Persistent in vivo cytogenetic effects of radioiodine therapy: a 21-year follow-up study using multicolor FISH

Persistent in vivo cytogenetic effects of radioiodine therapy: a 21-year follow-up study using... Journal of Radiation Research, Vol. 59, No. 1, 2018, pp. 10–17 doi: 10.1093/jrr/rrx049 Advance Access Publication: 21 September 2017 Persistent in vivo cytogenetic effects of radioiodine therapy: a 21-year follow-up study using multicolor FISH 1 1 2 Gordon K. Livingston , Maria Escalona , Alvis Foster 1, and Adayabalam S. Balajee Radiation Emergency Assistance Center and Training Site, Cytogenetic Biodosimetry Laboratory, Oak Ridge Associated Universities, Oak Ridge Institute for Science and Education, Oak Ridge, TN 37830, USA Indiana University Health, Ball Memorial Hospital, 2401 West University Avenue, Muncie, IN 47303, USA *Corresponding author: Cytogenetic Biodosimetry Laboratory, REAC/TS, ORAU, ORISE, Oak Ridge, TN 37830, USA. Tel: +1-865-574-1040; Fax: +1-865-574-1047; Email: Adayabalam.balajee@orau.org Received April 7, 2017; Revised July 28, 2017; Editorial Decision August 4, 2017 ABSTRACT Our previous studies demonstrated the cytogenetic effects in the peripheral blood lymphocytes of a 34-year-old male patient who received ablative radioactive iodine therapy (RIT) on two different occasions in 1992 and 1994. Assessment of RIT-induced chromosomal damage by the cytokinesis-blocked micronucleus assay (CBMN) showed the persistence of elevated micronucleus frequency in this patient for more than two decades since the first RIT. Subsequent cytogenetic analysis performed in 2012 revealed both stable and unstable aberrations, whose frequencies were higher than the baseline reported in the literature. Here, we report the findings of our recent cytogenetic analysis peformed in 2015 on this patient using the multicolor fluorescence in situ hybridization (mFISH) technique. Our results showed that both reciprocal and non-reciprocal translocations persisted at higher frequencies in the patient than those reported in 2012. Persistence of structural aberrations for more than two decades indicate that these aberrations might have originated from long-lived T-lymphocytes or hematopoietic stem cells. Our study suggests that the long-term persistence of chromosome translocations in circulating lymphocytes can be useful for monitoring the extent of RIT-induced chromosomal instability several years after exposure and for estimating the cumulative absorbed dose after multiple RITs for retrospective biodosimetry purposes. This is perhaps the first and longest follow-up study documenting the persistence of cytogenetic damage for 21 years after internal radiation exposure. KEYWORDS: in vivo exposure, Iodine, multicolor fluorescence in situ hybridization, chromosomal transloca- tions, micronuclei, telomere and centromere FISH analysis INTRODUCTION The use of radioiodine warrants further study for evaluation of bio- Cytogenetic assays are the methods of choice for radiation dose logical effects prospectively, since it has a short physical half-life assessment when intended or unexpected human exposures occur in (8 days) and an even shorter biological half-life (24 h) and decays the absence of physical dosimetry devices. It has been widely by emitting gamma rays and beta particles throughout the body. accepted that accurate dose estimation is critical for effectively plan- Patients undergoing therapy with medical isotopes such as I pro- ning appropriate medical/clinical management for mitigation of ill- vide a unique research opportunity for studying both the acute and ness and severity of radiation injuries. Radiopharmaceutical agents chronic biological effects of radiation administered under carefully are used widely in the fields of nuclear medicine and radiation controlled conditions, allowing prospective follow-up studies for oncology. Since its introduction in the 1940s for treatment of weeks, months, years and even decades after exposure. hyperthyroidism, the use of radioiodine has increased significantly Our previous studies [1, 2] demonstrated the cytogenetic effects and currently represents ~90% of all therapies in nuclear medicine. in the peripheral blood lymphocytes of a 34-year-old male patient © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re- use, please contact journals.permissions@oup.com � 10 Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent in vivo cytogenetic effects of radioiodine therapy � 11 who received ablative radioactive iodine therapy (RIT) on two dif- Review Board (IRB) protocol (ORAU000349). Aliquots of the ferent occasions in 1992 and 1994. Persistence of chromosomal aber- blood sample were cultured for a period of 48 h in RPMI medium rations in circulating lymphocytes of patients following radiation supplemented with 2% phytohaemagglutinin (PHA), 10% heat- exposure was first reported in 1962 by Bender and Gooch [3]. inactivated fetal bovine serum (Hyclone,GE,USA) and antibiotics, Studies on atomic bomb survivors showed elevated chromosome followed by the addition of colcemid (0.1 μg/ml) for the last 24 h. aberration rates in the exposed individuals that had persisted for at The cells were harvested at 48 h (from the initiation of culture with least two decades after the acute external exposures to thousands of PHA). Cells were treated with a hypotonic solution (0.56% KCl) individuals in 1945 [4–7]. The persistent cytogenetic effects were also for 18 min at 37°C and fixed in three changes of ice-cold acetic reported in Chernobyl accident victims in 1986 [8]as wellas inthe acid: methanol (1:3) mixture. An aliquot of fixed cells (25–30 μl) nuclear industry workers in the USA with accidental exposures to plu- was gently dropped at the center of the glass sides and air-dried for tonium [9–11]. Although persistence of chromosomal aberrations has multicolor fluorescence in situ hybridization (mFISH) and analysis. been reported in persons exposed to high-dose radiation, studies on long-term monitoring of cytogenetic damage following exposure to Analysis of micronuclei in binucleated cells low doses of medical/diagnostic radiation exposure are very limited. For the analysis of the MN frequency in binucleate cells, peripheral Here, we report a long-term cytogenetic study on a patient over a blood lymphocytes were grown in the presence of 2% PHA for period of 21 years using multiple assays ranging from the very earliest 44 h, followed by the addition of cytochalasin B (5 μg/ml) for the method (solid Giemsa staining) in the 1950s to the most advanced last 28 h; the cells were harvested 72 h after culture initiation. molecular method (mFISH) developed in the 21st century. Our study Binucleate cells with or without MN were imaged and analyzed demonstrates the persistence of structural chromosomal aberrations using the fluorescence microscope (Carl Zeiss Imager.Z2) equipped in this patient for more than two decades, which is likely due to RIT- with ISIS software (MetaSystems, Boston, MA) induced DNA damage inflicted either in long-lived T-lymphocytes or in hematopoietic stem cells.We suggest that stable structural chromo- some aberrations such as translocations are potentially useful for FISH and multi-color fluorescence in situ hybridization monitoring the long-term effects on chromosomal instability caused The procedure for the fluorescence in situ hybridization (FISH) by internal radiation exposure. technique using peptide nucleic acid–based human telomeric and centromeric DNA probes was essentially the same as described by the manufacturer (PNA Bio, Newbury Park, CA). For the present MATERIALS AND METHODS study, Tel C, a C-rich telomere probe for the leading strand with Patient history repeats of TAACCC and a pan centromeric probe for human alpha The patient was a healthy, non-smoking male who was diagnosed staellite repeats (AAACTAGACAGAAGCAT) were used. The with papillary thyroid carcinoma in