Transient cytotoxicity of ¹³¹I beta radiation in hyperthyroid patients treated with radioactive iodine

Material & Methods

Samples: The study included a total of 32 hyperthyroid patients (M: F = 14: 18), age range between 23-61 yr, mean 49 ± 7 yr who were under evaluation in Endocrinology department of Amrita Institute of Medical Sciences, Cochin, Kerala. Clinically hyperthyroid patients were sent to Nuclear medicine department of the same Institute where a Technetium thyroid scan was performed. Patients were selected consecutively from June 2007 to December 2008. Based on the thyroid scan findings of Graves′ disease, toxic multinodular goiter and autonomous toxic nodule empirical ¹³¹I dosage was decided as per the standard treatment protocol9. All patients were clinically and biochemically evaluated prior to Technetium thyroid scan by the endocrinologist. Basic blood tests along with serum free T4, T3 and TSH estimations were performed on their initial presentation to the endocrinologist. Control groups i.e., volunteers (who came for routine health checkup) (20 in number) were enrolled for this study. All of them were clinically normal with normal thyroid function tests and Technetium thyroid scan. They were not administered ¹³¹I and their blood samples were kept as control. All subjects were informed of the objective of the study and gave their consent. The institutional ethical committee approved the research procedures used in this study.

Inclusion criteria: Clinical, biochemical and scintigraphic confirmation of hyperthyroidism as Graves’ disease, toxic multinodular goiter and autonomous toxic nodule. Scintigraphic confirmation was done by Technetium thyroid scintigraphy in patients who were off antithyroid drugs for atleast five preceding days.

Exclusion criteria: (i) Scintigraphic diagnosis of subacute viral thyroiditis, (ii) pregnant women and those breastfeeding who could not wean off baby for eight weeks post-treatment with radioiodine, (iii) patients with free T4 levels > 5 ng/ml at the time of treatment were excluded as ¹³¹I administration may lead to a medical crises called thyroid storm with excess release of preformed hormones into circulation and (iv) patients with Graves’ ophthalmopathy.

The treatment consisted of ¹³¹I administration orally as an adjuvant radiation dose to destroy thyroid follicular cells. All patients recruited for this study were explained the details of the treatment, radiation safety measures to be followed for 10 days after the treatment. They were also instructed to stop iodised salt, seafoods and iodide containing medications atleast 3 weeks prior to this treatment. Antithyroid medications like carbimazole was stopped a week prior to the date of therapy.

Based on the trapping function of thyroid gland established by Technetium scan, patients were divided for empirical ¹³¹I dosage into 185 – 370 MBq, 370 – 555 MBq and above 555 MBq of ¹³¹I. Twenty five patients with Graves′ disease (GD), received a dose of 185 –370 MBq, five patients of toxic multinodular goiter (TMG) received between 370 – 555 MBq and remaining two patients of autonomous thyroid nodule (ATN) were administered more than 555 MBq of ¹³¹I therapy. Five ml of heparinised blood were collected from all these patients just before administering ¹³¹I low dose. Subsequent blood samples were collected from each patient one week and 30 days after the therapy to look for DNA changes.

Chromosomal analysis: RPMI1640 (Gibco, USA) foetal calf serum (Gibco) phytohaemagglutinin – M (Gibco) dimethyl sulphoxide 5 μl /ml (E.Merk, India) colchicines 0.20 μg/ml (Micro lab) and cytochalasin – B (Sigma, USA) were used for this analysis.

Peripheral blood cultures were prepared1011. Blood sample (0.5 ml) was inoculated under aseptic conditions into a culture vial containing 5.0 ml of culture medium, 1.0 ml of AB serum (Antibodies Inc. Washington, DC) and 0.2 ml of phytohaemagglutinin. The cultures were incubated at 37°C for 72 h. The dividing cells were arrested at the metaphase stage by adding 0.05 ml of colchicines solution (0.01%) 30 min before harvesting the culture. The contents of the vials were centrifuged at 200 g for 10 min at the end of colchicine treatment. The supernatant was discarded and 6 ml of hypotonic solution was added to the test tube after disturbing the cell button. The contents of the test tubes were incubated for 7 min and freshly prepared fixative (Methanol: glacial acetic acid 3:1v/v) was added and centrifuged at 200 g for 10 min. Later the supernatant was discarded and two or three changes of fixative were given to obtain colourless cell pellet. The slides bearing chromosome spreads were treated with 0.25 per cent trypsin for 3 to 10 sec and were stained in 4 per cent buffered Giemsa solution for 3 min. At least 100 metaphases were examined for the occurrence of different types of abnormality i.e., gaps, fragments, breaks, etc. Criteria to classify the different types of aberrations were in accordance with the recommendation of Environmental Health Criteria 46 for environmental monitory of human population12.

Micronucleus analysis: The lymphocytes were cultured according to the method of Fenech and Morley13. Pokeweed mitogen (Gibco BRL, USA) was used to stimulate the lymphocytes to proliferate in culture. A solution of cytochalasin B (Aldrich Chemical Co., India) was added 44 h after the commencement of the culture. The cultures were terminated 72 h after initiation. Scoring of MN was limited to bi-nucleated lymphocytes with preserved cytoplasm according to the criteria proposed by Countryman and Heddle14. The following are the criteria used for scoring the cell inclusions: (i) only cells with a distinct cytoplasm and distinct binucleation were analyzed for the presence of MN, (ii) only MN entirely inside the cytoplasm, separated from the main nucleus and with a diameter <1/3 of the size of the main nucleus were scored, and (iii) only MN staining the same as or lighter than the nucleus were scored. The suggested criteria for identifying MN were; (i) rounded smooth perimeter suggestive of a membrane; (ii) less than a third the diameter of the associated nucleus, but large enough to discern shape and colour; (iii) Feulgen positive, i.e., pink in bright field illumination; (iv) staining intensity similar to that of the nucleus; (v) texture similar to that of nucleus; (vi) same focal plane as nucleus; and (vii) absence of overlap with, or bridge to, the nucleus. The results are expressed as the average percentage of micro nucleated cells per binucleated cells.

