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Radiation Dose Assessment by Electron Spin Resonance Measurement on Tooth Enamel from Atomic-Bomb Survivors *
Nori Nakamura, Midori Iwasaki*, Chyuzo Miyazawa*, Katsumi Niwa*, Mitoshi Akiyama, Shozo Sawada** and Akio A.Awa
Departments of Genetics and Radiobioloqy, Radiation Effects Research Foundation, Hiroshima, Japan;
* - Departments of Dental Radiology and Preventive Dentistry, Ohu University, Koriyama, Japan;
** - Department of Radiobioloqy, Research Institute for Nuclear Medicine and Bioloqy, Hiroshima University, Hiroshima, Japan
Electron spin resonance (ESR) measurement was conducted for 11 teeth donated by 10 atomic-bomb survivors in Hiroshima. The ESR signal intensity and chromosome aberration frequency in lymphocytes of the tooth donors showed a good correlation. In one exceptional case, however, high chromosome aberration frequency failed to accompany any sign of radiation exposure in tooth enamel. The sample was found to be a wisdom tooth and the donor was 15 years of age at the time of bombing, raising a possibility that the enamel of the tooth was not completely developed at the time of radiation exposure. Our experience shows importance to collect detailed information on the donors whose biological materials were used for radiation dose reconstruction.
Оценка доз внешнего облучения лиц, переживших атомную бомбардировку, по ЭПР-спектроскопии эмали зубов
Нори Накамура, Мидори Ивасаки*, Чуизо Миязава*, Катсуми Нива*, Митоши Акияма, Шозо Савада**, Акио A.Ава
Фонд исследования радиационных эффектов, Хиросима, Япония;
* - Университет Оху, Корияма, Япония;
** - Институт ядерной медицины и биологии, Хиросима; Япония
Измерения спектров электронного парамагнитного резонанса (ЭПР) были проведены на 11 зубах, удаленных у 10 жителей Хиросимы, переживших атомную бомбардировку. Продемонстрирована хорошая корреляция интенсивности ЭПР-сигнала и частоты хромосомных аберраций в лимфоцитах доноров зубов. В одном случае, однако, высокая частота хромосомных аберраций не сопровождалась каким-либо признаком большой дозы облучения, определенной методом ЭПР по эмали зубов. Оказалось, что в этом случае измеряемый образец эмали приготовлен из зуба мудрости, эмаль которого на момент бомбардировки не полностью сформировалась (донору на момент бомбардировки было 15 лет). Результаты работы указывают на важность сбора детальной информации о донорах, чей биологический материал использовался для реконструкции дозы облучения.
1. Introduction
At Radiation Effects Research Foundation (RERF), former Atomic Bomb Casualty Commission, cytogenetic examination of peripheral blood lymphocytes from atomic-bomb (A-bomb) survivors has been conducted since late 1960s as a measure of radiation effects. Because the cytogenetic program was initiated more than 20 years after the radiation exposure, unstable chromosome aberrations (i.e., dicentrics, rings, fragments) had almost disappeared. Therefore, stable aberrations (i.e., translocations, inversions,
deletions) were the only useful endpoints to examine, but these are also known to require good skill for their proper detection. A cytogenetic laboratory was created and lymphocytes from a total of approximately 5000 blood samples from more than 2500 survivors have been hitherto examined. A part of the results is shown in Fig. 1. It is obvious from the figure that there is a considerable scatter in the dose response. At least two immediate explanations are possible. One is the individual variation of lymphocyte radiosensitivity, and the other is the error in dose estimation.
* Работа была представлена на российско-японском симпозиуме "Проблемы реконструкции индивидуальных поглощенных доз в результате крупномасштабных радиационных аварий и оценки радиационных рисков",
Москва, 20-21 октября, 1994 г.
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12 DS86 Bone marrow dose (Sv)
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Figure 1. Frequency of lymphocytes bearing stable chromosome aberrations against DS86 estimated dose for 181 Hiroshima survivors. r is the correlation coefficient between the estimated parameters.
The first hypothesis was tested recently by a dose-survival study using colony assay of peripheral blood lymphocytes in vitro. The lymphocytes were irradiated with X-rays (0 to 5 Gy) and then incubated for 2 weeks in 96-well microtiter plates with culture medium containing recombinant interleukin-2, a T cell growth factor, to allow the surviving cell to from colonies. We found that the variation in the dose response for 31 different individuals did not significantly differ from that for 28 repeated tests for a single donor [1]. Because unstable chromosome aberrations have been considered as the major cause of loss colony forming capability, the results strongly suggest that the first hypothesis is not the major cause.
Another source of evidence for the absence of large individual variation is obtained by in vitro experiments for the induction of dicentrics after X-irradiation of peripheral blood lymphocytes. Lymphocytes from those who appeared radiosensitive or resistant in Fig. 1 did not show different responses in vitro [2].
