Environmental Science and Pollution Research

, Volume 26, Issue 4, pp 3869–3881 | Cite as

Developmental toxicity and apoptosis in zebrafish embryos induced by low-dose γ-ray irradiation

  • Weichao Zhao
  • Nan Hu
  • Dexin DingEmail author
  • Dingxin Long
  • Sheng Li
  • Guangyue Li
  • Hui Zhang
Research Article


In this paper, the developmental toxicity and apoptosis in zebrafish (Danio rerio) embryos induced by 0.01, 0.05, and 0.10-Gy γ-ray irradiation were investigated and verified by single cell gel electrophoresis, acridine orange staining, flow cytometry, transmission electron microscopy, digital gene expression sequencing, and Western blot analysis. DNA damage, deformity rates, and apoptosis of zebrafish embryos were found to increase significantly with the increase of irradiation dose, and survival and hatching rates significantly decreased when the irradiation dose exceeds 0.10 and 0.05 Gy, respectively. Exposure to 0.10-Gy γ-ray irradiation resulted in the swelling of cell mitochondria of zebrafish embryos and changes in their intracellular vacuoles. mRNA and protein expression levels of Shh (sonic hedgehog 19 KDa) and Smo (smoothened 86 KDa) of Hh signaling pathway associated with the development of early embryos significantly increased with the increase of irradiation dose. Expression of the AKT (56 KDa) and PiK3r3 (55 KDa) genes, which are anti-apoptotic and involved with the PI3K/Akt signaling pathway, significantly decreased, while expression of the bada gene, which is pro-apoptotic, significantly increased. The results show that γ-ray irradiations of 0.01 and 0.05 Gy can induce developmental toxicity and apoptosis in zebrafish embryos via Hh and PI3K/Akt signaling pathways, respectively.


Low-dose γ-ray irradiation Zebrafish embryos Developmental toxicity Apoptosis Signaling pathway 



This project was supported by the Defense Industrial Technology Development Program (No. JCKY2016403C001) and Key Project of National Defense Basic Research (No. B3720132001).

Supplementary material

11356_2018_3893_MOESM1_ESM.docx (17 kb)
ESM 1 (DOCX 17 kb)


