULTS AND DISCUSSION No earthworm mortalities were recorded during the exposure periods at all the tested concentrations. DNA damage was expressed as olive tail moment (OTM) values. The background level of DNA damage (the control) was mainly low with a small number of cells with appreciable DNA damage. DNA damage in earthworm coelomocytes exposed to HgCl2 was higher than that of the control. The increase in DNA damage was shown to be dose dependent when

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Type of treatments

Comet assay

EFFECTS OF GAMMA RAYS AND HgCl2 IN EARTHWORMS Table 1. The values of OTMa (mean+ + SD) in E. fetida exposed to HgCl2 for 24 and 48 h. HgCl2 (mg kg21)

Experimental groups 24 hb

Control 10 20 40 80 160

48 h

First

Second

Third

First

Second

Third

0.15+0.20c 0.29+0.50 0.97+0.60 1.71+0.58 1.34+0.77 2.43+1.06

0.39+0.22 0.44+0.30 1.19+0.61 0.35+0.79 1.26+0.96 1.56+1.12

0.30+0.24 0.37+0.18 0.61+0.45 0.94+0.60 2.04+1.28 2.80+1.95

0.30+0.31 0.29+0.31 0.77+0.82 0.91+0.86 2.34+1.35 3.25+1.48

0.38+0.33 0.66+0.64 1.08+0.91 2.34+1.86 3.40+1.58 3.57+1.45

0.45+0.36 1.38+0.80 1.68+1.41 1.85+1.12 2.52+1.51 3.11+1.42

Triplicate values of OTM indicate the median mean of Olive tail moment. Exposure time (h). c Mean OTM+SD. b

Figure 1. DNA damage in E. fetida exposed to HgCl2 for 24 and 48 h, respectively. DNA damage in E. fetida treated with 10 and 80 mg kg21 during 48-h treatment was significantly higher than those of the groups exposed for 24 h. The estimation of OTM values used the following equation: OTM ¼ (tail mean 2 head mean) ` tail % DNA/100. Significant differences (*p , 0.005; **p , 0.001) from the controls are expressed in the figure.

exposed to HgCl2 during 24 and 48 h. DNA damage in the earthworm coelomocytes treated with the higher concentrations of HgCl2 was significantly higher than that treated with the lowest concentration of 10 mg kg21 (Table 1). Furthermore, it was clearly shown that DNA damage in the groups treated with 10 and 80 mg kg21 during 48-h treatment was significantly higher than those of the groups exposed for 24 h (Figure 1). In the comet assays of coelomocytes tested for 0, 12, 24, 48 and 72 h, after treatment with different concentrations of mercury, the OTM values decreased in a time-dependent manner (Figure 2). The estimated equation was confirmed in terms of the regression

coefficient. Linear equations for the treatments of 10, 20, 40, 80 and 160 mg kg21 showed correlation coefficients of 0.459, 0.673, 0.815, 0.844 and 0.879, respectively. The largest slope was 160 mg kg21, and the smallest slope was 10 mg kg21 on the linear regressions. Each of the experimental groups was remarkably comparable with the control group, 72 h after exposure to HgCl2. In particular, the DNA strand breaks decreased remarkably at 80 and 160 mg kg21 concentrations. The half repair times of the two experimental groups were 32.5 and 41.7 h, respectively. Eisenia fetida irradiated with 0 to 50 Gy showed that there was no mortality observed during the experiment process. At low doses of radiation, the number of cells remained unchanged, but at higher doses, most of the cells showed moderate or strong DNA damage (Figure 3). As a result, DNA damaging effects were enhanced with an increased exposure dose, and DNA migration showed a dose-response relationship. The values of OTM at an irradiation dose of 0, 2.5, 5, 10, 20 and 50 Gy were 0.18+0.22, 0.54+0.36, 1.28+0.59, 2.27+0.98, 3.8+1.25 and 8.99+1.98, respectively (Figure 4). Therefore, each experimental group showed 3, 7, 12, 21 and 50 times as heavy damage as the control group. The genotoxic effect of ionising radiation was detected at all doses as a dose-related increase in DNA migration. The DNA strand breaks decreased as accessed by in the SCGE assay with the lapse of time, and the OTM values of whole experimental groups were similar to those of the control group after 12 h. The values of OTM in the experimental groups were 0.3 (2.5 Gy) or 0.2 (5 Gy) after 2 h, 0.42 (10 Gy) or 0.3 (20 Gy) after 3 h and 0.26 (50 Gy) after a 12-h exposure. The results showed that the higher dose of radiation was irradiated, the longer repair time was required. According to Lankoff et al., in experiments with humans, the DNA of lymphocytes was completely repaired after irradiation with gamma rays 2 h later(20). This clearly