The second hypothesis for the presence of errors in physical dose evaluation has been suggested for many years. This is mainly due to the fact that all the necessary information for the computer calculation of dose is based on interview records that were collected between 5 to 10 years after the bombings. Imagine who can correctly recall the car accident that happened 5 to 10 years ago? Further, the interview records do not necessarily include all the information required for computer simulation. As a consequence, certain errors in physical dose evaluation cannot be avoided.
In order to provide an additional evidence in favor of the second hypothesis, we started collection of teeth from proximally exposed A-bomb survivors for electron spin resonance (ESR) measurement.
ESR assay detects radicals (unpaired electrons) caused by radiation exposure and tooth enamel and bones are suited for this purpose. Because bones are continuously remodeled and are not readily accessible, mainly teeth have been used in ESR studies to date. Enamel, the covering of the tooth surface, consists mostly of hydroxyapatite (a compound of crystalline structure consisting of calcium and phosphate) and is free from any metabolism. Tooth enamel is a unique inorganic body structure.
Although laboratory studies about ESR have been published since the 1960s, in the past decade ESR has received renewed attention. In Japan, M. Ikeya now at Osaka University has actively promoted ESR studies [3, 4]. A series of ESR studies on tooth enamel from mainly Nagasaki A-bomb survivors has been published by S. Okajima's research group at Nagasaki University [5, 6]. These results have hot been compared with individual Dosimetry System 1986 (DS86) dose estimates.
Current knowledge about ESR of tooth enamel is summarized as below.
1. It has been believed that CO3-3 radicals are being measured.
2. Photon-energy dependence is evident, e.g., 40 keV X-rays generate an signal more than 5 time greater than 60Co or 137Cs y-rays per unit dose [7, 8].
3. Compared to gamma-rays, neutrons are much less effective in producing ESR signals [9, 10].
4. Observed ESR signal intensity is linearly proportional (up to 30 mg) to the mass of enamel examined [11].
5. No dose-rate effect was observed after in vitro exposure to gamma rays with dose rates ranging from 225-0.33 R/min [12].
6. Irradiation of enamel samples in vitro, either in dry conditions or in water, produced identical ESR signal intensities [12].
7. Enamel grain sizes of 0.5-1.4 mm in diameter are preferred [13].
Several problems inherently associated with tooth ESR assay are also recognized. One is the adverse effect of dentine contamination. Because dentine gives rise to a larger background ESR signal compared with enamel, its contamination will obscure the presence of small radiation-related signal. In addition, dentine is quite ineffective in producing radiation-related signal. Consequently, after in vitro gamma-irradiation of known doses, the increase of radiation-related signal may well vary depending on the fraction of dentine in the preparation (e.g., compared with 100% enamel, the increase of ESR signal is expected to be only about one half if dentine contamination is 50%). Thus, careful isolation of enamel is recommended as much as possible.
Another problem is how to take into account the contribution of dental X-rays (which have an effective energy of 30 keV or less). As mentioned earlier, such low-energy photons are much more effective than 60Co y-rays and thus may contribute significantly to the ESR signal whereas actually contributing much less to the dose. We assume that most of the diagnostic dental X-ray came from outside of the tooth. Consequently, if we divide each tooth into two parts before separation of enamel - one half from the inside of the mouth and the other from the outside - the outer half would show radiation-related ESR signal equal to or larger than that of inner half. Whenever a larger ESR signal is seen in the outer half than in the inner half of the tooth, we need particular caution in interpreting the results for that donor.
2. Materials and methods
Over three hundred teeth were donated by Hiroshima A-bomb survivors during the past 7 years. Among these, one hundred teeth were selected according to the good condition (i.e., no severe decay) and to the DS86 estimated dose.
For the enamel separation, a disc-shaped diamond cutter was used with running water. This is totally a mechanical separation with no chemical treatment. The enamel was then crushed in an agate mortar until the grain size reached roughly homogeneous (0.5-1.4 mm in diameter).
ESR measurements were conducted at room temperature using an X-band spectrometer (JOEL RE-1X). A field modulation of 100 kHz in frequency and of 0.32 mT in width was used at a microwave of 5 mW, with a time constant of 0.1 sec and a field sweep of 10 mT in 16 min. The enamel sample and internally located manganese marker were measured simultaneously.
Two ml of peripheral blood is routinely used for cytogenetic examinations. After incubation for 48 hrs in the PHA-containing medium (colcemid was present during the last 2 hrs), metaphase preparation was made following the standard protocol [14]. The slides were stained with Giemsa and the frequency of lymphocytes bearing stable chromosome-type aberrations (mainly reciprocal translocations but include inversions and deletions as well) was assessed.
