Institute for Nuclear Research, Kiev, Ukraine The Radioecology Research Department COMPLEX RADIOBIOLOGICAL INVESTIGATIONS OF SMALL RODENTS FROM THE CHERNOBYL EXCLUSION ZONE Olena Burdo, Alla Lipska, Olena Sova, Natalia Ryabchenko 30 th August 2016, Chernihiv, Ukraine Outline of the report • Setting of the problem • Organization of the study – Place of study – Chernobyl exclusion zone – The object of study – murine rodents – Subject of study : cytogenetic, hematologic, morphophysiological parameters • The results of investigation – Cytogenetic effects – Hematologic effects – Morphophysiological effects – Link between the rate ​​of the radiation factor and cytogenetic effects . Conclusions 2 Setting of the problem • Research of the radiobiological effects is needed under the following conditions : – Low dosed-radiation – Prolonged irradiation (during the whole life cycle) – Combinated exposure (external + internal) – Different types of exposure (α-, β-, γ-) • Creating these conditions in the ordinary radiobiological experiment is difficult enough • We can use “in situ ” – contaminated sites. For example, Chernobyl exclusion zone 3 DESCRIPTION OF TEST SITES IN THE EXCLUSION ZONE Test site № 1 Yaniv γ 550 – 1400 μR/h 137Cs 3.6-20 MBq/m2 90Sr 0.68 – 2.1 MBq/m2 Control Test site γ 0,1 – 0,2 μR/h /h Test site № 2 Dityatki γ 20 – 22 μR/h 137Cs 0.18-0.27 MBq/m2 90Sr 0.013-0.015 MBq/m2 4 Algorithm of research Species: Species: .Clethrionomus •Cytogenetic parameters: (Myodes) glareolus •Mitotic index •Apoptosis •Micronucleus test •Binuclear cells •Premature chromosome condensation (PCC) •Flow cytometry Estimation of doses Radionuclide Contamination in animals organisms 5 Cesium 137 distribution in organs and tissues, % Gastrointestinal tract Heart Other Muscles Spleen Skeleton Skin kidneys Lungs Liver Strontium 90 distribution in organs and tissues, % Muscles Gastrointestinal tract About 90 % of Skin Other Strontium 90 is accumulated exactly in the tissues of skeleton Skeleton Results of micronucleus analysis of bone marrow cells C. glareolus The Frequency of Group Apoptotic cells Mitotic index cells with MN Reference 0,83±0,38 3,66±0,57 0.92±0, group Janiv 2008 *4,51±1,02 *6,13±0,63 1,31±0,44 Janiv 2009 1,86±1,04 4,20±0,56 1,83±0,67 Janiv 2012 0,64±0,16 *8,67±1,35 1,31±0,17 Janiv 2013 0,32±0,12 3,20±0,79 0,76±0,23 * - p <0,05 The level of Bone marrow cells with MN in relation to the reference group The relative amount cells with NM in the bone marrow cells 2,5 2 1,5 1 0,5 0 Reference group Janiv 2008 Janiv 2009 Janiv 2012 Janiv 2013 Janiv 2015 The level of binucleated cells in relation to the reference group The relative amount of binucleated cells ** 8 7 * 6 5 4 3 2 1 0 Reference Janiv 2008 Janiv 2009 Janiv 2012 Janiv 2013 Janiv 2015 group The level of cells with PCC relatively to the reference group The relative amount of cells with PСС 1,8 1,6 1,4 1,2 * 1 0,8 0,6 0,4 0,2 0 Reference group Janiv 2008 Janiv 2009 Janiv 2012 Janiv 2013 Janiv 2015 Distribution of cells by micronucleus number in C.glareolus 100 90 80 70 60 Reference group 50 40 Janiv 2008 30 Janiv 2009 20 Janiv 2012 10 Janiv 2013 0 1 MN 1 Janiv 2015 3 MN 3 5 MN 5 8 MN 8 Janiv2015 Janiv2013 Janiv2012 Janiv2009 Janiv2008 Referencegroup Results of micronucleus analysis of bone marrow cells C. glareolus 2015 Group of PCE with MN Cytotoxicity Apoptosis animals index Reference group 3,88±0,36 1,39±0,05 3,20±0,58 Dityatki *7,72±1,02 *4,27±0,48 2,25±0,36 Janiv *15,57±1,50 *3,90±0,66 *7,60±1,29 * - p <0,05 Micronuclei (MN) in