Are thyroid cancer patients sensitive to ionising radiation?
ABSTRACT To determine the ionising radiation sensitivity of peripheral blood lymphocytes in a group of differentiated thyroid cancer (DTC) patients.
A total of 53 thyroid cancer patients (26 women and 27 men) and 50 donors (23 women and 27 men) were included in the study. The cytokinesis-block micronucleus assay (CBMN) in G0 peripheral blood lymphocytes was carried out using the cytochalasin B technique. Four cultures were established per each donor, two were irradiated with 0.5 Gy 137Cs g-rays, while the other two remained untreated.
No significant differences were observed in the frequency of binucleated cells with micronuclei (BNMN) between patients and controls, for both spontaneous and after the irradiation frequencies. Nevertheless, a positive and significant correlation was found between the frequencies of both spontaneous and after irradiation DNA damage, for control and patient groups.
We have found that DTC patients do not present particular sensitivity to ionising radiation when an in vitro treatment is performed in G0 stage of the cell cycle, but this result does not discard the hypothesis about an increased sensitivity in other stages of the cell cycle in DTC patients.
- SourceAvailable from: Hannu Norppa[show abstract] [hide abstract]
ABSTRACT: At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.Environmental and Molecular Mutagenesis 02/2000; 35(3):167-72. · 3.71 Impact Factor
Article: The in vitro micronucleus technique.[show abstract] [hide abstract]
ABSTRACT: The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/2000; 455(1-2):81-95. · 3.90 Impact Factor
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ABSTRACT: The micronucleus (MN) assay in peripheral blood lymphocytes was applied to assess the genotoxic potential of a single dose of iodine-131 (131I) given to six patients for ablation of thyroid remnants after total thyroidectomy. Lymphocytes were taken at various times after 131I therapy (from 2 to 180 days), and evaluated for the presence of MN in the binucleated cells identified after blocking cytokynesis with cytochalasin B. The presence of ultrafiltered, low-molecular weight, clastogenic factor(s) (CFs) in the plasma of 11 patient undergoing 131I therapy was also sequentially assessed.A significantly increased MN frequency was observed in lymphocytes of patients as soon as the first sampling time (2 days after 131I therapy), multifactor analysis of variance (MANOVA): P<0.0001, peaking at day 7 at almost four-fold the spontaneous frequency observed in the pre-therapy samples. MN frequency slowly declined thereafter, reaching the baseline levels at the 180-day time point. When tested against peripheral blood lymphocytes from a healthy donor, the ultrafiltered CFs obtained from 11 patient's plasma induced a significant increase of the MN frequency peaking at day 15. Thereafter, a slow MN frequency decline was observed and the baseline frequency was reached after 180 days. A significant relationship was found between the MN frequency observed in lymphocytes of patients after 131I therapy and the genotoxic CFs activity present in their plasma (P=0.019). These findings suggest that 131I induces a significant increase in the MN frequency of peripheral blood lymphocytes, as well as the formation of transferable CFs which persist for at least 60 days after administration of the radionuclide. The presence of these CFs might be responsible of chromosome aberrations often observed in cultured lymphocytes following X-ray exposure. The possibility of reducing the genotoxic activity of radionuclide therapy by chemoprevention of CFs with antioxidant drugs remains to be explored.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/2002; 514(1-2):77-85. · 3.90 Impact Factor