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Biological dosimetry by chromosome analysis
Isamu Hayata
National Institute of Radiological Sciences, Japan
Биологическая дозиметрия с помощью хромосомного анализа
Исаму Хайата
Национальный институт радиологических наук, Япония
В работе на основе анализа литературных данных проводится оценка информативности и точности цитогенетических методов определения поглощенных доз ионизирующего излучения. Делается заключение, что диапазон доз, который может быть определен с помощью хромосомного анализа, лежит между 0.2-0.3 и 6-8 Гр в случае острого рентгеновского или у-облучения. Точная оценка доз может быть выполнена в пределах трех месяцев после облучения. Частота аберраций нестабильного типа (дицентрики и кольца) быстро падает в течение двух лет и после этого регистрировать их трудно. Теоретически оценка доз может быть проведена путем анализа аберраций стабильного типа (транслокации) с использованием метода специального окрашивания ^Ю^метод) в случае облучения свыше двух лет назад или хронического облучения. Подчеркивается необходимость внедрения автоматизированных систем цитогенетического анализа, поскольку подсчет аберраций при дозах ниже 0.2-0.3 Гр требует значительного объёма работы. Необходимо также определение стандартной кривой доза-эффект для FISH-метода.
Chromosome aberrations in the peripheral lymphocytes are an excellent indicator for the estimation of radiation dose. Figs. 1 and 2 show schematic figures of such aberrations. A multicentric with more then two centromeres occasionally seen in an irradiated cell is counted as two or more dicentrics in the dosimetry. The dicentrics, rings, and fragments are unstable, and are lost during cell division. The translocations and inversions are stable, and are inherited by the daughter cells. Both the unstable and stable types are thought to be produced in equal proportions, since the only difference between them is which partners join during the repair process as shown in Figs. 1 and 2.
Traditionally, unstable type aberrations are widely used for dosimetry because they can be detected without making a karyotype. The yield of the aberrations (Y) is related to dose (D) by an equation such as
Y = A + aD+ fi D2.
The coefficients A (background frequency), a(co-efficient related to aberrations each induced by a sin-
gle track), fi (coefficient related to aberrations each induced by two tracks) can be obtained by experimental study using irradiated peripheral blood.
The dose response curve described above has specificity depending on the type of radiation. In the case of low LET radiation, the frequency of ionization per unit track length is low. So, there is a low probability of producing a dicentric (or ring) by a single track and there is a higher probability of producing such an aberration by two independent tracks. As the LET of the radiation increases, there is a greater probability that two lesions within the target which result in a dicentric (or ring) will be induced by a single track.
The dose response curve is also affected by the dose rate. When the dose rate is low, the aberrations induced by two tracks decrease and the curve becomes less steep.
The lower limit of dose that can be estimated by chromosome analysis is considered to be about two cGy by X- or gamma-ray exposure according to [1, 2]. However, in such a low dose range an enormous number of cells has to be analyzed.
* 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.
Dicentric + F г figment
н
Тг ал sloe a.tio n
Fig. 1. Schematic figures of induction of a dicentric plus a fragment (unstable type aberrations) and a translocation (stable type aberration).
Fragment + Ring
Fig.2. Schematic figures of induction of an inversion (stable type aberration) and a fragment plus a ring (unstable type aberration).
cells per day in routine work. Therefore, it takes one technician about one week for the estimation of a 0.2-
0.3 Gy level. If one week's work load per case is assumed as a practical limit for analysis, it can be said that the lower dose limit that can be estimated by chromosome analysis is around this level.
The upper dose limit that can be estimated by chromosome analysis would be determined by the appearance of heavily damaged cells which have lost the capability to enter the metaphase stage in the cell cycle. A lack of dose response curve fitting to
Y - A + a D +fi • D2
becomes noticeable when doses higher that about 6 or 8 Gy of X-rays are used as shown in Table 2 [3, 4]. The upper limit for X-ray exposure would be 6-8 Gy.
