T-cell receptor assay and reticulocyte-micronuclei assay as biological dosimeters for ionizing radiation in humans [Elektronische Ressource] / vorgelegt von Stanislav Vershenya

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• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English

Extrait

Aus der Klinik und Poliklinik der Nuklearmedizin
der Universität Würzburg
Direktor: Prof. Dr. med. Chr. Reiners
T-cell receptor assay and reticulocyte-micronuclei assay as
biological dosimeters for ionizing radiation in humans
Inaugural – Disseration
zur Erlangung der Doktorwürde der
Medizinische Fakultät
der
Bayerischen Julius-Maximilians-Universität zu Würzburg
vorgelegt von
Stanislav Vershenya
aus Minsk, Weißrussland
Würzburg, Mai 2007Referent: Prof. Dr. med. Dr. rer. nat. K. Hempel
Koreferent: Prof. Dr. med. Chr. Reiners
Dekan: Prof. Dr. med. M. Frosch
Tag der mündlichen Prüfung: 17. September 2008
Der Promovend ist ArztI dedicate this work to my family and thank them for all their support and love
throughout all my years here, in Wuerzburg UniversityContents:
1. Introduction................................................................................................... 1
2. Method.......................................................................................................... 3
2.1. Thyroid cancer patients and radioiodine treatment (RIT).............................. 3
2.2. Assays.......................................................................................................... 5
2.2.1. Standard T-Cell receptor (TCR) assay...................................................... 5
2.2.2. Assay for latent TCR mutants ................................................................... 5
2.2.2.1. Culture of lymphocytes and detection of latent TCR mutants................ 6
2.2.2.2. Calibration of the assay in in vitro irradiated lymphocytes..................... 7
2.2.3. MN-Tf-Ret assay....................................................................................... 7
2.2.3.1. Isolation of transferrin receptor positive reticulocytes (Tf-Ret)............... 7
2.2.3.2. Fixation of cells...................................................................................... 8
2.2.3.3. Flow cytometric analysis........................................................................ 8
2.3. Irradiation and dosimetry .............................................................................. 9
2.3.1. Irradiation of lymphocytes ......................................................................... 9
2.3.2. Calculation of bone marrow radiation dose in radioiodine therapy............ 9
2.3.2.1. Retrospective dosimetry according to ICRP .......................................... 9
2.3.2.2. Individual dosimetry according to MIRD .............................................. 10
2.3.3. Radiation dose due to diagnostic ............................................................ 12
2.4. Statistics ..................................................................................................... 12
3. Results and Discussion .............................................................................. 13
3.1. Bone marrow radiation dose in RIT ............................................................ 13
3.2. Standard TCR assay .................................................................................. 14
3.2.1. Results (standard TCR assay) ................................................................ 14
3.2.1.1. TCR mutant frequency ( TCR-Mf ) in patients after RIT ..................... 14
3.2.1.2. A model for the description of the course of TCR-Mf after irradiation.. 18
3.2.2. Discussion ( Standard TCR Assay )........................................................ 20
3.2.2.1. Frequency of spontaneous TCR mutants ............................................ 20
3.2.2.2. Decline of TCR-Mf and its correlation with radiation dose ................... 20
3.2.2.3. TCR–Mf as a biological dosimeter....................................................... 223.3. Assay for latent TCR mutants..................................................................... 23
3.3.1. Results (latent TCR mutants).................................................................. 23
3.3.1.1. Lymphocyte culture and detection of latent TCR mutants ................... 23
3.3.1.2. Latent TCR mutants in patients treated with radioiodine ..................... 25
3.3.2. Discussion (latent TCR mutants) ............................................................ 27
3.3.2.1. Shortening of the latent period ............................................................ 27
3.3.2.2. Latent TCR mutants as a biodosimeter for recent exposure ............... 28
3.4. MN-Tf-Ret Assay ........................................................................................ 29
