Retrospective Evaluation of Cytogenetic Effects Induced by Internal Radioiodine Exposure: A 27-Year Follow-Up Study.

Radioiodine (131I) is widely used in the treatment of hyperthyroidism and as an effective ablative therapy for differentiated thyroid cancer. Radioiodine (131I) constitutes 90% of the currently used therapies in the field of nuclear medicine. Here, we report the cytogenetic findings of a long-term follow-up study of 27 years on a male patient who received two rounds of radioiodine treatment within a span of 26 months between 1992 and 1994 for his papillary thyroid cancer. A comprehensive cytogenetic follow-up study utilizing cytokinesis blocked micronucleus assay, dicentric chromosome assay, genome wide translocations and inversions was initiated on this patient since the first administration of radioiodine in 1992. Frequencies of micronuclei (0.006/cell) and dicentric chromosomes (0.008/cell) detected in the current study were grossly similar to that reported earlier in 2019. The mFISH analysis detected chromosome aberrations in 8.6% of the cells in the form of both unbalanced and balanced translocations. Additionally, a clonal translocation involving chromosomes 14p; 15q was observed in 2 of the 500 cells analyzed. Out of the 500 cells examined, one cell showed a complex translocation (involving chromosomes 9, 10, and 16) besides 5 other chromosome rearrangements. Collectively, our study indicates that the past radioiodine exposure results in long-lasting chromosome damage and that the persistence of translocations can be useful for both retrospective biodosimetry and for monitoring chromosome instability in the lymphocytes of radioiodine exposed individuals.

Cytogenetic follow-up studies on humans with internal and external exposure to ionizing radiation.

Cells exposed to ionizing radiation have a wide spectrum of DNA lesions that include DNA single-strand breaks, DNA double-strand breaks (DSBs), oxidative base damage and DNA-protein crosslinks. Among them, DSB is the most critical lesion, which when mis-repaired leads to unstable and stable chromosome aberrations. Currently, chromosome aberration analysis is the preferred method for biological monitoring of radiation-exposed humans. Stable chromosome aberrations, such as inversions and balanced translocations, persist in the peripheral blood lymphocytes of radiation-exposed humans for several years and, therefore, are potentially useful tools to prognosticate the health risks of radiation exposure, particularly in the hematopoietic system. In this review, we summarize the cytogenetic follow-up studies performed by REAC/TS (Radiation Emergency Assistance Center/Training site, Oak Ridge, USA) on humans exposed to internal and external radiation. In the light of our observations as well as the data existing in the literature, this review attempts to highlight the importance of follow-up studies for predicting the extent of genomic instability and its impact on delayed health risks in radiation-exposed victims.

Detection of Simple, Complex, and Clonal Chromosome Translocations Induced by Internal Radioiodine Exposure: A Cytogenetic Follow-Up Case Study after 25 Years.

Here, we report the findings of a 25-year cytogenetic follow-up study on a male patient who received 2 rounds of radioiodine treatment within a span of 26 months (1.78 GBq in 1992 and 14.5 GBq in 1994). The patient was 34 years old with a body mass index of 25 at the time of the first radioiodine treatment. Multicolor FISH and multicolor banding (mBAND) techniques performed on the patient detected inter- and intrachromosomal exchanges. Although the frequency of chromosome translocations remained essentially the same as reported in our earlier study (0.09/cell), the percentage of reciprocal (balanced) translocations increased from 54.38 to 80.30% in the current study. In addition to simple chromosome translocations, complex exchanges (0.29%) involving more than 2 chromosomes were detected for the first time in this patient. Strikingly, a clonal translocation involving chromosomes 14 and 15, t(14p;15q), was found in 7 of the 677 cells examined (1.03%). The presence of complex and clonal translocations indicates the onset of chromosomal instability induced by internal radioiodine exposure. mBAND analysis using probes specific for chromosomes 1, 2, 4, 5, and 10 revealed 5 inversions in a total of 717 cells (0.69%), and this inversion frequency is several-fold higher than the baseline frequency reported in healthy individuals using the classical G-banding technique. Collectively, our study suggests that stable chromosome aberrations such as translocations and inversions can be useful not only for retrospective biodosimetry but also for long-term monitoring of chromosomal instability caused by past radioiodine exposure.

Development of electronic training and telescoring tools to increase the surge capacity of dicentric chromosome scorers for radiological/nuclear mass casualty incidents.