polychromatophilic erythrocytes (PCE) of bone marrow cells Cells with MN 20 * 15 ‰ 10 * * - p <0,05 5 0 Reference group Dityatki Janiv 2015 year Cytotoxicity index * 5 * 4 3 ‰ 2 1 0 Reference group Dityatki Janiv 2015 year The level of apoptotic cells in voles bone marrow cells Apoptosis 10 * 8 6 ‰ 4 2 0 Reference group Dityatki Janiv 2015 year TheThe numberresults of bone of marrowresearch cells 14 * 12 10 * 9 8 10* 6 4 2 0 reference group Janiv2012 Janiv2013 Janiv2015 Group of animals/year The number of leukocytes The number of red blood cells 3 12 * 2,5 * 10 2 8 9 12 1,5 6 10* 10* * 1 4 0,5 2 0 0 reference group Janiv2012 Janiv2013 Janiv2015 reference group Janiv2012 Janiv2013 Janiv2015 Group of animals/year Group of animals/year * - p <0,05 The results of research The content of difference forms of leukocytes in peripheral blood of bank voles Stab Segmented Group Eosinophils Monocytes Basophils Lymphocytes neutrophils neutrophils Reference 4.17±0.65 20.67±2.44 4.00±0.58 2.50±0.43 0±0 68.33±3.40 group Janiv 2013 2.75±1.55 14.00±3.89 7.00±4.08 0±0 0.25±0.25 75.05±9.29 Janiv 2015 3.80±0.86 *14.80±1.56 6.20±0.97 *0.60±0.40 0±0 74.40±1.89 Segmented Group Stab neutropils Eosinophils Monocytes Basophils Lymphocytes neutrophils Reference 4.17±0.65 20.67±2.44 4.00±0.58 2.50±0.43 0±0 68.33±3.40 group Ditytatki 4,00±0,82 *14,75±1,80 5,25±1,10 *1,25±0,49 1,00±0,21 74,00±4,60 2013 Ditytatki 4,70±0,98 16,90±1,03 3,00±0,58 *0,10±0,10 1,00±0,21 73,90±1,83 2015 Changes in blood system of chronically exposed bank voles from the Chernobyl exclusion zone abnormal mitosis in fat inclusions hematopoiesis bone marrow formed in foci formed in spleen spleen immature erythrocytes in peripheral blood Group of The indices of weight, mg/g animals Liver Thymus Spleen Heart Kidney Adrenal Reference 66,01±4,83 0,681±0,038 3,92±0,57 6,27±0,12 13,74±0,39 0,56±0,088 group Janiv 2012 57,60±4,60 4,84±0,97 Janiv 2013 57,00±7,26 3,05±0,17 2,51±0,63 6,87±0,71 11,23±1,00 0,35±0,04 Janiv 2015 72,90±6,53 1,99±0,33 *1,81±0,08 6,09±0,20 *11,49±0,40 *0,26±0,03 The indices of weight, mg/g Group of animals Liver Thymus Spleen Heart Kidney Adrenal Reference 66,01±4,83 0,681±0,038 3,92±0,57 6,27±0,12 13,74±0,39 0,56±0,088 group Dityatki 2012 63,59±4,94 *2,55±0,28 Dityatki 2013 59,78±2,34 3,56±1,22 3,84±0,36 *5,54±0,15 *11,84±1,15 *0,23±0,009 Dityatki 2015 *55,63±2,58 1,19±0,12 3,91±0,74 6,42±0,25 *11,04±0,29 0,79±0,32 140 120 100 80 Janiv 2012 % Janiv 2013 60 Janiv 2015 40 20 0 Liver Spleen Heart Kidney Adrenal 160 140 120 100 Dityatki 2012 % 80 Dityatki 2013 60 Dityatki 2015 40 20 0 Liver Spleen Heart Kidney Adrenal Conclusion • Levels of Cs-137 and Sr-90 accumulation in the body of rodents from the Chernobyl exclusion zone were estimated; their high lability during the time after the accident and inter-individual were found. Radiation doses and structure of radiation loads on small rodents from contaminated sites were determined. • Qualitative changes in hematopoiesis of animals from the Chernobyl exclusion zone manifested in the disturbance of bone marrow cell maturation. Malfunction of hematopoiesis foci formation in liver and spleen was also found. Major changes were registered in erythroid lineage. High levels of genotoxic and cytotoxic effects in bone marrow was observed. • Differences between the reference and the exposed groups were found by the study of morphological parameters of voles internal organs, confirming the activation of the physiological stress in the exposed animals. Thank you for your attention ! 22.