  1. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. Nat Genet 25:25–29CrossRefGoogle Scholar
  2. Azqueta A, Collins AR (2013) The essential comet assay: a comprehensive guide to measuring DNA damage and repair. Arch Toxicol 87:949–968CrossRefGoogle Scholar
  3. Brenner DJ, Hall EJ (2007) Computed tomography-an increasing source of radiation exposure. N Engl J Med 357:2277–2284CrossRefGoogle Scholar
  4. Cadet J, Carell T, Cellai L, Chatgilialoglu C, Gimisis T, Miranda M, O’Neill P, Ravanat J, Robert M (2008) DNA damage and radical reactions: mechanistic aspects, formation in cells and repair studies. CHIMIA Int J Chem 62:742–749CrossRefGoogle Scholar
  5. Cadet J, Ravanat JL, TavernaPorro M, Menoni H, Angelov D (2012) Oxidatively generated complex DNA damage: tandem and clustered lesions. Cancer Lett 327:5–15CrossRefGoogle Scholar
  6. Cohen BL (2011) The cancer risk from low level radiation. In: Tack D, Kalra M, Gevenois P (eds) Radiation dose from multidetector CT. Medical radiology. Springer, Berlin, pp 61–79Google Scholar
  7. Datta K, Suman S, Fornace AJ (2014) Radiation persistently promoted oxidative stress, activated mTOR via PI3K/Akt, and downregulated autophagy pathway in mouse intestine. Int J Biochem Cell B 57:167–176CrossRefGoogle Scholar
  8. Dent P, Yacoub A, Contessa J, Caron R, Amorino G, Valerie K, Schmidt-Ullrich R (2003) Stress and radiation-induced activation of multiple intracellular signaling pathways. Radiat Res 159:283–300CrossRefGoogle Scholar
  9. di Magliano MP, Hebrok M (2003) Hedgehog signalling in cancer formation and maintenance. Nat Rev Cancer 3:903–911CrossRefGoogle Scholar
  10. Dimri M, Joshi J, Chakrabarti R, Sehgal N, Sureshbabu A, Prem KI (2015) Todralazine protects zebrafish from lethal effects of ionizing radiation: role of hematopoietic cell expansion. Zebrafish 12:33–47CrossRefGoogle Scholar
  11. Douki T, Ravanat JL, Pouget JP, Testard I, Cadet J (2006) Minor contribution of direct ionization to DNA base damage induced by heavy ions. Int J RadiatBiol 82:119–127CrossRefGoogle Scholar
  12. Fournier L, Laurier D, Caër-Lorho S, Laroche P, Le G, Bernard PF, Leuraud K (2016) P275 Risk of cancer mortality in a French cohort of nuclear workers when accounting for occupational, environmental and medical radiation exposure. Occup Environ Med 73(Suppl 1):A213–A213Google Scholar
  13. Gagnaire B, Cavalié I, Pereira S, Floriani M, Dubourg N, Camilleri V, Adam-Guillermin C (2015) External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio. Aquat Toxicol 169:69–78CrossRefGoogle Scholar
  14. Geisler R (2002) Zebrafish: a practical approach. The practical approach series 261:175–212Google Scholar
  15. Hallare AV, Schirling M, Luckenbach T, Köhler HR, Triebskorn R (2005) Combined effects of temperature and cadmium on developmental parameters and biomarker responses in zebrafish (Danio rerio) embryos. J ThermBiol 30:7–17Google Scholar
  16. Hamra GB, Richardson DB, Cardis E, Daniels RD, Gillies M, O’Haga JA, Moissonnier M (2016) Cohort profile: the international nuclear workers study (INWORKS). Int J Epidemiol 45:693–699CrossRefGoogle Scholar
  17. Han ZH, Wang QW, Fu J, Chen HS, Zhao Y, Zhou BS, Liu HL (2014) Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris (2-butoxyethyl) phosphate. Aquat Toxicol 150:175–181CrossRefGoogle Scholar
  18. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496:498–503CrossRefGoogle Scholar
  19. Hu M, Hu N, Ding DX, Zhao WC, Feng YF, Zhang H, Wan YD (2016) Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos. Radiat Environ Biophys 55:441–450CrossRefGoogle Scholar
  20. Hurem S, Gomes T, Brede DA, Lindbo EH, Mutoloki S, Fernandez C (2017a) Parental gamma irradiation induces reprotoxic effects accompanied by genomic instability in zebrafish (Danio rerio) embryos. Environ Res 159:564–578CrossRefGoogle Scholar
  21. Hurem S, Martín LM, DAnders B, Skjerve E, Nourizadeh-Lillabad R, Lind OC, Salbu B ((2017b)) Dose-dependent effects of gamma radiation on the early zebrafish development and gene expression. PLoS One 12:e0179259Google Scholar
