ABSTRACT. In this experimental study, investigators explored p53 tumor suppressor gene mutation induced by low and high doses of iodine-131 sodium iodide ...
Advances in Therapy®
Volume 23 No. 3 May/June 2006
Evaluation of the p53 Tumor Suppressor Gene Mutation in Normal Rat Salivary Gland Tissue After Radioiodine Application: An Experimental Study Bulent Turgut, MD, Assistant Professor Department of Nuclear Medicine
Ozturk Ozdemir, PhD, Associate Professor Department of Medical Genetics
Taner Erselcan, MD, Associate Professor Department of Nuclear Medicine Cumhuriyet University School of Medicine Sivas, Turkey
ABSTRACT In this experimental study, investigators explored p53 tumor suppressor gene mutation induced by low and high doses of iodine-131 sodium iodide (I-131) in salivary gland tissue in rats. Group 1 consisted of 10 rats; low and high I-131 doses were applied at a 1-wk interval. First, low doses of I-131 were injected. (The net injected dose was 47.5±9.2 µCi.) After 1 wk, high doses of I-131 were also injected. (The net injected dose was 1007.2±53 µCi.) Group 2 consisted of 5 rats, and only a low I-131 dose was applied. (The net injected dose was 52.7±5.5 µCi.) The Control Group consisted of 5 rats that did not receive I-131. Thyroidal I-131 uptakes were calculated for Groups 1 and 2 with the use of a gamma camera after 24 h of injections. Immediately after uptake was calculated, salivary glands were resected in all groups and DNA was extracted for genotyping. Genomic DNA of the p53 gene exon 5 was examined by polymerase chain reaction single-strand conformational polymorphism. In Group 1, thyroidal I-131 uptakes were calculated as 12.45%±4.14% and 9.66%±6.73% after low-dose and high-dose I-131 applications, respectively. In Group 2, thyroidal I-131 uptake was calculated as 13.12%±3.04%. In Group 1, p53 gene abnormality was seen in the salivary gland of only 1 of the rats. Double- and single-strand gene profiles showed that both alleles of this rat have a mutated
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Address reprint requests to Bulent Turgut, MD, Assistant Professor Department of Nuclear Medicine Cumhuriyet University School of Medicine P.K. 848, Campus 58140 Sivas, Turkey
single-strand conformational polymorphism profile of point mutation in the p53 gene exon 5. This rat received the highest low dose and the second highest total dose of I-131; its thyroidal uptakes were the second highest. In the other rats in Group 1, and in Group 2 and the Control Group, p53 gene abnormalities were not observed. In Groups 1 and 2, a significant relationship could not be discerned between thyroidal uptake of I-131 and p53 gene mutation in the salivary gland. No significant relationship was observed between thyroidal uptake alterations and p53 gene mutations in salivary glands in Group 1. A point mutation in the p53 gene exon 5 that was seen in only 1 of the rats in Group 1 seems related to the high-dose application of I-131, although coincidental occurrences could not be excluded. We believe that this topic is open to additional in vivo studies.
Keywords: iodine-131; salivary gland; rat; p53 gene; gene mutation INTRODUCTION In nuclear medicine, iodine-131 sodium iodide (I-131) has been used for years (1) to ablate thyroid tissue in well-differentiated thyroid carcinoma, (2) to treat patients with metastases or hyperthyroidism, and (3) to calculate iodine uptake and perform functional and morphologic imaging of the thyroid gland. It is well known that reversible and irreversible radiation damage occurs in thyroid tissue after consecutive therapeutic applications of radioiodine.1 After irradiation, 2 mechanisms of cell death have been defined: apoptosis and necrosis.2,3 Application of high-dose I-131 to thyroid tissue leads to tissue damage by necrosis; however, not enough evidence has been gathered on the role of apoptosis on the same occasion. Radioiodine therapy for differentiated thyroid carcinoma is known to be associated with several adverse effects in the salivary gland. Because the salivary gland is a highly radiosensitive organ, painful salivary gland swelling/radiation sialadenitis and parenchymal damage/functional impairment are well-recognized adverse effects after high-dose I-131 application for thyroid carcinoma.4-6 Although exposure to gamma radiation is the only well-established risk factor for salivary gland and thyroid gland cancers, the exact mechanisms remain unknown.6,7 On the other hand, it remains a question whether I-131 application (or thyroidal I-131 uptake) affects nearby salivary gland tissue in the development of “chromosome mutation effect.” Furthermore, some reports have described salivary gland cancer that developed after I-131 therapy for thyroid carcinoma.8,9 The p53 tumor suppressor (TS) gene is the mutated gene that is most frequently seen in human malignancies.10,11 The p53 protein is a key element in the control of genomic stability,12 and it has a role in controlling cellular proliferation and differentiation. After exposure to ultraviolet, gamma, and x-ray radiation, nuclear accumulation of p53 protein is induced in response to genotoxic stress. A high prevalence of mutation of the p53 gene has been seen in human undifferentiated and anaplastic thyroid tumors formed post irradiation.10 Although most examples are related to dietary and environmental sources and to occupational carcinogens,13 only a few papers have focused specifically on p53 mutations in tumors induced by radionuclide therapy.14
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The present study was undertaken in a rat model to investigate whether p53 gene mutation occurs after I-131 application in tissue that is near the thyroid gland, such as the submaxillary salivary gland. Investigators developed an in vivo rat model that simulated the application of diagnostic (low) and therapeutic (high) doses of I-131 as it is used in nuclear medicine practice. Thus, the p53 TS gene exon 5 mutation induced by ionizing radiation was investigated in in vivo conditions in normal rat salivary gland tissue through the application of low and high doses of I-131.