Metaphases were scored for CAs and included chromatid gaps, isochromatid gaps, breaks, fragments, etc. Gaps are true discontinuities of the chromosome structure15 and in this study metaphases were studied with and without gaps (Figs 1–3).

Close

Fig. 1

Binucleate cell with no micronuclei in a patient of Graves’ disease treated with 10 millicurie of ¹³¹I.

Export to PPT

Close

Fig. 2

Micronucleus in a binucleate cell of a patient with toxic multinodular goitre treated with more than 10 millicurie of ¹³¹I.

Export to PPT

Close

Fig. 3

Chromosomal aberrations seen in a patient with autonomous toxic nodule treated with 15 millicurie of ¹³¹I.

Export to PPT

Statistical analysis: Various statistical methods (Student’s t-test, Mann–Whitney test, Kolmogorov–Smirnov test and Pearson′s rank correlation analysis) were used depending on the nature of the data and the type of analysis needed. SPSS 11.01 statistical software (SPSS Inc., Chicago, IL) was used for the calculation of the results obtained.

The association of the frequency of micronuclei with the age and sex of subjects was tested with Pearson’s rank correlation analysis.

Results

In our study, female hyperthyroid patients were able to tolerate symptoms and signs for 1 to 2 years when compared to males, who had lower tolerance levels of around 6 months to 1 year. The predominant symptoms were weight loss followed by increased appetite, tremors, increased sweating, palpitations and mental disturbances.

In cytogenetic study, there was a moderate increase in chromosomal aberrations (CA) in day 7 post ¹³¹I therapy blood samples when compared to the pretreatment samples. A significant decrease of the same was observed in the day 30 post ¹³¹I therapy samples (Table I). Lymphocytes of control groups showed fewer MN and CA when compared to sample I (185-370 MBq).

In MN analysis, a moderate increase in micronuclei was observed in patients treated with higher dose of ¹³¹I. Similarly there was a transient increase in the number of MN (Table II). in day 7 compared to day 30 post-therapy samples. Patients receiving higher dosage of ¹³¹I showed both MN and abnormal metaphases in their blood samples. However there was no clear correlation between the micronuclei frequency and the therapeutic ¹³¹I dose used. In addition, age and sex did not show any influence on micronuclei frequency in either patients or control population (r=0.04, P>0.05).

Both MN and abnormal metaphases seen in hyperthyroid patients treated with ¹³¹I was insignificant when compared to control group.

Discussion

The results revealed that low dose ¹³¹I therapy induce chromosomal abnormalities in the form of increased CA and MN as early as 7 days after treatment. Similar observations on chromosomal abnormalities and structural aberrations in ¹³¹I hyperthyroid subjects have been reported earlier16. Ramirez et al17 reported a 1.8 fold increase in the frequency of 17p breaks after iodine-131 therapy in hyperthyroid patients. There is a minimal cytogenetic damage in the peripheral blood lymphocytes of ¹³¹I treated hyperthyroid patient in comparison with controls in their study. Similar results were observed by Guitizeez et al18.

Micronuclei are small nuclei like particles seen apart from the nucleus in an irradiated cell. These enclose acentric fragments or whole chromosomes that have not been included in the main nuclei at cell division and in the cytoplasm19. Therefore, the presence of micronuclei can indirectly denote chromosome breakage or impairment of the mitotic spindle. It has been shown that ionizing radiation induces micronuclei in human lymphocytes and, in spite of the variability of dose-response relations; there is a quantitative relationship between radiation dose and frequency of micronuclei, which can be used for biological dosimetry. The sensitivity of cytokinesis blocked micronuclei assay is well established20.

In our study, it was observed that the increase in micronuclei after therapy, was not statistically significant which is explained by the fact that the amount of radiation induced to cells may be minimal. Watanebe et al21 in their study reported an increase in MN after ¹³¹I therapy and they found that number of MN peaked at 3 days after ¹³¹I administration and gradually decreased.

Radiation induced ionization may act directly on the cellular component molecules indirectly or water molecules causing formation of water derived radicals. Radicals react with nearby molecules in a very short time resulting in breakage of chemical bonds or oxidation of the affected molecules. The DNA is a major target for radiation damages because DNA distraction can kill or mutate human cells. Double strand DNA breaks may play a major role in these biological effects822. The relatively low frequency of chromosomal aberrations and MN induced by ¹³¹I in vivo supports the contention that cytogenetic damage of this therapy in hyperthyroid patients was minimal transient and reversible.

To conclude, human peripheral blood lymphocytes are sensitive to ionizing effects of radiation, and low dose ¹³¹I therapy induces transient chromosomal abnormalities in the form of increased CA and MN in patients with hyperthyroidism as early as 7 days after treatment, which (P>0.5) reduces 30 days following therapy. These patients can be motivated to undertake this safe and easy procedure as a first line of therapy in the treatment of hyperthyroidism.