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Nobuyoshi Ishii and Nobuhiro Kaneko for technical support and helpful suggestions ... T. Saito, Y. Tsutsumi, A. Fujimori, S. Sato, K. Tatsumi, R. Araki and M. Abe,.
Effects of acute gamma irradiation on soil invertebrates in laboratory tests Taizo Nakamori1, Satoshi Yoshida1, Yoshihisa Kubota1, Tadaaki Ban-nai1, and Akira Fujimori1 1

National Institute of Radiological Sciences, 263-8555, Chiba, Japan.

INTRODUCTION As atomic power is increasingly recognised as a potential energy source to sustain future human development, radiological protection of the environment will become an even more important environmental safety concern. Thus, an understanding of the effects of ionising radiation on non-human biota is required by the International Commission on Radiological Protection for the radiological protection of the environment (International Commission on Radiological Protection (ICRP), 2003). Soil processes are vital to sustainable terrestrial ecosystems, and soil invertebrates play an important role in nutrient cycling by feeding on microbiota. Because of their ecological importance, soil invertebrates are used for ecological impact assessments of terrestrial ecosystem pollutants. For chemical substances, single-species laboratory tests are used to understand toxicity. Standard tests using earthworms and springtails have been developed by the Organisation for Economic Co-operation and Development (OECD) and the International Organization for Standardization (ISO) (Jänsch et al., 2005). Laboratory toxicity tests are also applicable in field contamination monitoring to determine if test organisms have been exposed to field-corrected soils. In such assays, gene expression as a biomarker has been receiving increased attention as it may produce fast, sensitive and diagnostic assays (e.g., Timmermans et al., 2007). A similar use of laboratory tests can be applied to assess the environmental impact of ionising radiation. The objectives of this study were to reveal dose–effect relationships for radiation on soil invertebrates and screen candidate biomarkers for radiation exposure. We applied standard test protocols to examine dose–effect relationships for gamma radiation on survival and reproduction in the earthworm Eisenia fetida (Oligochaeta) and the springtail Folsomia candida (Collembola). A novel technology, high-coverage expression profiling (HiCEP), was applied to detect radiation responsive genes as a biomarker in F. candida.

MATERIALS AND METHODS Animals The springtail F. candida is a soil-dwelling, unpigmented, eyeless, parthenogenic species belonging to the family Isotomidae. Adults reach 1.5–3 mm in body length. Stock cultures of springtails originating from a population in central Japan were reared on baker’s yeast. The earthworm E. fetida was obtained from a commercial source for composting and reared on wheat bran. Age-synchronised springtails and worms used in experiments were prepared according to ISO (1999) and OECD (2004), respectively. Toxicity tests Reproduction tests were carried out largely according to ISO (1999) and OECD (2004) with springtails and earthworms, respectively. When 10–12 days old, springtails were acutely irradiated at increasing doses of 137Cs gamma radiation and reared on moist substrates (plaster of Paris mixed with activated charcoal). Four weeks after irradiation, the number of neonates produced by irradiated springtails was examined (Nakamori et al., 2007). Earthworms at 7 months old were acutely irradiated at increasing doses of 137Cs gamma radiation and reared in OECD artificial soils (peat moss:kaolin clay:sand = 10:20:70 by weight). Four weeks after irradiation, worms were removed from the soils. Eight weeks after irradiation, the number of neonates in the soils was examined. Survival tests were carried out largely according to ISO (1999) and OECD (1984) with springtails and earthworms, respectively. Experimental procedures were as in reproduction tests except that 60Co gamma radiation was used and survival was examined after 4 and 2 weeks for springtails and earthworms, respectively. HiCEP The HiCEP method was applied to determine differentially expressed genes between irradiated and non-irradiated springtails. Irradiation experiments were carried out largely in the same manner as described above, except springtails were fixed 2 h after irradiation. HiCEP analyses were carried out as described in Fukumura et al. (2003). Briefly, total RNA was converted to cDNA with 5'-biotinylated oligo(dT) primers. Double-strand cDNA was prepared, digested with MspI and trapped by avidin bound to magnetic beads. After the fragments digested by MspI (except for most of the 3'-region bearing oligo(dT)-biotin) were washed off, a synthetic adaptor was ligated, and the trapped templates were digested by MseI. The resulting solution was used as a template for 256 runs of selective PCR. The products were denatured and loaded on an ABI PRISM 3100 electrophoresis system (Applied Biosystems). Transcript-derived fragments (TDFs) of interest were physically detected using polyacrylamide gel electrophoresis and sequenced.

