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FORMATION OF MICRONUCLEI IN HUMAN LYMPHOCYTES AFTER PARTIAL AND WHOLE BODY IRRADIATION

Objective

ONE OF THE PROBLEM ASSOCIATED WITH ACCIDENTS INVOLVING EXPOSURE TO IONIZING RADIATION REFERS TO A FAST, RELIABLE, AND SENSITIVE MONITORING OF THE EXPOSURE DOSES RECEIVED BY EACH INDIVIDUAL.
USUALLY, THE FORMATION OF DICENTRICS IN LYMPHOCYTES IS USED AS A BIOLOGICAL SYSTEM OF DOSIMETRY. HOWEVER, EVALUATION OF THIS PARAMETER IS TIME-CONSUMING AND NEEDS SKILFUL AND WELL TRAINED PERSONNEL.
EVALUATION OF MICRONUCLEI IN LYMPHOCYTES IS MUCH EASIER AND THE SOMEWHAT LOWER SENSITIVITY OF THIS TEST IS COMPENSATED FOR BY THE HIGHER NUMBERS OF CELLS THAT CAN BE SCORED. WITH FEW EXCEPTIONS, MOST STUDIES AIMED AT THE DETERMINATION OF RADIATION-INDUCED MICRONUCLEUS FORMATION IN LYMPHOCYTES HAVE BEEN CARRIED OUT BY IRRADIATING LYMPHOCYTES IN VITRO. IN ESSEN, THERE IS THE UNIQUE POSSIBILITY OF STUDYING MICRONUCLEUS FORMATION IN HUMAN LYMPHOCYTES AFTER PARTIAL AND WHOLE BODY IRRADIATION.
Methods have been developed using the micronucleus technique to determine radiation dose in accidentally exposed persons. Micronucleus expression is dependent on the number of mitoses carried out after radiation exposure, thus the fraction of lymphocytes that has divided must be determined. The method of Fenech and Morley was employed, in which cytochalasin B was used to prevent cytokinesis but not karyokinesis, and those lymphocytes which complete a mitosis show 2 cell nuclei per cell. The proliferation of lymphocytes in vitro after stimulation by phytohemagglutinin (PHA) showed considerable variation between individuals in terms of the fraction of proliferating cells and for the start of proliferation. Thus, continuous monitoring is necessary in order that cytochalasin B is added and cells harvested at optimal times. The spontaneous frequency of micronuclei was small in patients and not significantly different from health donors. Evaluation has been made of radiation induced micronuclei in lymphocytes of healthy donors exposed in vitro and in lymphocytes of patients exposed either in vitro or in vivo. Dose response equations have been determined and the quadratic coefficient was higher for health donors, due to a lower response of lymphocytes of patients after 5 Gy. No differences were detected for doses lower than 5 Gy. Binucleated lymphocytes were not obtained after exposure greater than 2.5 Gy in vivo, possibly resulting from heavy medication accompanying whole body irradiation. No differences were observed between in vitro and in vivo exposure after 1.25 Gy.
BLOOD WILL BE OBTAINED FROM CAREFULLY SELECTED PATIENTS WHO RECEIVE PARTIAL OR WHOLE BODY IRRADIATION. IN THE CASE OF WHOLE BODY EXPOSURE, PATIENTS (MAINLY WITH LEUKEMIA) RECEIVE 4 FRACTIONS WITH 2.5 GY (TOTAL DOSE 10 GY) ON 4 SUCCESSIVE DAYS. BLOOD WILL BE AVAILABLE BEFORE THE FIRST EXPOSURE, BETWEEN THE FRACTIONS, AND AFTER THE LAST EXPOSURE. IN THE CASE OF PARTIAL BODY IRRADIATION, PATIENTS WILL BE STUDIED WHO RECEIVE HALF BODY IRRADIATION (UPPER HALF) WITH ONE SINGLE DOSE OF 8.0 GY (CF. PATIENTS WITH BRONCHIAL CARCINOMA) OR WHO RECEIVE IRRADIATION OF LARGE VOLUMES OF BONE MARROW OR LYMPHATIC TISSUES (CF. PATIENTS WITH MORBUS HODGKIN).
IN THE LATTER CASES, FRACTIONATED IRRADIATION (5 X 2.0 GY PER WEEK, TOTAL DOSE 36 GY) IS USED. BLOOD WILL BE AVAILABLE BEFORE, DURING AND AFTER RADIOTHERAPY.

MICRONUCLEI WILL BE DETERMINED IN STIMULATED LYMPHOCYTES. IF NECESSARY, SUB-POPULATIONS OF LYMPHOCYTES WILL BE CULTURED SEPARATELY. THE INTERPHASES AFTER ONE OR TWO POST-RADIATION MITOSES WILL BE USED FOR ANALYSIS. THE CORRESPONDING METAPHASES WILL ALSO BE PREPARED FOR STUDIES OF CHROMOSOMAL ABERRATIONS IN ORDER TO COMPARE THE NUMBER OF MICRONUCLEI WITH THE NUMBER OF CHROMOSOME ABERRATIONS IN THE SAME CELL POPULATION. RADIATION INDUCED MICRONUCLEI CAN BE FORMED FROM ACENTRIC CHROMOSOME FRAGMENTS OR FROM COMPLETE CHROMOSOMES WITH A CENTROMERE. THESE TWO POSSIBILITIES CAN BE DISTINGUISHED WITH THE AID OF CENTROMERE-SPECIFIC ANTIBODIES. SUCH AN ANALYSIS WILL BE CARRIED OUT.

TO AVOID WRONG CONCLUSIONS, IT IS NECESSARY TO OBTAIN INFORMATION ON THE CELL PROLIFERATION OF STIMULATED LYMPHOCYTES. THIS WILL BE DONE USING TWO PARAMETER ANALYSIS OF FLOW CYTOMETRIC DATA. THE DNA CONTENT WILL BE DETERMINED AFTER STAINING WITH A FLUORESCENT DYE, AND THE EXTENT OF DNA-SYNTHESIS WILL BE MEASURED AFTER INCORPORATION OF 5-BUDR WHICH IS THEN DETECTED WITH A SPECIFIC ANTIBODY. FROM THESE TWO PARAMETERS THE DISTRIBUTION OF CELLS IN THE CELL CYCLE WILL BE OBTAINED AS WILL THE NUMBER OF POST IRRADIATION MITOSES WHICH THE CELLS HAVE GONE THROUGH.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Universitätsklinikum Essen
Address
Hufelandstraße 55
45147 Essen
Germany