November 1991 followed by a mFISH technique was performed essentially as described before total thyroidectomy in December 1991 at 34 years of age. The patient [12, 13]. Briefly, slides were treated for 1 min with 0.001% acidic is a health physicist by profession and and did not have any history of pepsin solution (0.01N HCl) at 37°C for 1–2 min, followed by two occupational or medical exposure to ionizing radiation prior to the washes of 5 min each in phosphate-buffered saline. The slides were diagnosis of thyroid cancer in 1991. Prior to medical diagnosis of his post-fixed for 10 min in a solution of formaldehyde/MgCl (1% for- pathological condition, the patient actually participated as a donor in maldehyde/50 mM MgCl in PBS). The slides after denaturation a study on assessment of micronuclei (MN) formation after ex vivo (2X SSC at 70°C for 20 min and after cooling to ambient tempera- irradiation with X-rays. This enabled us to determine his baseline MN ture 1 min in 0.07N NaOH) were dehydrated in a graded series of frequency before the radioiodine treatment. In mid-January of 1992, 131 ethanol (30%, 70%, 90% and 100%) and air dried. The mBAND the patient received 1.78 GBq (48 mCi) of I as ablative therapy for probe was denatured separately by incubation at 75°C for 5 min, residual tumor cells. The MN analysis on this patient was undertaken followed by incubation at 37°C for 30 min to allow the annealing of 11 days after the radioiodine treatment and continued for 14 serial repetitive DNA sequences. An aliquot of the 10 μl probe was placed monthly samples, followed by quarterly sampling up to 2 years. Due on the slide and covered with a coverslip. The slides were kept in a to a suspicious lesion in the lung after 26 months of radioiodine treat- humidified hybridization chamber at 37°C for at least 72 h. The ment, the patient was subjected to a second radioiodine treatment unbound probe was removed by washing the slides in pre-warmed with 14.5 GBq (392 mCi) in mid-March, 1994. The MN analysis (75°C) 1X SSC (pH 7.0–7.5) for 5 min, followed by incubation in continued during this period and 6 months after the second treat- 4XSSCT (4X SSC with 0.1% Tween 20) for 5 min. Indirectly ment. The blood sample collected from this patient in April of 2015 labeled probe (Cy5), if needed, was amplified by incubation for was utilized in the current study for the analyses of MN, dicentric 30 min with each of the antibodies. The slides were incubated first chromosomes and translocations. with a 1: 50 dilution of Cy5-conjugated streptavidin in blocking buf- fer (5% non-fat dried milk prepared in 4X SSC with 0.1% Tween Lymphocyte culture and metaphase chromosome 20), followed by a 1:50 dilution of biotinylated anti-streptavidin in preparations blocking buffer and Cy5-conjugated streptavidin (1:50 dilution). All A 10 ml sample of whole blood was obtained from the donors (35 the antibodies were purchased from Invitrogen, Carlsbad, CA. The and 55 years of age), and the blood collection was performed with slides were washed three times for 5 min each in 4XSSCT after the donor’s consent in strict compliance with the Institutional incubation with each of the antibodies to remove the unbound Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 12 � G.K. Livingston et al. probe. The slides, after a final wash in 1X PBS for 5 min, were air in a 34-year-old male patient who received ablative I therapy on dried at ambient temperature. The nuclei were counterstained with two different occasions in 1992 and 1994 [1, 2]. In this study, we DAPI (Vectashield Laboratories, Burlingame, CA, USA). Metaphase examined the frequency of structural chromosomal aberrations in the cells were detected by the metaphase finder (MetaSystems, Boston, same patient 21 years after the second radioiodine exposure by using MA) program, and the mFISH images were captured under a ×63 the state of the art technique, mFISH. Representative pictures of oil immersion objective lens, using the Zeiss epifluorescence micro- mFISH-labeled aberrant metaphase cells with one- and two-way trans- scope. Image analysis was performed using the ISIS software locations are shown in Fig. 1. Out of the 600 metaphase cells analyzed, (MetaSystems, Boston, MA, USA) essentially according to the pub- 65 cells (10.83%) were found to be aberrant. Translocations were lished procedure. Normal and aberrant chromosomes were identi- found in 8.83% of the total cells (53 of 600 cells analyzed), with 57 fied by unique chromosome-specific processed color generated by translocations [57 translocations (reciprocal 31 and non-reciprocal 26) the ISIS software based on pixel intensities of combinatorial labeling in 600 cells (9.5%)], with a frequency of 0.095 ± 0.012 (mean ± SE) of the five fluorochromes (FITC, Spectrum Orange, Texas Red, translocations/metaphase cell. Among the 53 aberrant cells with trans- DEAC and Cy5). Translocations (one-way and two-way) were iden- locations, 4 metaphase cells showed a total of 9 non-reciprocal translo- tified by color junctions in 600 well-spread metaphase cells. cations; two translocations in each of the three cells and three translocations in one cell. Chromosomes 1, 2, 3 and 4 were found to be involved in 36 of the 57 translocations with other chromosomes, Statistical analysis accounting for 57.9% of the total yield. It is interesting to note that Data obtained on MN, dicentric chromosomes and translocations reciprocal translocations involving chromosomes 2 and 7 were found are expressed as mean ± standard error of the mean (SEM). The in 3 cells. Among all the chromosomes, chromosome 4 was involved Student paired t test was used to analyze the differencs between the in greatest number of translocations (11 out of 57 translocations) with means. A P value of <0.05 was considered to be significant. other chromosomes, followed by chromosome 10 (10 of 57 transloca- tions), chromosome 2 (9 of 57 translocations) and chromosome 1 (6 RESULTS out of 57 translocations). Our earlier conventional FISH study using a Elevated frequency of structural chromosome aberrations cocktail of three chromosome-specificprobes(1, 4and 12)revealeda genome equivalent of 41 ± 7 total translocations (inclusive of com- detected by mFISH Our earlier studies detected the persistence of elevated chromosomal plete and incomplete exchanges) in 1000 cells, compared with 95 translocations (frequency adjusted for 1000 cells) detected by mFISH damage in the form of MN, dicentric chromosomes and translocations Fig. 1. (A) Representative mutlicolor FISH pictures of aberrant metaphases with one way and two way translocations are shown. The translocated chromosomes are marked with numbers. (B) Chromosomes involved in translocation were identified by pseudocolor patterns generated by ISIS software based on the intensities of five different fluorochromes used for the combinatorial labeling of human chromosomes. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent in vivo cytogenetic effects of radioiodine therapy � 13 methodology in this study. The frequency of translocations detected after the first radioiodine in vivo exposure) by both conventional by the mFISH technique was significantly higher than that detected by Giemsa staining and FISH techniques revealed frequencies of the cocktail of 3 whole chromosome-specificprobes. Usingthe 10 ± 3.2 and 6 ± 1.3 in 1000 metaphase cells, respectively. Since the mFISH technique, we found that only 21 of the 57 total translocations dicentric chromsosome analysis was not performed until 2011, involved chromosomes 1, 4 and 12. The frequencies of all types of data on the initial dicentric frequency immediately or after the two stable and unstable chromosome aberrations are shown in Table 1. radioiodine treatments were not available. Considering the accepted baseline frequency of 1 dicentric chromosome in 1000 cells, it was concluded that the dicentric frequency observed in the radioiodine patient was significantly higher than the background Elevated frequency of unstable chromosome aberrations level. However, our recent FISH analysis on the peripheral blood In addition to translocations, three chromatid breaks (chromosomes lymphocytes collected from this patient in 2015 (21 years after the 2, 5 and X), one chromosome break (Chromosome 4) and 12 acen- two radioiodine therapies) revealed a further elevation in dicentric tric fragments (3 of chromosome 1, 2 of chromosome 4, 2 of chromosome frequency (5 out of 251 cells, 0.02 dicentrics/per chromosome 5, 2 of chromosome 20, 1 of each of chromosomes 3, metaphase cells; 20 per 1000 cells). The representative aberrant 9 and 19) were observed. Observation of increased frequencies of metaphases with dicentric chromosomes detected by FISH using stable and unstable aberrations suggests an elevated level of human telomere- and centromere-specific peptide nucleic acid chromosomal instability in the radiotherapy patient. The frequencies (PNA) probes are shown in Fig. 2. Although the precise reason for of all types of stable and unstable aberrations were found to be the elevated dicentric frequency is not known, it may be due to 0.121/metaphase cell (73 aberrations in 600 cells). A total of 8 the persistence of dicentric-bearing long-lived lymphocytes, or to hypodiploid (<46 chromosomes) and 12 hyperdiploid (>46 chro- the lymphocytes generated from damaged stem/progenitor cells. mosomes) cells were detected during the analysis of 600 metaphase Alternately, delayed genomic instability triggered by radioiodine cells by the mFISH technique. Although chromatid breaks, hypo- exposure can also contribute to an elevation in dicentric chromo- and hyperdiploidy as well as other chromosome anomalies are not some frequency. directly induced by radiation, they can be considered as suitable indices for assessing the extent of genomic instability [14, 15]. The dicentric chromosome analysis performed for the first Elevation of micronuclei frequency time on the blood lymphocytes of this patient in 2012 (20 years The baseline frequency of MN estimated in 1991 was found to be 6.0 ± 1.7 per 1000 binucleate cells (0.6% of cells with MN) for this Table 1. Stable and unstable chromosomal aberrations patient before he was diagnosed with thyroid cancer and subse- observed in the radioiodine-treated patient quently received ablative radioiodine therapy. The MN frequency determined at a monthly interval for 12 months in 1992 after the Stable aberrations first radioiodine treatment varied from 0.018 to 0.035/binucleate cell (1.8% to 3.5% of cells with MN). The MN frequency measured Total number of metaphase cells analyzed 600 at 2-month intervals in 1994 after the second I treatment ranged Normal cells 535 (89.16%) from 0.059 to 0.102/binucleate cell, with a peak value of 0.102 in September of 1994. The highest frequency of MN (0.11/binucleate Aberrant cells 65 (10.83%) cell) ever recorded in this patient after the second I treatment One-way translocations 26 (4.33%) was in January of 1995. This frequency (0.11) was 18.33-fold more than that of the baseline frequency (0.006) observed in this patient Two-way translocations 31 (5.16%) before the radioiodine treatment. Subsequent MN analysis per- formed from 1995 through 1997 at 3- and 6-month intervals Total translocations 57 (9.5%) showed small fluctuations, and a MN frequency of 0.073 was Unstable aberrations observed in January of 1997. The increase in MN frequency caused by the radioiodine treatment was found to be highly statistically sig- Total number of metaphase cells analyzed 600 nificant (P < 0.05) when compared with the baseline frequency. Chromatid breaks 3 (0.5%) MN analysis performed in March 2012 showed a frequency of 0.016/binculeate cell, which was still 2.66-fold more than the base- Chromosome breaks 1 (0.16%) line frequency. MN analysis performed in April 2015 revealed a Acentric fragments 12 (2%) similar MN frequency of 11 in 695 binucleate cells (0.015/cell) in the patient relative to 3 MN in 526 binucleate cells cells (0.005/ Dicentrics 5 (2%) cell) in the sample from a 35-year-old healthy male donor. The MN frequencies (averaged for multiple sampling times in a year) Rings 1 (0.39%) observed over the years from 1991 through 2015 in this patient are 251 cells were analyzed by FISH using centromere- and telomere-specific PNA summarized in Fig. 3. High frequencies of MN observed after probes. A total of 8 hypodiploid (<46 chromosomes) and 12 hyperdiploid (>46 almost 21 years of radioiodine exposure indicates the persistence of chromosomes) cells were detected during the analysis of 600 metaphase cells by the mFISH technique. chromosomal instability in this patient. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 14 � G.K. Livingston et al. Fig. 2. Detection of dicentric chromosomes in the patient’s sample by FISH using human centromere- (green color) and telomere- (red) specific peptide nucleic acid (PNA) probes. Arrows indicate the R-Ring chromosomes with two centromeric spots (green color). The metaphase chromosomes were counterstained with DAPI (blue color). Fig. 3. (A) Frequencies of dicentric chromosomes and translocations detected in the patient blood samples collected in 2012 and 2015. Note that the frequencies of both dicentric chromosomes and translocations are elevated in the sample analyzed in 2015 relative to that of 2012. The translocation frequency detected by mFISH was almost doubled in 2015. (B) Frequency of MN detected in the patient from 1991 (pre-radioiodine treatment) through 2015 (post-treatment) is shown. Bars represent the SE of the mean. Asterisks indicate that the P values are statistically significant (P > 0.05). that the frequency of persistent chromosomal damage correlates DISCUSSION Therapeutic application of radioiodine for hyperthyroidism and thy- with absorbed radiation dose in exposed humans [16–18]. Increased frequencies of MN, dicentric chromosomes and transloca- roid cancer in humans provides an unique opportunity for assessing the effects of internal radiation exposure with a feasibility of per- tions were reported by us in a patient 20 years after the in vivo exposure to radioiodine [1, 2]. Both stable and unstable forming long-term follow-up studies. Earlier reports demonstrated Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent in vivo cytogenetic effects of radioiodine therapy � 15 chromosomal aberrations were observed in this patient at higher fre- retain the cytogenetic damage owing to their quiescent stage. An quencies than those reported for similarly aged human populations equally likely possibility is that the DNA damage initially induced [1, 2]. In this study, we monitored the long-term effects of RIT on by the radioiodine treatments persisted in a subset of stem/progeni- the frequency of structural chromosomal aberrations in the entire tor cells, giving rise to different types of stable and unstable human genome by the mFISH technique, following two rounds of chromosomal aberrations in mature lymphocytes. Observation of RITs in 1992 and 1994. The mFISH technique is distinctly advanta- clonal translocations in A-bomb survivors suggests that a single geous over conventional FISH because it alleviates the need for con- stem cell of an adult can generate long-lived myeloid and lymphoid verting the frequencies to genome equivalents, and therefore progeny, accounting for a substantial fraction of circulating lympho- eliminates the associated statistical uncertainties, if any, with such a cytes and hematopoietic progenitors [29, 30]. It is worth noting conversion. The mFISH revealed that the frequency of both recipro- that no clonal translocations were observed in the current study. cal and non-reciprocal translocations was 0.095/metaphase cell, Further evidence for the long-time persistence of stable chromo- which was 2-fold more than that detected by FISH in 2012 (0.041/ somal aberrations in hematopoietic stem and progenitor cells came metaphase cell), using a cocktail of three chromosome-specific from the study of Kreja et al. [31], who examined the hematopoietic probes. Of interest, mFISH detected the involvement of chromo- colonies derived from the peripheral blood samples of 9 highly somes 1, 2, 3 and 4 with other chromosomes in 57.89% of the total exposed individuals after 7–25 years of radiation accidents that translocations. Similar to translocations, the frequency of dicentric occurred in Moscow (1971), Kazan (1975) and Chernobyl (1986). chromosomes detected by FISH in the present study was also high- Interestingly, no unstable aberrations were detected in this study. er than that detected in 2012. Similar observations have been Persistence of translocations was also reported in 8 radiotherapy reported by Puerto et al. [19] in 10 radioiodine therapeutic thyroid patients (total radiotherapy dose ranged from 40–80 Gy) with dif- cancer patients by FISH, using a cocktail of chromosome-specific ferent types of cancer over a 5-year period [32]. In an 8-year fol- probes for chromosomes 1, 4 and 10. low-up study, Natarajan et al. [33] did not find any trend for the Khvostunov et al. [20] recently reported the yield of unstable persistence of translocations, as varying levels of increase and and stable chromosomal aberrations in 24 differentiated thyroid decrease in translocations were found among the radiation accident cancer patients who were either treated for the first time or mul- victims of Goiânia. tiple times with radioiodine. In corroboration with our study, fre- The frequencies of dicentric chromosomes and translocations quencies of both stable and unstable aberrations had increased were distinctly higher in the blood sample analyzed in 2015 com- considerably in these patients before and after radioiodine therapy pared with those analyzed in our earlier study in 2012. We believe when compared with the baseline frequencies reported in the lit- that the fluctuations observed in the frequencies of chromosomal erature. Using the elevated frequencies of chromosomal aberra- aberrations may be due to varying number of damaged cells being tions, the authors were able to estimate the cumulative whole-body present in the circulating blood during the collection times. dose after multiple radioiodine treatments [18]. Using the micro- Alternately, the increased frequency of stable chromosomal aberra- −1 nucleus assay, a biodose ranging from 0.089 mGy.MBq to tions detected by mFISH may also have been due to delayed effects −1 0.197 mGy.MBq was reported by a few studies [21–23]. M’Kacher of genomic instability triggered by radioiodine-induced DNA damage −1 et al. [24] reported a mean dose of 0.146 mGy.MBq using the accumulation. Further studies are required to clarify whether or not chromosome aberration assay, and the activity of I was 3.7 GBq in the DNA damage inflicted in the adult stem cells by radioiodine all of these above-mentioned studies. A biodose of 0.23 mGy/MBq therapy is the root cause for the ensuing chromosomal aberrations at was obtained according to the Medical Internal Radiation Dose enhanced levels in this patient. The current study indicates that the (MIRD) procedures [25]. On the basis of these values in the litera- persistence of stable and unstable aberrations induced by RIT can be ture, we estimate that our patient might have absorbed a whole-body of use, not only in long-term monitoring of genomic instability, but dose in the range of 1.3–2.9 Gy by the second treatment with also in predicting the absorbed dose by retrospective biodosimetry, 14.5 GBq of I[2]. as recently demonstrated by Khvostunov et al. [20]. However, I, due to its emission of gamma rays and beta particles during extreme caution is necessary when extrapolating the initial yield of decay, is extremely hazardous when absorbed into cells and tissues. stable aberrations to equate absorbed radiation dose when the thera- Considering the half-life of radioiodine, which is ~8 days, it is logical peutic radiation exposure is high and non-uniform in nature. to expect that the absorbed radioiodine would have been completely eliminated from the patient during the follow-up study period, which was more than 21 years from the second administration of CONCLUSIONS radioiodine in 1994. In this study, T-lymphcoytes stimulated by Our 21-year cytogenetic follow-up study suggests that the internal PHA were utilized for the detection of chromosomal damage in the exposure to gamma rays and β-particles emitted during the decay of patient. It has been suggested that naïve T cells that frequently cir- radioiodine can cause long-lasting cytogenetic damage in humans, culate between blood and lymph can survive for prolonged periods and that the persistence of stable chromosomal aberrations is likely of time by being in a resting stage, thereby maintaining the due to the long-lived nature of a subset of damaged T lymphocytes. immunological responsiveness to new antigens [26–28]. It is cur- Our study suggests the possibility that stable chromosomal aberra- rently unclear which T cells (naïve or memory) are preferentially tions can be effectively used for retrospective biodosimetry for stimulated by PHA, and whether or not long-lived naïve T cells internal radiation exposure. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 16 � G.K. Livingston et al. 12. Piao CQ, Liu L, Zhao YL et al. Immortalization of human small ACKNOWLEDGEMENTS airway epithelial cells by ectopic expression of telomerase. This study was performed under the contract #DE-AC05- Carcinogenesis 2005;26:725–31. 06OR23100 between Oak Ridge Associated Universities (ORAU) 13. Shao G, Balajee AS, Hei TK et al. p16INK4a downregulation is and the U.S. Department of Energy (U.S. DOE). REAC/TS is an involved in immortalization of primary human prostate epithe- organizational program of the Oak Ridge Institute for Science and lial cells induced by telomerase. Mol Carcinog 2008;47:775–83. Education and is operated by ORAU for the U.S.DOE. The content 14. Tanaka K, Kohda A, Toyokawa T et al. Chromosome aberra- is solely the responsibility of the authors and does not reflect the tion frequencies and chromosome instability in mice after long- official views or opinions of the U.S. DOE. term exposure to low-dose-rate gamma-irradiation. Mutat Res 2008;657:19–25. 15. Tanaka K, Kumaravel TS, Ihda S et al. Characterization of late- CONFLICT OF INTEREST The authors declare that there are no conflicts of interest. arising chromosome aberrations in human B-cell lines estab- lished from alpha-ray– or gamma-ray–irradiated lymphocytes. Cancer Genet Cytogenet 2008;187:112–24. 16. Hande MP, Azizova TV, Burak LE et al. Complex chromosome FUNDING The study was supported by funding from the United States aberrations persist in individuals many years after occupational exposure to densely ionizing radiation: an mFISH study. Genes Department of Energy (US DOE; Contract# DE-AC05-06OR23100). Chromosomes Cancer 2005;44:1–9. 17. Livingston GK, Falk RB, Schmid E. Effect of occupational radi- REFERENCES ation exposures on chromosome aberration rates in former plu- tonium workers. Radiat Res 2006;166:89–97. 1. Livingston GK, Foster AE, Elson HR. Effect of in vivo exposure to iodine-131 on the frequency and persistence of micronuclei 18. Matsumoto K, Ramsey MJ, Nelson DO et al. 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Estimating the number 32. Tawn EJ, Whitehouse CA. Persistence of translocation frequencies of hematopoietic or lymphoid stem cells giving rise to clonal in blood lymphocytes following radiotherapy: implications for chromosome aberrations in blood T lymphocytes. Radiat Res retrospective radiation biodosimetry. JRadiolProt 2003;23:423–30. 2004;161:273–81. 33. Natarajan AT, Santos SJ, Darroudi F et al. Cesium-induced 31. Kreja L, Greulich KM, Fliedner TM et al. Stable chromosomal chromosome aberrations analyzed by fluorescence in situ aberrations in haemopoietic stem cells in the blood of radiation hybridization: eight years follow up of the Goiania radiation accident victims. Int J Radiat Biol 1999;75:1241–50. accident victims. Mutat Res 1998;400:299–312. 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Persistent in vivo cytogenetic effects of radioiodine therapy: a 21-year follow-up study using multicolor FISH