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Figure 2. Dose-dependent DNA repair kinetics of E. fetida after exposure to HgCl2 (0–160 mg kg21). DNA damage is expressed as olive tail moment (OTM) values. OTM values decreased with time in artificial clean soil after exposure. Each of the experimental groups was remarkably comparable with the control group 72 h after exposure to HgCl2.

showed that the DNA repair time of earthworms is very similar to that of humans. To investigate the combined effects on radiation and mercury in E. fetida, the worms were irradiated with 0–50 Gy gamma rays after 0–160 mg kg21

HgCl2 treatment. There was no mortality observed during the experiment process. When a combination of HgCl2 and gamma radiation was applied, the OTM values were much higher than those treated with HgCl2 or radiation alone, which indicated that the

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EFFECTS OF GAMMA RAYS AND HgCl2 IN EARTHWORMS

Figure 3. The comet images of nuclei isolated from coelomocytes of E. fetida treated with ionising radiation. Unexposed control coelomocyte (a), coelomocyte irradiated with 5 Gy (b) and 20 Gy gamma rays (c). Higher dose of radiation-induced strong DNA damage is shown as the longer comet tail.

genotoxic effects were increased after combined treatment of mercury and radiation (Figure 5). It has to be noted that the combination of lowest concentration of HgCl2 (10 mg kg21) with the smallest radiation dose (2.5 Gy) could result in the greatest synergistic effect except for the combination of 50 Gy of gamma rays and 160 mg kg21 of HgCl2. All data points in Figure 5 indicate the synergistic effect of mercury and radiation. These results are in agreement with reports that the combination of mercury and radiation enhanced synergistic effects on HeLa and PLHC-1 cells(21, 22). Eisenia fetida treated with 20-Gy gamma rays alone or with exposure ionising radiation combined with 40 mg kg21 HgCl2 showed that levels of DNA damage decreased with time (Figure 6). The repair time of the damaged DNA was calculated at 12 h, only when irradiated with 20 Gy radiations. On the other hand, when 40 mg kg21 HgCl2 and 20 Gy gamma rays were treated together, the repair time of damaged DNA was 96 h. Therefore, the repair time in the animals treated with the combination of HgCl2 and ionising radiation was nearly eight times longer than that in the animals treated with ionising radiation alone. In

Figure 5. DNA damage in E. fetida irradiated with gamma rays (0– 20 Gy) after the treatments of HgCl2 (0–40 mg kg21) for 48 h. The filled triangles indicate the additive values of OTM value for mercury treatment and that for irradiation. The open triangles show OTM values from combined treatment with HgCl2 and gamma rays. When a combination of HgCl2 and gamma radiation was applied, the OTM values were higher than those treated with HgCl2 or radiation alone.

Figure 6. DNA damage and repair kinetics of E. fetida irradiated with gamma rays (20 Gy), with or without the presence of HgCl2 (40 mg kg21). The repair time of DNA damage in E. fetida treated with the combination of HgCl2 and ionising radiation was nearly eight times longer than that in E. fetida treated with ionising radiation alone.

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Figure 4. DNA damage and repair kinetics in E. fetida irradiated with gamma rays. DNA damage decreased with time lapse after irradiation, and the OTM values of whole experimental groups after 12 h repair reached to similar value of the control group. Significant differences (*p , 0.005; **p , 0.001) from the controls are expressed in the figure.