  22. Hurem S, Martín LM, Lindeman L, Brede DA, Salbu B, Lyche JL, AleströmP KJH (2018a) Parental exposure to gamma radiation causes progressively altered transcriptomes linked to adverse effects in zebrafish offspring. Environ Pollut 234:855–863CrossRefGoogle Scholar
  23. Hurem S, Gomes T, Brede DA, Mayer I, Lobert VH, Mutoloki S, Gutzkow KB, Teien HC, Oughton D, Aleström P, Lyche PJ ((2018b)) Gamma irradiation during gametogenesis in young adult zebrafish causes persistent genotoxicity and adverse reproductive effects. Ecotox Environ Safe 154:19–26Google Scholar
  24. ICRP (1990) Annex B Biological effects of ionising radiations. ICRP Publication 60. Pergamon Press, OxfordGoogle Scholar
  25. Jarvis R, Knowles J (2003) DNA damage in zebrafish larvae induced by exposure to low-dose rate γ-radiation: detection by the alkaline comet assay. Mutat Res-Gen ToxEn 541:63–69CrossRefGoogle Scholar
  26. Kalueff AV, Stewart AM, Gerlai R (2014) Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol Sci 35:63–75CrossRefGoogle Scholar
  27. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2007) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36(S1):480–484CrossRefGoogle Scholar
  28. Kosmehl T, Hallare AV, Braunbeck T, Hollert H (2008) DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay. Mutat Res-Gen ToxEn 650:1–14CrossRefGoogle Scholar
  29. Kumar MKP, Shyama SK, Kashif S, Dubey SK, Sonaye BH, Samit BK, Chaubey RC (2017) Effects of gamma radiation on the early developmental stages of Zebrafish (Danio rerio). Ecotox Environ Safe 142:95–101CrossRefGoogle Scholar
  30. Leuraud K, Richardson DB, Cardis E, Daniels RD, Gillie M, O’Haga JA, Moissonnie M (2015) Ionising radiation and risk of death from leukaemia and lymphoma in radiation-monitored workers (INWORKS): an international cohort study. Lancet Haematol 2:e276–e281CrossRefGoogle Scholar
  31. Matsumura H, Yoshida K, Luo S, Kimur E, Fujibe T, Albertyn Z, Schroth GP (2010) High-throughput Super SAGE for digital gene expression analysis of multiple samples using next generation sequencing. PLoS One 5:e12010CrossRefGoogle Scholar
  32. Michiue T, Yamamoto T, Yasuoka Y, Goto T, Ikeda T, Nagura K, Nakayama T, Taira M, Kinoshita T (2017) High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis. Dev Biol 426:270–290CrossRefGoogle Scholar
  33. Miglioretti DL, Johnson E, Williams AG, Robert T, Weinmann S, Solberg LI, Vanneman N (2013) The use of computed tomography in pediatrics and the associated radiation exposure and estimated cancer risk. JAMA Pediatr 167:700–707CrossRefGoogle Scholar
  34. Nasevicius A, Ekker SC (2000) Effective targeted gene “knockdown” in zebrafish. Nat Genet 26:216–220CrossRefGoogle Scholar
  35. Olive PL, Banáth JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat Res 122:86–94CrossRefGoogle Scholar
  36. Pereira S, Bourrachot S, Cavalie I, Plaire D, Dutilleul M, Gilbin R, Adam-Guillermin C (2011) Genotoxicity of acute and chronic gamma-irradiation on zebrafish cells and consequences for embryo development. Environ Toxicol Chem 30:2831–2837CrossRefGoogle Scholar
  37. Pereira S, Malard V, Ravanat JL, Davin AH, Armengaud J, Foray N, Adam-Guillermin C (2014) Low doses of gamma-irradiation induce an early bystander effect in zebrafish cells which is sufficient to radioprotect cells. PLoS One 9:e92974CrossRefGoogle Scholar
  38. Porter JA, Young KE, Beachy PA (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science 274:255–259CrossRefGoogle Scholar
  39. Postlethwait JH, Woods IG, Ngohazelett P, Yan YL, Kelly PD, Chu F, Huang H, Hill-Force A, Talbot WS (2000) Zebrafish comparative genomics and the origins of vertebrate chromosomes. Genome Res 10:1890–1902CrossRefGoogle Scholar
  40. Rasooly RS, Henken D, Freeman N, Tompkins L, Badman D, Briggs J, Hewitt AT (2003) Genetic and genomic tools for zebrafish research: the NIH zebrafish initiative. Dev Dyn 228:490–496CrossRefGoogle Scholar