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Journal of Radiation Research, Vol. 59, No. 1, 2018, pp. 10–17 doi: 10.1093/jrr/rrx049 Advance Access Publication: 21 September 2017 Persistent in vivo cytogenetic effects of radioiodine therapy: a 21-year follow-up study using multicolor FISH 1 1 2 Gordon K. Livingston , Maria Escalona , Alvis Foster 1, and Adayabalam S. Balajee Radiation Emergency Assistance Center and Training Site, Cytogenetic Biodosimetry Laboratory, Oak Ridge Associated Universities, Oak Ridge Institute for Science and Education, Oak Ridge, TN 37830, USA Indiana University Health, Ball Memorial Hospital, 2401 West University Avenue, Muncie, IN 47303, USA *Corresponding author: Cytogenetic Biodosimetry Laboratory, REAC/TS, ORAU, ORISE, Oak Ridge, TN 37830, USA. Tel: +1-865-574-1040; Fax: +1-865-574-1047; Email: Adayabalam.balajee@orau.org Received April 7, 2017; Revised July 28, 2017; Editorial Decision August 4, 2017 ABSTRACT Our previous studies demonstrated the cytogenetic effects in the peripheral blood lymphocytes of a 34-year-old male patient who received ablative radioactive iodine therapy (RIT) on two different occasions in 1992 and 1994. Assessment of RIT-induced chromosomal damage by the cytokinesis-blocked micronucleus assay (CBMN) showed the persistence of elevated micronucleus frequency in this patient for more than two decades since the first RIT. Subsequent cytogenetic analysis performed in 2012 revealed both stable and unstable aberrations, whose frequencies were higher than the baseline reported in the literature. Here, we report the findings of our recent cytogenetic analysis peformed in 2015 on this patient using the multicolor fluorescence in situ hybridization (mFISH) technique. Our results showed that both reciprocal and non-reciprocal translocations persisted at higher frequencies in the patient than those reported in 2012. Persistence of structural aberrations for more than two decades indicate that these aberrations might have originated from long-lived T-lymphocytes or hematopoietic stem cells. Our study suggests that the long-term persistence of chromosome translocations in circulating lymphocytes can be useful for monitoring the extent of RIT-induced chromosomal instability several years after exposure and for estimating the cumulative absorbed dose after multiple RITs for retrospective biodosimetry purposes. This is perhaps the first and longest follow-up study documenting the persistence of cytogenetic damage for 21 years after internal radiation exposure. KEYWORDS: in vivo exposure, Iodine, multicolor fluorescence in situ hybridization, chromosomal transloca- tions, micronuclei, telomere and centromere FISH analysis INTRODUCTION The use of radioiodine warrants further study for evaluation of bio- Cytogenetic assays are the methods of choice for radiation dose logical effects prospectively, since it has a short physical half-life assessment when intended or unexpected human exposures occur in (8 days) and an even shorter biological half-life (24 h) and decays the absence of physical dosimetry devices. It has been widely by emitting gamma rays and beta particles throughout the body. accepted that accurate dose estimation is critical for effectively plan- Patients undergoing therapy with medical isotopes such as I pro- ning appropriate medical/clinical management for mitigation of ill- vide a unique research opportunity for studying both the acute and ness and severity of radiation injuries. Radiopharmaceutical agents chronic biological effects of radiation administered under carefully are used widely in the fields of nuclear medicine and radiation controlled conditions, allowing prospective follow-up studies for oncology. Since its introduction in the 1940s for treatment of weeks, months, years and even decades after exposure. hyperthyroidism, the use of radioiodine has increased significantly Our previous studies [1, 2] demonstrated the cytogenetic effects and currently represents ~90% of all therapies in nuclear medicine. in the peripheral blood lymphocytes of a 34-year-old male patient © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re- use, please contact journals.permissions@oup.com � 10 Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent in vivo cytogenetic effects of radioiodine therapy � 11 who received ablative radioactive iodine therapy (RIT) on two dif- Review Board (IRB) protocol (ORAU000349). Aliquots of the ferent occasions in 1992 and 1994. Persistence of chromosomal aber- blood sample were cultured for a period of 48 h in RPMI medium rations in circulating lymphocytes of patients following radiation supplemented with 2% phytohaemagglutinin (PHA), 10% heat- exposure was first reported in 1962 by Bender and Gooch [3]. inactivated fetal bovine serum (Hyclone,GE,USA) and antibiotics, Studies on atomic bomb survivors showed elevated chromosome followed by the addition of colcemid (0.1 μg/ml) for the last 24 h. aberration rates in the exposed individuals that had persisted for at The cells were harvested at 48 h (from the initiation of culture with least two decades after the acute external exposures to thousands of PHA). Cells were treated with a hypotonic solution (0.56% KCl) individuals in 1945 [4–7]. The persistent cytogenetic effects were also for 18 min at 37°C and fixed in three changes of ice-cold acetic reported in Chernobyl accident victims in 1986 [8]as wellas inthe acid: methanol (1:3) mixture. An aliquot of fixed cells (25–30 μl) nuclear industry workers in the USA with accidental exposures to plu- was gently dropped at the center of the glass sides and air-dried for tonium [9–11]. Although persistence of chromosomal aberrations has multicolor fluorescence in situ hybridization (mFISH) and analysis. been reported in persons exposed to high-dose radiation, studies on long-term monitoring of cytogenetic damage following exposure to Analysis of micronuclei in binucleated cells low doses of medical/diagnostic radiation exposure are very limited. For the analysis of the MN frequency in binucleate cells, peripheral Here, we report a long-term cytogenetic study on a patient over a blood lymphocytes were grown in the presence of 2% PHA for period of 21 years using multiple assays ranging from the very earliest 44 h, followed by the addition of cytochalasin B (5 μg/ml) for the method (solid Giemsa staining) in the 1950s to the most advanced last 28 h; the cells were harvested 72 h after culture initiation. molecular method (mFISH) developed in the 21st century. Our study Binucleate cells with or without MN were imaged and analyzed demonstrates the persistence of structural chromosomal aberrations using the fluorescence microscope (Carl Zeiss Imager.Z2) equipped in this patient for more than two decades, which is likely due to RIT- with ISIS software (MetaSystems, Boston, MA) induced DNA damage inflicted either in long-lived T-lymphocytes or in hematopoietic stem cells.We suggest that stable structural chromo- some aberrations such as translocations are potentially useful for FISH and multi-color fluorescence in situ hybridization monitoring the long-term effects on chromosomal instability caused The procedure for the fluorescence in situ hybridization (FISH) by internal radiation exposure. technique using peptide nucleic acid–based human telomeric and centromeric DNA probes was essentially the same as described by the manufacturer (PNA Bio, Newbury Park, CA). For the present MATERIALS AND METHODS study, Tel