Dicentric chromosome assay (DCA) is most frequently used for estimating the absorbed radiation dose in the peripheral blood lymphocytes of humans after occupational or incidental radiation exposure. DCA is considered to be the "gold standard" for estimating the absorbed radiation dose because the dicentric chromosome formation is fairly specific to ionizing radiation exposure and its baseline frequency is extremely low in non-exposed humans. However, performance of DCA for biodosimetry is labor intensive and time-consuming making its application impractical for radiological/nuclear mass casualty incidents. Realizing the critical need for rapid dose estimation particularly after radiological/nuclear disaster events, several laboratories have initiated efforts to automate some of the procedural steps involved in DCA. Although metaphase image capture and dicentric chromosome analysis have been automated using commercially available platforms, lack or an insufficient number of these platforms may pose a serious bottleneck when hundreds and thousands of samples need to be analyzed for rapid dose estimation. To circumvent this problem, a web-based approach for telescoring was initiated by our laboratory, which enabled the cytogeneticists around the globe to analyze and score digital images. To further increase the surge capacity of dicentric scorers, we recently initiated a dicentric training and scoring exercise involving a total of 50 volunteers at all academic levels without any prerequisite for experience in radiation cytogenetics. Out of the 50 volunteers enrolled thus far, only one outlier was found who overestimated the absorbed radiation dose. Our approach of training the civilians in dicentric chromosome analysis holds great promise for increasing the surge capacity of dicentric chromosome scorers for a rapid biodosimetry in the case of mass casualty scenarios.

Extension of lymphocyte viability for radiation biodosimetry: Potential implications for radiological/nuclear mass casualty incidents.

Dicentric chromosome assay (DCA) is routinely used for estimating the absorbed radiation dose in exposed humans. Optimal lymphocyte viability is crucial for reliable dose estimation and most cytogenetic laboratories prefer the receipt of blood samples within 24 to 36 hours after collection. Delays in the shipment/receipt of samples can occur sometimes under certain unforeseen circumstances: (1) Adverse weather conditions, (2) distant location of blood collection sites, and (3) shipping and handling of a large number of samples after radiological/nuclear mass casualty incident(s). To circumvent some of these limitations, we evaluated the suitability of ex vivo irradiated blood samples stored in the presence of phytohemagglutinin (PHA) for 7 days at ambient temperature (22-24°C) for radiation biodosimetry. Blood samples stored in the presence of PHA for up to 7 days showed a higher mitotic index than blood samples stored without PHA. To verify the use of stored blood samples for DCA, frequencies of X-rays induced dicentric chromosomes were analyzed in the blood samples that were cultured either 24 hours after exposure or 7 days later after storage. Our results indicate that storage of ex vivo irradiated blood samples in the presence of PHA at ambient temperature was found optimal for DCA and that the radiation doses estimated by dicentric chromosome frequencies were grossly similar between the fresh and stored blood samples. Our study suggests that reliable and accurate biodosimetry results can be obtained for triage using blood samples stored for up to a week at ambient temperature in the presence of PHA.

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

Our previous studies demonstrated the cytogenetic effects in the peripheral blood lymphocytes of a 34-year-old male patient who received ablative radioactive 131iodine 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.

Cytogenetic effects of radioiodine therapy: a 20-year follow-up study.

The purpose of this study was to compare cytogenetic data in a patient before and after treatment with radioiodine to evaluate the assays in the context of biological dosimetry. We studied a 34-year-old male patient who underwent a total thyroidectomy followed by ablation therapy with (131)I (19.28 GBq) for a papillary thyroid carcinoma. The patient provided blood samples before treatment and then serial samples at monthly intervals during the first year period and quarterly intervals for 5 years and finally 20 years after treatment. A micronucleus assay, dicentric assay, FISH method and G-banding were used to detect and measure DNA damage in circulating peripheral blood lymphocytes of the patient. The results showed that radiation-induced cytogenetic effects persisted for many years after treatment as shown by elevated micronuclei and chromosome aberrations as a result of exposure to (131)I. At 5 years after treatment, the micronucleus count was tenfold higher than the pre-exposure frequency. Shortly after the treatment, micronucleus counts produced a dose estimate of 0.47 ± 0.09 Gy. The dose to the patient evaluated retrospectively using FISH-measured translocations was 0.70 ± 0.16 Gy. Overall, our results show that the micronucleus assay is a retrospective biomarker of low-dose radiation exposure. However, this method is not able to determine local dose to the target tissue which in this case was any residual thyroid cells plus metastases of thyroidal origin.