MATERIALS AND METHODS In this experimental study, adult male Wistar rats (7–12 mo of age; body weight >300 g) were divided into 2 groups. Group 1 consisted of 10 rats (mean body weight, 396±43 g); low and high I-131 doses were applied to this group at a 1-wk interval. Group 2 consisted of 5 rats (mean body weight, 381±24.1 gr); only low-dose I-131 was applied to this group. A Control Group, which consisted of 5 rats (mean body weight, 336±24.1 gr), was also included in the study. Rats were housed conventionally at a constant temperature of 22ºC, with lights on between 07.00 and 19.00 h, and with food and water freely available. Because it is known that retention of I-131 in the thyroid is closely related to food and water intake,15 food and water were not restricted during the study. All animals were fed standard commercial pelleted rat food (diet without iodine addition, purchased from Aytekinler Ltd., Konya, Turkey) and water ad libitum during the study. Animal experimentation was performed in accordance with the recommendations of the local Experimental Animal Ethical Committee. In Group 1, at first, low-dose I-131 was injected intraperitoneally (IP) under sterile conditions. The net injection dose was calculated as 47.5±9.2 µCi (net dose range, 29.3–61.6 µCi in this group [Table]). After 24 h, all animals were anesthetized with 5 mg/kg of xylazine (Rhompun; Bayer, Istanbul, Turkey) and 40 mg/kg of ketamine HCl (Ketalar; Eczacibasi, Istanbul, Turkey), and thyroidal I-131 uptakes were calculated by scintigraphic techniques with a gamma camera. Rats were fixed to the plate in the supine position, 1 at a time, and anterior scintigraphic images were obtained with the use of a single-head gamma camera (Toshiba GCA-7100A; Toshiba Medical Systems Corporation, Tokyo, Japan) that was equipped with a pinhole collimator and interfaced to a dedicated computer. For image acquisition, a peak energy setting at 364 kiloelectronvolts (keV) with a 20% window was used. Each of the images was acquired for 1 min 4 cm away from the neck region of the rat with a 256×256 matrix and zoom 1. Immediately after this process was completed, a point source was prepared as a “standard,” and a scintigraphic image of the standard was obtained with use of the same acquisition parameters and the same gamma camera. In all rats, focal I-131 accumulations were detected within the thyroid localization in the neck. Activity counts from thyroid and background localizations were calculated according to regions of interest (ROIs). Average counts per pixel of circular ROIs placed over the thyroid localization and boxed ROIs placed over the background area in anterior static images were used for calculating thyroidal uptake of I-131. Activity counts were also obtained from “standard” images with use of the same ROI placements. All ROI placements were done by the same person.