C, a C-rich telomere probe for the leading strand with Patient history repeats of TAACCC and a pan centromeric probe for human alpha The patient was a healthy, non-smoking male who was diagnosed staellite repeats (AAACTAGACAGAAGCAT) were used. The with papillary thyroid carcinoma in November 1991 followed by a mFISH technique was performed essentially as described before total thyroidectomy in December 1991 at 34 years of age. The patient [12, 13]. Briefly, slides were treated for 1 min with 0.001% acidic is a health physicist by profession and and did not have any history of pepsin solution (0.01N HCl) at 37°C for 1–2 min, followed by two occupational or medical exposure to ionizing radiation prior to the washes of 5 min each in phosphate-buffered saline. The slides were diagnosis of thyroid cancer in 1991. Prior to medical diagnosis of his post-fixed for 10 min in a solution of formaldehyde/MgCl (1% for- pathological condition, the patient actually participated as a donor in maldehyde/50 mM MgCl in PBS). The slides after denaturation a study on assessment of micronuclei (MN) formation after ex vivo (2X SSC at 70°C for 20 min and after cooling to ambient tempera- irradiation with X-rays. This enabled us to determine his baseline MN ture 1 min in 0.07N NaOH) were dehydrated in a graded series of frequency before the radioiodine treatment. In mid-January of 1992, 131 ethanol (30%, 70%, 90% and 100%) and air dried. The mBAND the patient received 1.78 GBq (48 mCi) of I as ablative therapy for probe was denatured separately by incubation at 75°C for 5 min, residual tumor cells. The MN analysis on this patient was undertaken followed by incubation at 37°C for 30 min to allow the annealing of 11 days after the radioiodine treatment and continued for 14 serial repetitive DNA sequences. An aliquot of the 10 μl probe was placed monthly samples, followed by quarterly sampling up to 2 years. Due on the slide and covered with a coverslip. The slides were kept in a to a suspicious lesion in the lung after 26 months of radioiodine treat- humidified hybridization chamber at 37°C for at least 72 h. The ment, the patient was subjected to a second radioiodine treatment unbound probe was removed by washing the slides in pre-warmed with 14.5 GBq (392 mCi) in mid-March, 1994. The MN analysis (75°C) 1X SSC (pH 7.0–7.5) for 5 min, followed by incubation in continued during this period and 6 months after the second treat- 4XSSCT (4X SSC with 0.1% Tween 20) for 5 min. Indirectly ment. The blood sample collected from this patient in April of 2015 labeled probe (Cy5), if needed, was amplified by incubation for was utilized in the current study for the analyses of MN, dicentric 30 min with each of the antibodies. The slides were incubated first chromosomes and translocations. with a 1: 50 dilution of Cy5-conjugated streptavidin in blocking buf- fer (5% non-fat dried milk prepared in 4X SSC with 0.1% Tween Lymphocyte culture and metaphase chromosome 20), followed by a 1:50 dilution of biotinylated anti-streptavidin in preparations blocking buffer and Cy5-conjugated streptavidin (1:50 dilution). All A 10 ml sample of whole blood was obtained from the donors (35 the antibodies were purchased from Invitrogen, Carlsbad, CA. The and 55 years of age), and the blood collection was performed with slides were washed three times for 5 min each in 4XSSCT after the donor’s consent in strict compliance with the Institutional incubation with each of the antibodies to remove the unbound Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 12 � G.K. Livingston et al. probe. The slides, after a final wash in 1X PBS for 5 min, were air in a 34-year-old male patient who received ablative I therapy on dried at ambient temperature. The nuclei were counterstained with two different occasions in 1992 and 1994 [1, 2]. In this study, we DAPI (Vectashield Laboratories, Burlingame, CA, USA). Metaphase examined the frequency of structural chromosomal aberrations in the cells were detected by the metaphase finder (MetaSystems, Boston, same patient 21 years after the second radioiodine exposure by using MA) program, and the mFISH images were captured under a ×63 the state of the art technique, mFISH. Representative pictures of oil immersion objective lens, using the Zeiss epifluorescence micro- mFISH-labeled aberrant metaphase cells with one- and two-way trans- scope. Image analysis was performed using the ISIS software locations are shown in Fig. 1. Out of the 600 metaphase cells analyzed, (MetaSystems, Boston, MA, USA) essentially according to the pub- 65 cells (10.83%) were found to be aberrant. Translocations were lished procedure. Normal and aberrant chromosomes were identi- found in 8.83% of the total cells (53 of 600 cells analyzed), with 57 fied by unique chromosome-specific processed color generated by translocations [57 translocations (reciprocal 31 and non-reciprocal 26) the ISIS software based on pixel intensities of combinatorial labeling in 600 cells (9.5%)], with a frequency of 0.095 ± 0.012 (mean ± SE) of the five fluorochromes (FITC, Spectrum Orange, Texas Red, translocations/metaphase cell. Among the 53 aberrant cells with trans- DEAC and Cy5). Translocations (one-way and two-way) were iden- locations, 4 metaphase cells showed a total of 9 non-reciprocal translo- tified by color junctions in 600 well-spread metaphase cells. cations; two translocations in each of the three cells and three translocations in one cell. Chromosomes 1, 2, 3 and 4 were found to be involved in 36 of the 57 translocations with other chromosomes, Statistical analysis accounting for 57.9% of the total yield. It is interesting to note that Data obtained on MN, dicentric chromosomes and translocations reciprocal translocations involving chromosomes 2 and 7 were found are expressed as mean ± standard error of the mean (SEM). The in 3 cells. Among all the chromosomes, chromosome 4 was involved Student paired t test was used to analyze the differencs between the in greatest number of translocations (11 out of 57 translocations) with means. A P value of <0.05 was considered to be significant. other chromosomes, followed by chromosome 10 (10 of 57 transloca- tions), chromosome 2 (9 of 57 translocations) and chromosome 1 (6 RESULTS out of 57 translocations). Our earlier conventional FISH study using a Elevated frequency of structural chromosome aberrations cocktail of three chromosome-specificprobes(1, 4and 12)revealeda genome equivalent of 41 ± 7 total translocations (inclusive of com- detected by mFISH Our earlier studies detected the persistence of elevated chromosomal plete and incomplete exchanges) in 1000 cells, compared with 95 translocations (frequency adjusted for 1000 cells) detected by mFISH damage in the form of MN, dicentric chromosomes and translocations Fig. 1. (A) Representative mutlicolor FISH pictures of aberrant metaphases with one way and two way translocations are shown. The translocated chromosomes are marked with numbers. (B) Chromosomes involved in translocation were identified by pseudocolor patterns generated by ISIS software based on the intensities of five different fluorochromes used for the combinatorial labeling of human chromosomes. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent in vivo cytogenetic effects of radioiodine therapy � 13 methodology in this study. The frequency of translocations detected after the first radioiodine in vivo exposure) by both conventional by the mFISH technique was significantly higher than that detected by Giemsa staining and FISH techniques revealed frequencies of the cocktail of 3 whole chromosome-specificprobes. Usingthe 10 ± 3.2 and 6 ± 1.3 in 1000 metaphase cells, respectively. Since the mFISH technique, we found that only 21 of the 57 total translocations dicentric chromsosome analysis was not performed until 2011, involved chromosomes 1, 4 and 12. The frequencies of all types of data on the initial dicentric frequency immediately or after the two stable and unstable chromosome aberrations are shown in Table 1. radioiodine treatments were not available. Considering the accepted baseline frequency of 1 dicentric chromosome in 1000 cells, it was concluded that the dicentric frequency observed in the radioiodine patient was significantly higher than the background Elevated frequency of unstable chromosome aberrations level. However, our recent FISH analysis on the peripheral blood In addition to translocations, three chromatid breaks (chromosomes lymphocytes collected from this patient in 2015 (21 years after the 2, 5 and X), one chromosome break (Chromosome 4) and 12 acen- two radioiodine therapies) revealed a further elevation in dicentric tric fragments (3 of chromosome 1, 2 of chromosome 4, 2 of chromosome frequency (5 out of 251 cells, 0.02 dicentrics/per chromosome 5, 2 of chromosome 20, 1 of each of chromosomes 3, metaphase cells; 20 per 1000 cells). The representative aberrant 9 and 19) were observed. Observation of increased frequencies of metaphases with dicentric chromosomes detected by FISH using stable and unstable aberrations suggests an elevated level of human telomere- and centromere-specific peptide nucleic acid chromosomal instability in the radiotherapy patient. The frequencies (PNA) probes are shown in Fig. 2. Although the precise reason for of all types of stable and unstable aberrations were found to be the elevated dicentric frequency is not known, it may be due to 0.121/metaphase cell (73 aberrations in 600 cells). A total of 8 the persistence of dicentric-bearing long-lived lymphocytes, or to hypodiploid (<46 chromosomes) and 12 hyperdiploid (>46 chro- the lymphocytes generated from damaged stem/progenitor cells. mosomes) cells were detected during the analysis of 600 metaphase Alternately, delayed genomic instability triggered by radioiodine cells by the mFISH technique. Although chromatid breaks, hypo- exposure can also contribute to an elevation in dicentric chromo- and hyperdiploidy as well as other chromosome anomalies are not some frequency. directly induced by radiation, they can be considered as suitable indices for assessing the extent of genomic instability [14, 15]. The dicentric chromosome analysis performed for the first Elevation of micronuclei frequency time on the blood lymphocytes of this patient in 2012 (20 years The baseline frequency of MN estimated in 1991 was found to be 6.0 ± 1.7 per 1000 binucleate cells (0.6% of cells with MN) for this Table 1. Stable and unstable chromosomal aberrations patient before he was diagnosed with thyroid cancer and subse- observed in the radioiodine-treated patient quently received ablative radioiodine therapy. The MN frequency determined at a monthly interval for 12 months in 1992 after the Stable aberrations first radioiodine treatment varied from 0.018 to 0.035/binucleate cell (1.8% to 3.5% of cells with MN). The MN frequency measured Total number of metaphase cells analyzed 600 at 2-month intervals in 1994 after the second I treatment ranged Normal cells 535 (89.16%) from 0.059 to 0.102/binucleate cell, with a peak value of 0.102 in September of 1994. The highest frequency of MN (0.11/binucleate Aberrant cells 65 (10.83%) cell) ever recorded in this patient after the second I treatment One-way translocations 26 (4.33%) was in January of 1995. This frequency (0.11) was 18.33-fold more than that of the baseline frequency (0.006) observed in this patient Two-way translocations 31 (5.16%) before the radioiodine treatment. Subsequent MN analysis per- formed from 1995 through 1997 at 3- and 6-month intervals Total translocations 57 (9.5%) showed small fluctuations, and a MN frequency of 0.073 was Unstable aberrations observed in January of 1997. The increase in MN frequency caused by the radioiodine treatment was found to be highly statistically sig- Total number of metaphase cells analyzed 600 nificant (P < 0.05) when compared with the baseline frequency. Chromatid breaks 3 (0.5%) MN analysis performed in March 2012 showed a frequency of 0.016/binculeate cell, which was still 2.66-fold more than the base- Chromosome breaks 1 (0.16%) line frequency. MN analysis performed in April 2015 revealed a Acentric fragments 12 (2%) similar MN frequency of 11 in 695 binucleate cells (0.015/cell) in the patient relative to 3 MN in 526 binucleate cells cells (0.005/ Dicentrics 5 (2%) cell) in the sample from a 35-year-old healthy male donor. The MN frequencies (averaged for multiple sampling times in a year) Rings 1 (0.39%) observed over the years from 1991 through 2015 in this patient are 251 cells were analyzed by FISH using centromere- and telomere-specific PNA summarized in Fig. 3. High frequencies of MN observed after probes. A total of 8 hypodiploid (<46 chromosomes) and 12 hyperdiploid (>46 almost 21 years of radioiodine exposure indicates the persistence of chromosomes) cells were detected during the analysis of 600 metaphase cells by the mFISH technique. chromosomal instability in this patient. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 14 � G.K. Livingston et al. Fig. 2. Detection of dicentric chromosomes in the patient’s sample by FISH using human centromere- (green color) and telomere- (red) specific peptide nucleic acid (PNA) probes. Arrows indicate the R-Ring chromosomes with two centromeric spots (green color). The metaphase chromosomes were counterstained with DAPI (blue color). Fig. 3. (A) Frequencies of dicentric chromosomes and translocations detected in the patient blood samples collected in 2012 and 2015. Note that the frequencies of both dicentric chromosomes and translocations are elevated in the sample analyzed in 2015 relative to that of 2012. The translocation frequency detected by mFISH was almost doubled in 2015. (B) Frequency of MN detected in the patient from 1991 (pre-radioiodine treatment) through 2015 (post-treatment) is shown. Bars represent the SE of the mean. Asterisks indicate that the P values are statistically significant (P > 0.05). that the frequency of persistent chromosomal damage correlates DISCUSSION Therapeutic application of radioiodine for hyperthyroidism and thy- with absorbed radiation dose in exposed humans [16–18]. Increased frequencies of MN, dicentric chromosomes and transloca- roid cancer in humans provides an unique opportunity for assessing the effects of internal radiation exposure with a feasibility of per- tions were reported by us in a patient 20 years after the in vivo exposure to radioiodine [1, 2]. Both stable and unstable forming long-term follow-up studies. Earlier reports demonstrated Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent in vivo cytogenetic effects of radioiodine therapy � 15 chromosomal aberrations were observed in this patient at higher fre- retain the cytogenetic damage owing to their quiescent stage. An quencies than those reported for similarly aged human populations equally likely possibility is that the DNA damage initially induced [1, 2]. In this study, we monitored the long-term effects of RIT on by the radioiodine treatments persisted in a subset of stem/progeni- the frequency of structural chromosomal aberrations in the entire tor cells, giving rise to different types of stable and unstable human genome by the mFISH technique, following two rounds of chromosomal aberrations in mature lymphocytes. Observation of RITs in 1992 and 1994. The mFISH technique is distinctly advanta- clonal translocations in A-bomb survivors suggests that a single geous over conventional FISH because it alleviates the need for con- stem cell of an adult can generate long-lived myeloid and lymphoid verting the frequencies to genome equivalents, and therefore progeny, accounting for a substantial fraction of circulating lympho- eliminates the associated statistical uncertainties, if any, with such a cytes and hematopoietic progenitors [29, 30]. It is worth noting conversion. The mFISH revealed that