Evaluation of the annual Canadian biodosimetry network intercomparisons.

PURPOSE: To evaluate the importance of annual intercomparisons for maintaining the capacity and capabilities of a well-established biodosimetry network in conjunction with assessing efficient and effective analysis methods for emergency response. MATERIALS AND METHODS: Annual intercomparisons were conducted between laboratories in the Canadian National Biological Dosimetry Response Plan. Intercomparisons were performed over a six-year period and comprised of the shipment of 10-12 irradiated, blinded blood samples for analysis by each of the participating laboratories. Dose estimates were determined by each laboratory using the dicentric chromosome assay (conventional and QuickScan scoring) and where possible the cytokinesis block micronucleus (CBMN) assay. Dose estimates were returned to the lead laboratory for evaluation and comparison. RESULTS: Individual laboratories performed comparably from year to year with only slight fluctuations in performance. Dose estimates using the dicentric chromosome assay were accurate about 80% of the time and the QuickScan method for scoring the dicentric chromosome assay was proven to reduce the time of analysis without having a significant effect on the dose estimates. Although analysis with the CBMN assay was comparable to QuickScan scoring with respect to speed, the accuracy of the dose estimates was greatly reduced. CONCLUSIONS: Annual intercomparisons are necessary to maintain a network of laboratories for emergency response biodosimetry as they evoke confidence in their capabilities.

Management of ionizing radiation injuries and illnesses, Part 3: Radiobiology and health effects of ionizing radiation.

Ionizing radiation exposure can induce profound changes in intracellular components, potentially leading to diverse health effects in exposed individuals. Any cellular component can be damaged by radiation, but some components affect cellular viability more profoundly than others. The ionization caused by radiation lasts longer than the initial inciting incident, continuing as 1 ionization incident causes another. In some cases, damage to DNA can lead to cellular death at mitosis. In other cases, activation of the genetic machinery can lead to a genetic cascade potentially leading to mutations or cell death by apoptosis. In the third of 5 articles on the management of injuries and illnesses caused by ionizing radiation, the authors provide a clinically relevant overview of the pathophysiologic process associated with potential exposure to ionizing radiation.

Role of dicentric analysis in an overarching biodosimetry strategy for use following a nuclear detonation in an urban environment.

In the moments immediately following a nuclear detonation, casualties with a variety of injuries including trauma, burns, radiation exposure, and combined injuries would require immediate assistance. Accurate and timely radiation dose assessments, based on patient history and laboratory testing, are absolutely critical to support adequately the triage and treatment of those affected. This capability is also essential for ensuring the proper allocation of scarce resources and will support longitudinal evaluation of radiation-exposed individuals and populations. To maximize saving lives, casualties must be systematically triaged to determine what medical interventions are needed, the nature of those interventions, and who requires intervention immediately. In the National Strategy for Improving the Response and Recovery for an Improvised Nuclear Device (IND) Attack, the U.S. Department of Homeland Security recognized laboratory capacity for radiation biodosimetry as having a significant gap for performing mass radiation dose assessment. The anticipated demand for radiation biodosimetry exceeds its supply, and this gap is partly linked to the limited number and analytical complexity of laboratory methods for determining radiation doses within patients. The dicentric assay is a key component of a cytogenetic biodosimetry response asset, as it has the necessary sensitivity and specificity for assessing medically significant radiation doses. To address these shortfalls, the authors have developed a multimodal strategy to expand dicentric assay capacity. This strategy includes the development of an internet-based cytogenetics network that would address immediately the labor intensive burden of the dicentric chromosome assay by increasing the number of skilled personnel to conduct the analysis. An additional option that will require more time includes improving surge capabilities by combining resources available within the country's 150 clinical cytogenetics laboratories. Key to this intermediate term effort is the fact that geneticists and technicians may be experts in matters related to identifying chromosomal abnormalities related to genetic disorders, but they are not familiar with dosimetry for which training and retraining will be required. Finally, long-term options are presented to improve capacity focus on ways to automate parts of the dicentric chromosome assay method.

A modified system for analyzing ionizing radiation-induced chromosome abnormalities.

