Clinical Investigations
Impaired DNA Repair as Assessed by the “Comet” Assay in Patients with Thyroid Tumors After a History of Radiation Therapy: A Preliminary Study

Part of the manuscript was presented at the 10th International Congress of Endocrinology, June 12–15, 1996, San Francisco, CA.
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Abstract

Purpose: Patients with a history of head and neck irradiation in childhood are at risk to develop thyroid tumors. The aim of this study was to determine if an impairement of DNA strand breaks repair could account for this observation.

Methods and Materials: Circulating unstimulated lymphocytes of a group of 13 patients who developed thyroid tumors after radiotherapy were submitted to the alkaline single-cell gel electrophoresis assay (SCGE or “comet” assay) after in vitro exposure to 2 and 5 Gy of γ-rays. A control group of 8 healthy donors and 2 cases with a history of neck irradiation who did not develop a thyroid tumor were also analysed. The immediate response was compared to that observed after 15, 30, and 60 min of postexposure incubation periods.

Results: Induction of DNA strand breaks is a dose–dependent process. The SCGE assay parameters did not differ significantly between patients and controls immediately (t = 0) after irradiation at the two doses used. As compared to healthy donors, a slower kinetics of repair was found in the patients. The proportion of residual damage at 60 min postirradiation was significantly (p < 0.01) higher in patients than in controls, at both doses analysed. Flow cytometric analysis of apoptosis and p53 protein status studied before and after irradiation showed no apparent relationship with the repair capacity.

Conclusion: This preliminary study suggests that a subgroup of patients who develop thyroid tumors after a history of irradiation are partially defective in the late restitution of in vitro radiation-induced DNA strand breaks. This deficiency could be a predisposing factor to radiation-associated thyroid tumorigenesis. Detection of susceptible individuals using the simple and rapid comet assay, especially children receiving radiotherapeutic treatment, may allow a preventive surveillance for radiation-associated epithelial thyroid tumor development.

Introduction

Several epidemiological studies have provided extensive evidence for a positive correlation between thyroid tumors and an accidental or therapeutic exposure to ionizing radiation 1, 2, 3, 4, 5, 6. It is estimated that the excess relative risk of developing a thyroid cancer following a radiation dose of 1 Gy to the thyroid in childhood is equal to 7.7 [5]. Compared to the general population, a 53-fold increased risk of secondary thyroid cancer is suggested after therapeutic irradiation at a young age [6]. The specific sensitivity to radiation of the thyroid is stressed by the fact that no effect of chemotherapy was found on the risk of thyroid tumor production [6].

Activation of oncogenes and inactivation of tumor suppressor genes by mutations provide unscheduled growth signals that are involved in tumorigenesis (for a review, see [7]). Alterations of such genes in benign or malignant human thyroid tumors have been described [8]. For instance, ras or gsp point mutations were found in both spontaneous and radiation-induced thyroid tumors 9, 10. This suggested that mutations in these genes may be involved in thyroid tumorigenesis independently of previous radiation exposure. On the contrary, activation of the ret oncogene is strongly associated with the papillary type of thyroid carcinomas predominantly observed after irradiation [11]. A high frequency of ret rearrangements, that activate the gene, has been reported in thyroid tumors occurring in children contaminated as a consequence of the Chernobyl accident 12, 13. This type of genomic alteration is likely to be produced as a consequence of an abnormal repair of the DNA strand breaks induced by radiation. The relevance of such mechanisms in predisposition to cancer development is suggested by the high cancer proneness observed in diseases associated with defective processing of DNA lesions, such as xeroderma pigmentosum [14], ataxia telangiectasia [15], or familial nonpolyposis colon cancer [16].

Consequently, it was of interest to investigate if alterations in DNA repair could play a role in the occurrence of radiation-associated thyroid tumors in patients with a history of irradiation. In this preliminary study, we examined, using the single cell gel electrophoresis (SCGE) assay, the capability of restitution of in vitro radioinduced DNA strand breaks of peripheral blood unstimulated lymphocytes (G0 cells) from 13 patients in comparison to those of 8 healthy donors.

Section snippets

Patients and Blood Collection

Among thyroid patients followed at the Institut Gustave Roussy (Villejuif, France), we examined 13 non smoking patients irradiated during childhood, who developed thyroid tumors (Table 1 ). The highest cumulative estimated dose to the thyroid was 48 Gy. Eight of the cases with radiation-associated thyroid tumor also received chemotherapy for treatment of their primary tumor. Eight age- and gender-matched healthy donors and 2 patients who were treated with neck irradiation for a primary

SCGE Assay

Clinical characteristics of patients who developed thyroid tumors after a history of radiotherapy (13 cases) and of individuals with no secondary tumor despite previous neck irradiation (2 cases) are provided in Table 1.

SCGE assay detected DNA damage in peripheral blood lymphocytes of all subjects examined immediately after irradiation at 2 Gy or 5 Gy. The magnitude of this damage is evaluated in individual cells by the increase in the tail moment immediately after irradiation (t = 0). The

Discussion

The sensitivity of the thyroid gland to the carcinogenic effects of ionizing radiations is well documented 1, 2, 3, 4, 5, 6. The influence of dose level, age, and gender has been emphasized 5, 6. Little is known about other individual risk factors predisposing to thyroid tumor development after radiation exposure, although familial susceptibility has been reported [22]. In the present study, we used the SCGE test to determine if a defect in DNA strand breaks rejoining could be associated to the

Acknowledgements

This work, done in UMR 218 CNRS, Institut Curie-Recherche, was supported by grants of Association pour la Recherche sur le Cancer (H. G. S., A. S., and E. M.), Electricité de France (H. G. S., M. S. and F. R.), Commissariat à l’Energie Atomique (CEA) (E. M.), Ligue Nationale Française contre le Cancer (E. M.), and Fondation pour la Recherche Médicale (F. L.).

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