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Net µCi
gr
470 410 380 340 420 380 460 360 370 370 396 43 350 400 400 395 360 381 24.1 350 310 360 350 310 336 24.1
Rat
1 2 3 4 5 6 7 8 9 10 Mean SD 1 2 3 4 5 Mean SD C1 C2 C3 C4 C5 Mean SD
47.4 37 29.3 47 53.58 55.85 49.5 48.76 61.65 45.2 47.5 9.2 57.12 56.57 52.17 43.5 54.2 52.7 5.5 Saline Saline Saline Saline Saline
1st Inj Act,
Body Weight,
0.10 0.09 0.08 0.14 0.13 0.15 0.11 0.14 0.17 0.12 0.12 0.04 0.16 0.14 0.13 0.11 0.15 0.14 0.02
µCi/gr
Activity,
8.82 12.22 16.17 8.28 12.61 7.61 8.56 18.65 18.11 13.49 12.45 4.14 12.35 9.82 11.2 17.54 14.7 13.12 3.04
%
1st Uptake,
1019.2 928 966 1031.7 984.5 990.2 1050.3 952.8 1045.9 1103.8 1007.2 52.9
Net µCi
2nd Inj Act,
2.17 2.26 2.54 3.03 2.34 2.61 2.28 2.65 2.83 2.98 2.57 0.3
Activity, µCi/gr 3.21 3.38 4.24 3.33 11.12 8.04 24.4 13.24 13.96 11.72 9.66 6.73
%
2nd Uptake,
1066.6 965 995.3 1078.7 1038.1 1046.1 1099.8 1001.6 1107.6 1149 1054.8 57
µCi
Total Act,
Experimental and Control Group Characteristics, I-131 Doses, Uptakes, and p53 Results
2.27 2.35 2.62 3.17 2.47 2.75 2.39 2.78 2.99 3.11 2.7 0.32
µCi/gr
Total Act,
36.3 27.6 26.2 40.2 88.1 105.6 285.0 70.9 77.0 86.8 84.37 75.88
%
2nd/1st Uptake Alteration,
Normal Normal Normal Normal Normal
Normal Normal Normal Normal Normal
Normal Normal Normal Normal Normal Normal Normal Normal Mutation (+) Normal
Tissue Results
One week after low-dose injections were given, high doses of I-131 were injected into the same rats. The mean injection dose was 1007.2±52.9 µCi (net dose range, 928–1103.8 µCi) in this group (Table). Twenty-four hours after this process was completed, second scintigraphic images were obtained with same gamma camera parameters, and thyroidal uptakes of high doses of I-131 were calculated again by means of the same technique. Thyroidal I-131 uptakes were calculated with use of the following related formula: Thyroid ROI (cpm) – Uptake (%) =
Number of thyroid pixels x background ROI (cpm) Number of background pixels Net injected dose (µCi)
×
Standard activity (µCi) Standard ROI (cpm)
× 100
In Group 2 rats, only low doses of I-131 were injected IP under sterile conditions. The mean injection dose was 52.7±5.5 µCi for low-dose I-131 in Group 2 (net dose range, 43.5–57.1 µCi). After 24 h, thyroidal uptakes were calculated through the same scintigraphic technique. Immediately after the uptake was calculated, submaxillary salivary gland tissues were resected by an anterior approach in both groups. In Control Group rats, I-131 was not applied. Only 1 mL of physiologic saline was applied IP to this group. Living conditions of the Control Group were the same as those of other groups. Submaxillary salivary gland tissues of this group were also resected. All resected tissues were placed separately in sterile Eppendorf tubes that contained pure alcohol; these were quickly frozen and stored at –80ºC. Genomic DNA was extracted from the salivary gland tissues of controls and all experimental groups for genotyping through appropriate techniques.16 The polymerase chain reaction (PCR)-based single-stranded conformational polymorphism (SSCP) technique was used to amplify fragments and screen for possible mutations. Lyophilized primers (Thermo Electron, Bremen, Germany) were used to amplify the 249-base pair (bp) fragment of the TS gene p53 exon 5 gene pair domain. Primer sequences for PCR amplification were as follows: Forward: 5’-GAT TCT TTC TCC TCT CCT AC-3’ Reverse: 5’-CCC TGG ACA ACC AGT TCT AA-3’ Thermocycle (GeneAmp PCR System 9700; Applied Biosystems, Foster City, Calif) settings consisted of profiles for 35-cycle amplification reactions, which were as follows: initial denaturation at 94ºC for 5 min, denaturation at 94ºC for 1 min, annealing at 54ºC for 1 min, extension at 72ºC for 1 min, and final extension at 72ºC for 7 min. Amplified gene products were genotyped and compared under optimal conditions of 10% nondenaturating polyacrylamide gel (37.5:1 acrylamide-to-bisacrylamide cross-linking) electrophoresis technique.17 Agarose and denaturating gels were visualized with the Imaging Densitometer System (Vilber Lourmat, Marne-la-Vallée Cedex, France). Images were stored in a computer system. The “size marker” image, which includes known base pairs, was used for comparison with these chromosome images.
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Statistical Analysis Uptake results are presented as mean±SD (standard deviation). Statistical differences in first uptake values between Groups 1 and 2 were assessed by the Mann Whitney U test. The significance of differences in 1st and 2nd uptake calculations was evaluated with a paired t test in Group 1. A P value