The analysis of dicentric chromosomes in human peripheral blood lymphocytes (PBLs) by Giemsa staining is the most established method for biological dosimetry. However, this method requires a well-trained person because of the difficulty in detecting aberrations rapidly and accurately. Here, we applied a fluorescence in situ hybridization (FISH) technique, using telomere and centromere peptide nucleic acid (PNA) probes, to solve the problem of biological dosimetry in radiation emergency medicine. A comparison by a well-trained observer found that FISH analysis of PBLs for the dose estimation was more accurate than the conventional Giemsa analysis, especially in samples irradiated at high doses. These results show that FISH analysis with centromeric/telomeric PNA probes could become the standard method for biological dosimetry in radiation emergency medicine.

Biological dosimetry by the triage dicentric chromosome assay: potential implications for treatment of acute radiation syndrome in radiological mass casualties.

Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.

WHO 1st consultation on the development of a global biodosimetry laboratories network for radiation emergencies (BioDoseNet).

The World Health Organization (WHO) held a consultation meeting at WHO Headquarters, Geneva, Switzerland, December 17-18, 2007, to develop the framework for a global biodosimetry network. The WHO network is envisioned to enable dose assessment using multiple methods [cytogenetics, electron paramagnetic resonance (EPR), radionuclide bioassays, etc.]; however, the initial discussion focused on the cytogenetic bioassay (i.e., metaphase-spread dicentric assay). Few regional cytogenetic biodosimetry networks have been established so far. The roles and resources available from United Nations (UN) agencies that provide international cooperation in biological dosimetry after radiological emergencies were reviewed. In addition, extensive reliance on the use of the relevant International Standards Organization (ISO) standards was emphasized. The results of a WHO survey of global cytogenetic biological dosimetry capability were reported, and while the survey indicates robust global capability, there was also a clear lack of global leadership and coordination. The expert group, which had a concentrated focus on cytogenetic biodosimetry, formulated the general scope and concept of operations for the development of a WHO global biodosimetry laboratory network for radiation emergencies (BioDoseNet). Follow-on meetings are planned to further develop technical details for this network.

Interlaboratory comparison of the dicentric chromosome assay for radiation biodosimetry in mass casualty events.

This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose-effect calibration curve for the yield of dicentrics for (60)Co gamma rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + alphaD + betaD(2). For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.

Effect of occupational radiation exposures on chromosome aberration rates in former plutonium workers.

A fluorescence in situ hybridization (FISH) method was used to measure chromosome aberration rates in lymphocytes of 30 retired plutonium workers with combined internal and external radiation doses greater than 0.5 Sv along with 17 additional workers with predominantly external doses below 0.1 Sv. The former group was defined as high-dose and the latter as low-dose with respect to occupational radiation exposure. The two groups were compared to each other and also to 21 control subjects having no history of occupational radiation exposure. Radiation exposures to the high-dose group were primarily the result of internal depositions of plutonium and its radioactive decay products resulting from various work-related activities and accidents. The median external dose for the high-dose group was 280 mSv (range 10-730) compared to a median of 22 mSv (range 10-76) for the low-dose group. The median internal dose to the bone marrow for the high-dose group was 168 mSv (range 29-20,904) while that of the low-dose group was considered negligible. Over 200,000 metaphase cells were analyzed for chromosome aberrations by painting pairs 1, 4 and 12 in combination with a pancentromeric probe. Additionally, 136,000 binucleated lymphocytes were analyzed for micronuclei in parallel cultures to assess mitotic abnormalities arising from damaged chromosomes. The results showed that the frequency of structural aberrations affecting any of the painted chromosomes in the high-dose group correlated with the bone marrow dose but not with the external dose. In contrast, the frequency of micronuclei did not vary significantly between the study groups. The total translocation frequency per genome equivalent x 10(-3) +/- SE was 4.0 +/- 0.6, 9.0 +/- 1.1 and 17.0 +/- 2.1 for the control, low-dose and high-dose groups, respectively. Statistical analysis of the data showed that the frequency of total translocations and S-cells correlated with the bone marrow dose, with P values of 0.005 and 0.004, respectively. In contrast, these two end points did not correlate with the external dose, with P values of 0.45 and 0.39, respectively. In conclusion, elevated rates of stable chromosome aberrations were found in lymphocytes of former workers decades after plutonium intakes, providing evidence that chronic irradiation of hematopoietic precursor cells in the bone marrow induces cytogenetically altered cells that persist in peripheral blood.