The number of cells (N) to be scored is given by the following equation
Z -_____________S________
95 Ip• (1 - p) '
V N
where Z95 is the 95% confidence level;
p is the probability of chromosome aberrations;
S is the relative statistical error.
Examples of the number of cells to be scored are given in Table 1.
The aberration frequency level induced by acute exposure of X- or gamma-rays at a dose of 0.2-0.3 Gy is p = 0.01. It is said that one can analyze 300-350
Table 1
Number of cells to be scored in dosimetry
s % P
0.1 | °.01 0.001
± 10 3457 38832 383776
± 20 864 9508 95944
± 50 138 1521 15351
Table 2
Results of fitting data of Norman and Sasaki (1966) showing the effect of removing the higher dose levels
The lifespan of the majority of lymphocytes is long, with a half-life of about three years. J.G. Brewen et al. [5] showed that the frequency of cells with unstable type aberrations did not decrease significantly during five weeks after exposure. The decrease was not found during three months after the exposure. It was showed that about one third of the cells with unstable type aberrations were lost in two years [6, 7]. The frequency of the cells with stable type aberrations did not change for many years.
In the case of chronic exposure new unstable type aberrations are produced regularly, while some of the aberrations are lost constantly. If the duration of exposure is long, the yield of unstable type aberrations is influenced more the dose rate than by the total dose accumulated. In such cases, theoretically, a stable type aberrations is a better indicator for estimating the total dose.
The stable type chromosome aberrations such as translocations can now be easily defected using a Painting method. D.Pinkel et al. [8] succeeded for the first time in coloring the whole of some chromosome pairs by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes. When translocation occurred between a painted chromosome and a non-painted chromosome, the aberrant chromosome shows mosaic pattern so that the aberration can be detected with ease. According to [9], genomic translocation frequency can be obtained by the equation
Fp - 2.05 • fp • (1 - fp)• Fg,
where Fp is the translocation frequency measured using FISH;
Fg is the genomic translocation frequency;
fp is the fraction of the genome covered by the fluorescent probe.
For example, J.N.Lucas et al. [9] demonstrated in the case of A-bomb survivors that the result obtained by the painting method was in accord with that obtained by karyotyping [10].
The painting method has been confirmed to be an easy and useful technique for demonstrating structural rearrangements of chromosomes. It will produce more accurate data if some improvements are made in the following areas.
1) It is not always easy to distinguish stable type aberrations (translocations) from unstable type aberrations (dicentrics) in this method even if a dual staining supplemented by the centromeric heterochromatin specific probe were used.
2) Experimental results do not confirm the theoretically predicted ratio of translocations vs. dicentrics of 1:1. Translocation seems to have occurred excessively in the exposed cells [11-20, and our unpublished data].
3) Chromosome rearrangements do not occur at random relative to the chromosome lengths [20]. So, the equation
Fp - 2.05 • fp • (1 - fp)• Fg
may not give the exact genomic frequency of translocations.
4) The background frequency of translocations is 5 to 10 times higher than that of dicentrics. In addition, since the aberrations can be detected in only a part of genome (usually one-fifth to one-third) by the painting method, five to three time more cells than those analysed by the traditional method have to be scored in order to achieve statistically significant data by the painting method. Therefore, the estimation of low dose exposure by translocation becomes difficult.
It is necessary to make more studies to find ways of effectively applying the painting method.
In summary, the range of doses that can be estimated by chromosome analysis is between 0.2-0.3 and 6-8 Gy of an acute exposure to X- or gamma-rays. Dose estimation can be made accurately within three months after the exposure. The frequency of unstable type aberrations (dicentrics and rings) drops rapidly in two years and after that detecting them becomes difficult. Theoretically, dose estimation can be done by analyzing stable type aberrations (translocations) using the painting method in cases of exposures more than 2 years old or of chronic exposures. As described above, applying the painting method does not result in any reduction of the actual work load for estimating low doses. Therefore, the introduction of an automated system is urgently recommended because it will cope with the enormous amount of work that will be required for scoring aberrations at doses lower than 0.2-0.3 Gy. It is also necessary to establish a standard dose response curve for the painting method.
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