Quantitative PCR Irradiation experiments were carried out largely in the same way as in HiCEP analysis. Quantitative polymerase chain reaction (PCR) was performed using the PRISM7500 Real-time PCR system with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and TDF-specific primers. The data were normalised in relation to the expression level of the β-actin gene. RESULTS AND CONCLUSIONS The 50% effective doses (ED50) for reproduction were 22 and 11 Gy in F. candida (Nakamori et al., 2007) and E. fetida, respectively, and the 50% lethal doses were 1356 and 825 Gy in F. candida (Nakamori et al., 2007) and E. fetida, respectively. Reproduction was more sensitive to gamma radiation than survival in both F. candida and E. fetida. The higher sensitivity of reproduction is consistent with findings in other invertebrates (e.g., Watanabe et al., 2006). A HiCEP analysis showed that several TDFs were up-regulated by irradiation in F. candida, and sequencing the TDFs revealed that a few of them were similar to genes relating to DNA repair and response to oxidative stress. For the other TDFs, no similarity was found in the gene database, probably because of the limited length of TDFs or limited genome information in springtails. These findings suggest that HiCEP is effective at discovering both known and unknown TDFs, even in non-genomic model organisms such as F. candida. Thus, HiCEP is useful in ecotoxicology in which various non-genomic model organisms are involved. Expression of TDFs found to be up-regulated in the HiCEP analysis was also examined using a quantitative PCR method. Most of the TDFs were also found up-regulated in quantitative PCR analysis. Some TDFs were up-regulated more than tenfold by acute gamma irradiation at ED50 for reproduction, whereas additional experiments revealed that the TDFs showed little response to cadmium exposure at 50% effective concentration. Therefore, the findings suggest that these TDFs are candidates for discriminative biomarkers between radiation and cadmium exposure, although differences existed in exposure and experimental conditions. In conclusion, the present analysis establishes important baselines for the radiological protection of terrestrial ecosystems, and indicates that gene expression in soil invertebrates may be useful for distinguishing between the impacts of radiation and chemicals in contaminated soils.

ACKNOWLEDGEMENTS We thank Ayako Kojima, Keiji Kinoshita, Syunsuke Ando, Yoshito Watanabe, Shoichi Fuma, Nobuyoshi Ishii and Nobuhiro Kaneko for technical support and helpful suggestions, and Makiko Hasegawa and Ryosaku Itoh for supplying the F. candida. This work was partly supported by a grant from the Ministry of Environment, Japan. Expression profiling in this study was performed by the HiCEP unit team founded by an initiative of the president of the National Institute of Radiological Sciences, Japan. REFERENCES Fukumura, R., H. Takahashi, T. Saito, Y. Tsutsumi, A. Fujimori, S. Sato, K. Tatsumi, R. Araki and M. Abe, 2003. A sensitive transcriptome analysis method that can detect unknown transcripts. Nucleic Acids Res. 31: e94. ICRP, 2003. A framework for assessing the impact of ionizing radiation on non-human species. Publication 91, Annals of the ICRP 33, Pergamon Press, Oxford. ISO, 1999. Soil quality—inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants, no. 11267, ISO, Geneva. Jänsch, S., M.J. Amorim and J. Römbke, 2005. Identification of the ecological requirements of important terrestrial ecotoxicological test species. Environ. Rev. 13: 51–83. Nakamori, T., S. Yoshida, Y. Kubota, T. Ban-nai, N. Kaneko, M. Hasegawa and R. Itoh, 2007. Effects of acute gamma irradiation on Folsomia candida (Collembola) in a standard test. Ecotoxicol. Environ. Saf. doi: 10.1016/j.ecoenv.2007.10.029. OECD, 1984. Guideline for testing of chemicals 207. Earthworm Acute Toxicity Tests. OECD, Paris. OECD, 2004. Guideline for testing chemicals 222. Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei). OECD, Paris. Timmermans, M.J.T.N., M.E. de Boer, B. Nota, T.E. de Boer, J. Mariën, R.M. Klein-Lankhorst, N.M. van Straalen and D. Roelofs, 2007. Collembase: a repository for springtail genomics and soil quality assessment. BMC Genomics, 8:341. Watanabe, M., T. Sakashita, A. Fujita, T. Kikawada, D.D. Horikawa, Y. Nakahara, S. Wada, T. Funayama, N. Hamada, Y. Kobayashi and T. Okuda, 2006. Biological effects of anhydrobiosis in an African chironomid, Polypedium vanderplanki, on radiation tolerance. Int. J. Radiat. Biol. 82: 587–592.