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CONCLUSIONS The increase in DNA damage linearly increases with radiation dose and HgCl2 concentration. The more the oxidative stress induced by radiation or mercury, the longer the repair time is required. The level of DNA damage significantly increases after the combined treatment of HgCl2 and ionising radiation. In addition, longer repair time is required when DNA damage is induced by the combined treatment of two factors. The results suggest that the mercury has deleterious effects on the DNA repair. Influence of mercury on the DNA repair mechanisms can be confirmed by the comet assays used in this study. ACKNOWLEDGEMENTS This study has been carried out under the National R&D Program by the ministry of Science, ICT and Future Planning (MSIP) of Korea. FUNDING This study was funded by the National Research Foundation (NRF) of Korea. REFERENCES 1. Meek, M. E., Boobis, A. R., Crofton, K. M., Heinemeyer, G., Raaij, M. V. and Vickers, C. Risk assessment of combined exposure to multiple chemicals: A WHO/IPCS framework. Regul. Toxicol. Pharm. 60, S1–S14 (2011). 2. Silins, I. and Ho¨gberg, J. Combined toxic exposures and human health: biomarkers of exposure and effect. Int. J. Environ. Res. Public Health. 8, 629– 647 (2011).

3. Esnault, M., Legue, F. and Chenal, C. Ionizing radiation: advances in plant response. Environ. Exp. Bot. 68, 231– 237 (2010). 4. Sterpons, S. and Cozzi, R. Influence of XRCC1 genetic polymorphisms on ionizing radiation-induced DNA damage and repair. J. Nucl. Acids. (2010) doi:10.4061/ 2010/780369. 5. Yablokov, A. V., Nesterenko, V. B. and Nesterenko, A. V. Chernobyl: chapter III. 02162;. Consequences of the Chernobyl catastrophe for the environment. Ann. NY Acad. Sci. 1181, 221–286 (2009). 6. International Commission on Radiological Protection. A framework for assessing the impact of ionizing radiation on non-human species. ICRP Publication 91. Ann ICRP 33, (2003). 7. International Commission on Radiological Protection. Environmental protection—the concept and use of reference animals and plants. ICRP Publication 108. Ann ICRP 38, (2008). 8. Organization for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals, Section 2: effects on Biotic Systems Test No. 207: Earthworm Acute Toxicity Tests. OECD publishing, (1984) ISBN 9789264070042. 9. Jensen, J. and Pedersen, M. B. Ecological risk assessment of contaminated soil. Rev. Environ. Contam. Toxicol. 186, 73– 105 (2006). 10. Sanchez-Hernandez, J. Earthworm biomarkers in ecological risk assessment. Rev. Environ. Contam. Toxicol. 188, 85–126 (2006). 11. Peijnenburg, W. J. G. M., Baerselman, R., de Groot, A. C., Tjalling, J., Posthuma, L. and Van Veen, R. P. M. Relating environmental availability to bioavailability: soil type-dependent metal accumulation in the oligochaete Eisenia andrei. Ecotoxicol. Environ. Safe. 44, 294– 310 (1999). 12. Giovanetti, A., Fesenko, S., Cozzella, M. L., Asencio, L. D. and Sansone, U. Bioaccumulation and biological effects in the earthworm Eisenia fetida exposed to natural and depleted uranium. J. Environ. Radioactiv. 101, 509– 516 (2010). 13. Pandey, S., Kumar, R., Sharma, S., Nagpure, N. S., Srivastava, S. K. and Verma, M. S. Acute toxicity bioassays of mercuric chloride and malathion on air-breathing fish Channa punctatus (Bloch). Ecotox. Environ. Safe. 61, 114–120 (2005). 14. Palmeira, C. M. and Madeira, V. M. C. Mercuric chloride toxicity in rat liver mitochondria and isolated hepatocytes. Environ. Toxicol. Pharm. 3, 229–235 (1997). 15. Crespo-Lo´pez, M. E., Maceˆdo, G. L., Pereira, S. I. D., Arrifano, G. P. F., Picanco-Diniz, D. L. W., do nascimento, J. L. M. and Herculano, A. M. Mercury and human genotoxicity: critical considerations and possible molecular mechanisms. Pharmacol. Res. 60, 212– 220 (2009). 16. Edwards, J. C., Chapman, D., Cramp, W. A. and Yatvin, M. B. The effects of ionizing radiation on biomembrane structure and function. Prog. Biophys. Mol. Bio. 43, 71– 93 (1984). 17. Espinoza-Navarro, O. and Bustos-Obrego´n, E. Sublethal doses of malathion alter male reproductive parameters of Eisenia foetida. Int. J. Morphol. 22, 297– 302 (2004).