3.4.1. Results (MN-Tf-Ret Assay) ..................................................................... 29
3.4.1.1. Flow cytometric measurements of micronucleated Tf-Ret................... 29
3.4.1.2. Short time memory of MN-Tf-Ret Assay.............................................. 29
3.4.1.3. MN-Tf-Ret mutant frequency during RIT ............................................. 31
3.4.1.4. Red marrow radiation dose and f(MN-Tf-Ret) ..................................... 33
3.4.2. Discussion (MN-Tf-Ret assay) ................................................................ 39
3.4.2.1. Transferrin positive reticulocytes in blood as a part of erythron........... 39
3.4.2.2. Role of lifespan of Tf-Ret in peripheral blood ...................................... 39
3.4.2.3. Radiation dose per cycle and f(MN-Tf-Ret) in blood............................ 40
3.4.2.4. Model describing the time- and dose-dependence of f(MN-Tf-Ret)..... 42
3.4.2.5. Adaptation of the model to MN in polychromatic erythrocytes in mice. 45
3.4.2.6. Features of the MN-Tf-Ret assay ........................................................ 47
3.5. Biological dosimetry in radioiodine treated patients.................................... 48
4. Summary .................................................................................................... 51
5. References ................................................................................................. 54
6. Abbreviations.............................................................................................. 631. Introduction
After the Chernobyl disaster the frequency of thyroid cancer has
dramatically increased in children from Belarus [1-4]. The treatment for thyroid
cancer is surgical removal of the tumour (thyroidectomy) followed by one or more
treatments with radioiodine. In about a quarter of the Belarussian patients
radioiodine therapy is performed at the Clinic for Nuclear Medicine of the University
of Würzburg thanks to the financial support of the association of the German
Electricity Companies (VDEW) and the ‘Medizinische Hilfe für Tschernobyl-Kinder’
Association.
The main risks of radioiodine treatment (RIT) are the induction of lung
fibrosis and secondary cancers as a consequence of the genotoxic effects of the
radioiodine radiation [5]. The frequency of somatic mutations in basic genes of
erythrocytes and lymphocytes has been used as an indicator of the genotoxic
effect of radiation [6-11]. The corresponding assays are 1) hypoxanthine-guanine
phosphoribosyl transferase (HPRT), 2) glycophorin A (GPA), 3) T-cell receptor
(TCR) assays.
In the present investigation the TCR assay was applied to the thyroid cancer
patients to quantify the genotoxic effect of the radioiodine therapy and to detect
those patients who might have an increased cancer risk. The patients were
represented by young patients from Belarus who were undergoing the radioiodine
therapy in the Clinic of Nuclear Medicine. After the irradiation with the
increase in frequency of mutant TCR receptors could be seen after a six month
latent period. In order to shorten this time, for some samples the cell culture of
lymphocytes had been applied before TCR assay was done.
Another method of biological dosimetry used was the reticulocyte-
micronucleus assay. This method is long known and used in mice as a MN assay
in polychromatic erythrocytes [12]. In humans the application of this assay was
problematic because all the aberrant (in our case micronucleated) cells are quickly
removed by the spleen. Therefore, in humans the assay was restricted to
spleenectomized subjects. Recently the flow-cytometric in vivo micronucleus assay
1has been adapted for use in humans [13]. This is done by immunomagnetic
separation of immature transferrin-positive reticulocytes (Tf-Ret) present in the
peripheral blood. The isolated Tf-Ret are then analysed for the presence of
micronuclei. The results of this previous study indicated that micronuclei in human
Tf-Ret could be a sensitive biomarker for chromosome damage.
In order to investigate the utility and sensitivity of this method for the
detection of radiation-induced chromosome damage, we have now performed a
study of MN in Tf-Ret in 46 patients undergoing radioiodine therapy. These
131patients received defined activities of I, precise dosimetry during their
hospitalisation, and sequential determinations of the individual frequencies of MN-
Tf-Ret. It was worth mentioning that this study was a first attempt of using MN-Tf-
Ret assay as a biological dosimeter.
22. Method
2.1. Thyroid cancer patients and radioiodine treatment (RIT)
In most assays blood from thyroid cancer patients was used. Blood was
sampled with informed consent. The thyroid cancer was caused by radioi