the frequency of both recipro- that no clonal translocations were observed in the current study. cal and non-reciprocal translocations was 0.095/metaphase cell, Further evidence for the long-time persistence of stable chromo- which was 2-fold more than that detected by FISH in 2012 (0.041/ somal aberrations in hematopoietic stem and progenitor cells came metaphase cell), using a cocktail of three chromosome-specific from the study of Kreja et al. [31], who examined the hematopoietic probes. Of interest, mFISH detected the involvement of chromo- colonies derived from the peripheral blood samples of 9 highly somes 1, 2, 3 and 4 with other chromosomes in 57.89% of the total exposed individuals after 7–25 years of radiation accidents that translocations. Similar to translocations, the frequency of dicentric occurred in Moscow (1971), Kazan (1975) and Chernobyl (1986). chromosomes detected by FISH in the present study was also high- Interestingly, no unstable aberrations were detected in this study. er than that detected in 2012. Similar observations have been Persistence of translocations was also reported in 8 radiotherapy reported by Puerto et al. [19] in 10 radioiodine therapeutic thyroid patients (total radiotherapy dose ranged from 40–80 Gy) with dif- cancer patients by FISH, using a cocktail of chromosome-specific ferent types of cancer over a 5-year period [32]. In an 8-year fol- probes for chromosomes 1, 4 and 10. low-up study, Natarajan et al. [33] did not find any trend for the Khvostunov et al. [20] recently reported the yield of unstable persistence of translocations, as varying levels of increase and and stable chromosomal aberrations in 24 differentiated thyroid decrease in translocations were found among the radiation accident cancer patients who were either treated for the first time or mul- victims of Goiânia. tiple times with radioiodine. In corroboration with our study, fre- The frequencies of dicentric chromosomes and translocations quencies of both stable and unstable aberrations had increased were distinctly higher in the blood sample analyzed in 2015 com- considerably in these patients before and after radioiodine therapy pared with those analyzed in our earlier study in 2012. We believe when compared with the baseline frequencies reported in the lit- that the fluctuations observed in the frequencies of chromosomal erature. Using the elevated frequencies of chromosomal aberra- aberrations may be due to varying number of damaged cells being tions, the authors were able to estimate the cumulative whole-body present in the circulating blood during the collection times. dose after multiple radioiodine treatments [18]. Using the micro- Alternately, the increased frequency of stable chromosomal aberra- −1 nucleus assay, a biodose ranging from 0.089 mGy.MBq to tions detected by mFISH may also have been due to delayed effects −1 0.197 mGy.MBq was reported by a few studies [21–23]. M’Kacher of genomic instability triggered by radioiodine-induced DNA damage −1 et al. [24] reported a mean dose of 0.146 mGy.MBq using the accumulation. Further studies are required to clarify whether or not chromosome aberration assay, and the activity of I was 3.7 GBq in the DNA damage inflicted in the adult stem cells by radioiodine all of these above-mentioned studies. A biodose of 0.23 mGy/MBq therapy is the root cause for the ensuing chromosomal aberrations at was obtained according to the Medical Internal Radiation Dose enhanced levels in this patient. The current study indicates that the (MIRD) procedures [25]. On the basis of these values in the litera- persistence of stable and unstable aberrations induced by RIT can be ture, we estimate that our patient might have absorbed a whole-body of use, not only in long-term monitoring of genomic instability, but dose in the range of 1.3–2.9 Gy by the second treatment with also in predicting the absorbed dose by retrospective biodosimetry, 14.5 GBq of I[2]. as recently demonstrated by Khvostunov et al. [20]. However, I, due to its emission of gamma rays and beta particles during extreme caution is necessary when extrapolating the initial yield of decay, is extremely hazardous when absorbed into cells and tissues. stable aberrations to equate absorbed radiation dose when the thera- Considering the half-life of radioiodine, which is ~8 days, it is logical peutic radiation exposure is high and non-uniform in nature. to expect that the absorbed radioiodine would have been completely eliminated from the patient during the follow-up study period, which was more than 21 years from the second administration of CONCLUSIONS radioiodine in 1994. In this study, T-lymphcoytes stimulated by Our 21-year cytogenetic follow-up study suggests that the internal PHA were utilized for the detection of chromosomal damage in the exposure to gamma rays and β-particles emitted during the decay of patient. It has been suggested that naïve T cells that frequently cir- radioiodine can cause long-lasting cytogenetic damage in humans, culate between blood and lymph can survive for prolonged periods and that the persistence of stable chromosomal aberrations is likely of time by being in a resting stage, thereby maintaining the due to the long-lived nature of a subset of damaged T lymphocytes. immunological responsiveness to new antigens [26–28]. It is cur- Our study suggests the possibility that stable chromosomal aberra- rently unclear which T cells (naïve or memory) are preferentially tions can be effectively used for retrospective biodosimetry for stimulated by PHA, and whether or not long-lived naïve T cells internal radiation exposure. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/10/4209302 by Ed 'DeepDyve' Gillespie user on 16 March 2018 16 � G.K. Livingston et al. 12. Piao CQ, Liu L, Zhao YL et al. Immortalization of human small ACKNOWLEDGEMENTS airway epithelial cells by ectopic expression of telomerase. This study was performed under the contract #DE-AC05- Carcinogenesis 2005;26:725–31. 06OR23100 between Oak Ridge Associated Universities (ORAU) 13. Shao G, Balajee AS, Hei TK et al. p16INK4a downregulation is and the U.S. Department of Energy (U.S. DOE). REAC/TS is an involved in immortalization of primary human prostate epithe- organizational program of the Oak Ridge Institute for Science and lial cells induced by telomerase. Mol Carcinog 2008;47:775–83. Education and is operated by ORAU for the U.S.DOE. The content 14. Tanaka K, Kohda A, Toyokawa T et al. Chromosome aberra- is solely the responsibility of the authors and does not reflect the tion frequencies and chromosome instability in mice after long- official views or opinions of the U.S. DOE. term exposure to low-dose-rate gamma-irradiation. Mutat Res 2008;657:19–25. 15. Tanaka K, Kumaravel TS, Ihda S et al. Characterization of late- CONFLICT OF INTEREST The authors declare that there are no conflicts of interest. arising chromosome aberrations in human B-cell lines estab- lished from alpha-ray– or gamma-ray–irradiated lymphocytes. Cancer Genet Cytogenet 2008;187:112–24. 16. Hande MP, Azizova TV, Burak LE et al. Complex chromosome FUNDING The study was supported by funding from the United States aberrations persist in individuals many years after occupational exposure to densely ionizing radiation: an mFISH study. Genes Department of Energy (US DOE; Contract# DE-AC05-06OR23100). Chromosomes Cancer 2005;44:1–9. 17. Livingston GK, Falk RB, Schmid E. Effect of occupational radi- REFERENCES ation exposures on chromosome aberration rates in former plu- tonium workers. Radiat Res 2006;166:89–97. 1. Livingston GK, Foster AE, Elson HR. Effect of in vivo exposure to iodine-131 on the frequency and persistence of micronuclei 18. Matsumoto K, Ramsey MJ, Nelson DO et al. 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Journal of Radiation ResearchOxford University Press

Published: Jan 1, 2018

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