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addition, E. fetida exposed to mercury showed a statistically lower repair efficiency of radiation-induced DNA damage. An interesting study was done on workers intoxicated by mercury vapour by Cebulska-Wasilewska et al.(23). The authors analysed the DNA repair efficiency in the primary cultures of blood lymphocytes irradiated with X-rays or UV-C light. The low concentrations of the metal would interfere with the recombination and base excision repair mechanisms but not with the nucleotide excision repair mechanisms(24). In addition to an indirect action on the DNA repair system, mercury can also be directly bound to the ‘Zinc fingers’ core of DNA repair enzymes, affecting their activity(25, 26). The permissible levels of mercury can even have deleterious effects on the DNA repair system. Therefore, the influence of mercury on the DNA repair mechanisms has been confirmed through this experiment.

EFFECTS OF GAMMA RAYS AND HgCl2 IN EARTHWORMS 22. Han, M., Hyun, K. M., Nili, M., Hwang, I. Y. and Kim, J. K. Synergistic effects of ionizing radiation and mercury chloride on cell viability in fish hepatoma cells. Korean J. Environ. Biol. 27, 140– 145 (2009). 23. Cebulska-Wasilewska, A., Panek, A., Z˙abin´ski, Z., Moszczyn´ski, P. and Au, W. W. Occupational exposure to mercury vapour on genotoxicity and DNA repair. Mut. Res. 586, 102– 114 (2005). 24. Christie, N., Cantoni, O., Sugiyama, M., Cattabeni, F. and Costa, M. Differences in the effects of Hg (II) on DNA repair induced in Chinese Hamster ovary by ultraviolet or X-rays. Mol. Pharm. 24, 173–178 (1985). 25. Stohs, S. J. and Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radical Bio. Med. 18, 321–336 (1995). 26. Asmuss, M., Mullenders, L. H. F. and Hartwig, A. Interference by toxic metal compounds with isolated zinc finger DNA repair proteins. Toxicol. Lett. 112– 113, 227–231 (2000).

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18. Eyambe, G. S., Goven, A. J., Fitzpatrick, L. C., Venables, B. J. and Cooper, E. L. A non-invasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies. Lab. Animal. 25, 61– 67 (1991). 19. Singh, N. P., McCoy, M. T., Tice, R. R. and Schneider, E. L. A simple technique for quantization of low levels of DNA damage in individual cells. Exper. Cell Res. 175, 184 –191 (1988). 20. Lankoff, A., Bialczyk, J., Dziga, D., Carmichael, W. W., Gradzka, I., Lisowska, H., Kuszewski, T., Gozdz, S., Piorun, I. and Wojcik, A. The repair of gamma-radiation-induced DNA damage is inhibited by microcystinLR, the PP1 and PP2A phosphatase inhibitor. Mutagenesis. 21, 83–90 (2006). 21. Woo, H. J., Kim, J. H., Cebulska-Wasilewska, A. and Kim, J. K. Evaluation of DNA damage by mercury chloride (II) and ionizing radiation in HeLa Cells. Korean J. Environ. Biol. 24, 46–52 (2006).

Genotoxicity in earthworm after combined treatment of ionising radiation and mercury.

This study was performed to investigate the acute genotoxic effects of mercury and radiation on earthworms (Eisenia fetida). The levels of DNA damage ...
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