  41. Scholz S, Fischer S, Gündel U, Küster E, Luckenbach T, Voelker D (2008) The zebrafish embryo model in environmental risk assessment-applications beyond acute toxicity testing. Environ Sci PollutR 15:394–404CrossRefGoogle Scholar
  42. Schuurbiers OCJ, Kaanders JHAM, van der Heijden HFM, Dekhuijzen RPN, Oyen WJG, Bussink J (2009) The PI3-K/AKT-pathway and radiation resistance mechanisms in non-small cell lung cancer. J Thorac Oncol 4:761–767CrossRefGoogle Scholar
  43. Socol Y, Dobrzyński L, Doss M, Janiak MK, Miller ML, Sanders CL, Vaiserman A (2014) Commentary: ethical issues of current health-protection policies on low-dose ionizing radiation. Dose-Response 12:342–348CrossRefGoogle Scholar
  44. Suman S, Kallakury BVS, Fornace Jr Albert J, Datta K (2015) Protracted upregulation of leptin and IGF1 is associated with activation of PI3K/Akt and JAK2 pathway in mouse intestine after ionizing radiation exposure. Int J BiolSci 11:274–283CrossRefGoogle Scholar
  45. Tang Y, Gholamin S, Schubert S, Willardson MI, Lee A, Bandopadhayay P, Vue N (2014) Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition. Nat Med 20:732–740CrossRefGoogle Scholar
  46. Tucker B, Lardelli M (2007) A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos. Zebrafish 4:113–116CrossRefGoogle Scholar
  47. UNSCEAR (2010) Summary of low-dose radiation effects on health. UNSCEAR Publication 90. English, Publishing and Library Section, United Nations Office at Vienna, New YorkGoogle Scholar
  48. Veinotte CJ, Dellaire G, Berman JN (2014) Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era. Dis Model Mech 7:745–754CrossRefGoogle Scholar
  49. Wang T, Zhang Y, Xu HM, Jiang JQ, Fan LG (2006) Individual external dose monitoring at QNPC from 1993 to 2005. Radiat Protect Bull 26:38–42 (in Chinese)Google Scholar
  50. Wang T, Xu W, Deng CG, Xua SX, Li FH, Wu YJ, Wu LJ, Bian P (2016) A pivotal role of the jasmonic acid signal pathway in mediating radiation-induced bystander effects in Arabidopsis thaliana. Mutat Res-FundMol M 791-792:1–9CrossRefGoogle Scholar
  51. Wang XZ, Zhi J, Yang HH, Liu YJ (2018) Dibenzoxanthenes induce apoptosis and autophagy in HeLa cells by modeling the PI3K/Akt pathway. J Photochem Photobiol B 187:76–88CrossRefGoogle Scholar
  52. Watkins DN, Berman DM, Burkholder SG, Wang B (2003) Hedgehog signaling within airway epithelial progenitors and in small-cell lung cancer. Nature 422:313–317CrossRefGoogle Scholar
  53. Weichselbaum R, Shafaee Z, Du W (2005) Combination therapy of hedgehog inhibitors, radiation and chemotherapeutic agents. U.S. Patents Application 11(/576):310Google Scholar
  54. Wu G (2008) The calculation method for effective dose of worker personnel in uranium mines. Ura Min Met 27:77–80 (in Chinese)Google Scholar
  55. Yang LL, Wang RJ, Gao YB, Xu XP, Fu KF, Wang SX, Peng RY (2014) The protective role of interleukin-11 against neutron radiation injury in mouse intestines via MEK/ERK and PI3K/Akt dependent pathways. Dig Dis Sci 59:1406–1414CrossRefGoogle Scholar
  56. Yang W, Liu YY, Gao RL, Yu HQ, Sun T (2018) HDAC6 inhibition induces glioma stem cells differentiation and enhances cellular radiation sensitivity through the SHH/Gli1 signaling pathway. Cancer Lett 415:164–176CrossRefGoogle Scholar
  57. Zhou B, Tan PP, Liu SJ, Zhao WP, Wang HW (2018) PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells. Chemosphere 199:297–302CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Weichao Zhao
    • 1
    • 3
  • Nan Hu
    • 2
  • Dexin Ding
    • 2
    Email author
  • Dingxin Long
    • 3
  • Sheng Li
    • 2
  • Guangyue Li
    • 2
  • Hui Zhang
    • 2
  1. 1.School of Environment Protection and Safety EngineeringUniversity of South ChinaHengyangChina
  2. 2.Key Discipline Laboratory for National Defense for Biotechnology in Uranium Mining and HydrometallurgyUniversity of South ChinaHengyangChina
  3. 3.School of Public HealthUniversity of South ChinaHengyangChina

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