3. Results and discussion
Chromosome aberration frequency in lymphocytes from the tooth donors is plotted against DS86 estimated dose (Fig. 2). A considerable scatter is apparent as seen in Fig. 1. Eleven tooth samples from 10 donors were so far examined by ESR; 5 donors are close to the average and the remaining 5 are so called "outliers" (2 are high above and 3 are far below the average aberration frequency).
Examples of the ESR signals are shown in Fig. 3 and the frequency of chromosome aberrations in lymphocytes is indicated on the right. The summary of chromosome aberration frequency vs. ESR signal intensity is shown in Fig. 4.
Present ESR measurements do not include any in vitro gamma-irradiation of known doses. Thus, the results may not necessarily correspond linearly with dose because each tooth may respond to radiation somewhat differently. Nevertheless, several interesting features are apparent. First, the two tooth samples derived from the same donor gave similar results. Second, the chromosome-aberration data correlated fairly well with the ESR data and outliers appear to be no more a serious problem. Third, there was one exceptional case, however. The tooth of a survivor whose DS86 dose is 1.4 Gy and who has a 35% frequency of lymphocytes with chromosome aberration(s) (the first case shown in Fig. 3) showed no sign of radiation exposure! That particular sample was found to be a wisdom tooth, known to be formed much later than other human teeth, and the tooth donor was 15 years of age at the time of bombing. Because individual variation in the development of wisdom teeth is quite wide, we do not know if the tooth in question was really underdeveloped at the time of exposure in 1945. We hope to examine another tooth other than wisdom tooth from this donor in the future.
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DS86 dose (Gy)
Figure 2. Lymphocyte chromosome aberration frequency of tooth donors against DS86 estimated dose.
The closed circles represent cases for whom tooth ESR measurements were performed.
Figure 3. Examples of ESR signal of tooth enamel.
On the left is shown the total amount of enamel examined and on the right the frequency of lymphocytes with stable chromosome aberration(s). The third and fourth samples are derived from a single donor. The inversed signals on both ends represent the third and fourth signals of manganese used for internal marker. Triangles show the position of radiation-related signal.
ESR signal intensity/100mg enamel
Figure 4. Frequency of lymphocyte chromosome aberration plotted against ESR signal intensity of tooth enamel.
The black circles represent five cases that are close to the average in chromosome-aberration dose response in the larger cohort. The five open circles represent so called outliers. The two black circles connected with a line represent the results of two teeth from the same donor. The ESR signal shown here is for outer halves of the samples.
4. Future prospects
In view of the applicability of ESR for dating fossil teeth, it seems very likely that tooth enamel can properly accumulate radiation doses imparted at extremely low dose rates. This means that human teeth may be distinctive natural biodosimeters not only for acute exposures such as atomic-bomb radiation but also for repeated small doses or chronic y-ray exposures such as radiation workers and people residing in contaminated environment. One serious disadvantage of tooth ESR is that the availability of teeth totally depends on chance. In contrast, blood samples are readily available from virtually any people, and thus cytogenetic examination is superior for dose reconstruction on each individual.
The drawback to use chromosome aberration data for dose reconstruction of chronically exposed people is the lack of information on the dose rate factor. In theory, linear quadratic model predicts that the quadratic component disappears when the dose rate is very reduced. Thus, one may use the fitted linear coefficient of acute dose response curve of chromo-
some aberration induction in vitro. However, it is desirable to collect information on both tooth ESR and chromosome aberration data on the same donors to substantiate the theory.
Then, another problem arises: most of the chromosome aberrations remaining in human body many years after the radiation exposure are stable type aberrations. These aberrations are known to require good skill for proper detection, and interlaboratory comparison cannot be made without careful standardisation of the technique. Fortunately, recently developed fluorescence-in-situ-hybridisation (FISH) technique is very suited for objective scoring of stable-type aberrations, although it is quite expensive. We would initiate concurrent measurements of ESR and FISH for A-bomb survivors, and urge similar studies to be undertaken for chronically exposed people as well so that both sets of data may be compared in the future for proper assessment of radiation dose under various dose rates.
Last, it is noted that the conventional staining method (i.e., simple Giemsa staining method) can detect as much as 70-80 % of stable chromosome
aberrations when compared with FISH method (manuscript in preparation). Because the conventional method does not require any expensive reagents and fluorescence microscopes, training of cytogenetic eye is an alternative choice for large scale survey.
Acknowledgments
We are grateful to the donors of teeth for their cooperation, M. Miura and K. Muramoto for their excellent technical assistance in cytogenetic analyses, M. Utaka for preparation of the manuscript. The study is based on research protocol RP1-92 concerning research performed at RERF, Hiroshima and Nagasaki, Japan. RERF is a private foundation funded equally by the Japanese Ministry of Health and Welfare and the US Department of Energy through the National Academy of Sciences.
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