

ORIGINAL ARTICLE 

Year : 2016  Volume
: 7
 Issue : 1  Page : 2 

Calibration curve for dicentric chromosomes induced in human blood lymphocytes exposed to gamma rays at a dose rate of 12.5 mgy/s
Tran Que, Pham Ngoc Duy, Bui Thi Kim Luyen
Department of Biotechnology, Biodosimetry Section, Nuclear Research Institute, Vietnam Atomic Energy Commission, VINATOM, Dalat, Vietnam
Date of Web Publication  30Dec2016 
Correspondence Address: Tran Que Department of Biotechnology, Biodosimetry Section, Nuclear Research Institute, Vietnam Atomic Energy Commission, VINATOM, No. 1, Nguyen Tu Luc, Dalat Vietnam
Source of Support: None, Conflict of Interest: None  Check 
DOI: 10.4103/20419414.197171
To develop a calibration curve for induction of dicentric chromosomes by radiation, we have used a 60Co gammaray source with dose rate of 12.5 mGy/s. Whole blood from 15 healthy donors was collected. Whole blood from each donor was divided equally into 8 parts for exposing to supposedly physical doses 0, 0.30, 0.50, 1.00, 1.50, 2.00, 3.00 and 4.00 Gy for a independent calibration curve. Whole blood from 15 donors was used to calibrate dose – effect and statistical for general calibration curve. Using Poisson test (Utest) for the distribution of dicentric chromosomes in the metaphases to determine the uniformity of the radiation field. The average from 15 independent calibration curves of linear correlated coefficient was determined to be r (y, d) = 0.5136 ± 0.0038. The model equation derived is y = aD + bD2 + C. The calibration equation of doseeffect was y = 1.01D + 4.43D2 + 0.56. Keywords: Exposed dose point, gamma rays, general curve, independent curve, uniform radiation field
How to cite this article: Que T, Duy PN, Luyen BT. Calibration curve for dicentric chromosomes induced in human blood lymphocytes exposed to gamma rays at a dose rate of 12.5 mgy/s. Genome Integr 2016;7:2 
How to cite this URL: Que T, Duy PN, Luyen BT. Calibration curve for dicentric chromosomes induced in human blood lymphocytes exposed to gamma rays at a dose rate of 12.5 mgy/s. Genome Integr [serial online] 2016 [cited 2019 Sep 19];7:2. Available from: http://www.genomeintegrity.org/text.asp?2016/7/1/2/197171 
Introduction   
The tools of biodosimetry for monitoring individuals exposed to environmental, occupational, clinical, or accidental radiation are background dataset and calibration dose–effect dataset. These datasets need to be created by each biodosimetry laboratory to strengthen their preparedness in response to radiation emergencies. In addition, calibration or dose–response curve can be generated with good regression coefficients but are characterized by linear energy transfer (LET) and dose rate of radiation source. Therefore, choosing an appropriate calibration dose–effect curve close to radiation source to which victim exposed in terms of LET and dose rate is required. The calibration of dose–effect for ^{60} Co gammaray source with dose rate of 12.5 mGy/s was chosen for this study.
Materials and Methods   
Blood was drawn from 15 healthy donors (22–45 years of age) and each blood sample was divided into eight parts for exposing it to radiation doses 0, 0.3, 0.5, 1.0, 1.5, 2.0, 3.0, or 4.0 Gy. A ^{60} Co gamma ray source (gamma cell – Isscledavatel, Russia) was used to irradiate the blood at the dose rate of 12.5 mGy/s.
The component of radiations and the component of radiation dose were also determined by physical dosimetry method and chemical method (Fricke) at Nuclear Research Institute, Vietnam. The absorbed dose values were counted for each exposed times of all supposed dose points, mean that 105 exposed dose values and 15 controls had presented for this calibration. Following radiation exposure, lymphocytes were cultured in RPMI 16040 medium supplemented with phytohemagglutinin and fetal calf serum (Sigma), incubated for 48 h/37°C. Colcemid was added 2 h before harvesting to prepare metaphase spreads. Dicentric chromosome aberration analysis relied on centromere, number of fragments, and according to classification of chromosome aberration in the first cell cycle.^{[1],[2],[3],[4],[5],[6]} Data generated were subjected to ttest for comparisons. In addition, Poisson test was used to estimate dicentric chromosome distributions induced in exposed lymphocytes. The hypothesis of Poisson distribution was done according to the standard of Chisquare. The samples are exposed in uniform radiation field if Chisquare value fits hypothesis value.^{[2],[7],[8]} The correlation coefficient r (y, d) was used to find model equation, and recurrent equation was solved to find recurrent coefficients for independent curves, statistical and presenting general calibration dose–effect curve.^{[6],[7],[8],[9],[10]}
Results and Discussion   
The dicentric data from 15 independent samples were used to generate a composite calibration curve. In this case, we notice that there are individual variations among the different samples. However, 120 data couples on related dose–dicentric were used to find Poisson distribution, linear correlated coefficients, model equation, experimental regressive coefficients, and general calibration equation of dose–effect curve.
Testing of Poisson distribution
Poisson distribution of dicentric chromosomes among metaphases was used to check the uniformity of the radiation field. A total of 548 dicentrics distributed in 758 cells with the number dicentric in each cell are presented in [Table 1].  Table 1: Distribution of dicentric per cell in 10 random samples of 15 independent curves exposed to gamma radiation
Click here to view 
Hypothesis of Poisson distribution was tested according to the standard of Chisquare with formula χ^{2}= Σ_{i}^{k}χ_{i}^{2}= Σ_{i}^{k} ([m_{i} − np_{i}] ^{2}/np_{i}) (i:i = 0, 1, 2, 3 … is natural number showed number dicentric in a cell; m_{i} is the number of cells have i dicentric; n is the total cells analyzed; p_{i} is the theory numerical value of Poisson, p_{i} = N'x/N = e ^{−}^{λ}. λ^{x}/x!; λ: Average frequency of dicentric per cell, λ = Σm_{i}/n). The parameters of Chi square were λ = Σmi/Σn = 548/758 = 0.72; e ^{−}^{λ}= 0.48; Σ_{χ}_{i}^{2} = 1.21 + 0.53 + 1.47 + 0.06 + 0.33 = 3.60. Consulting Chisquare table with k = 5, α = 0.05 had χ^{2}_{k−1} (α) = 9.19. The data presented Σχ_{i}^{2}< χ^{2}_{k−1} (α) which essentially means that dicentrics induced in the cells fited in Poisson distribution. This result ensured the reliability of the samples exposed in the uniform radiation field.
Finding model equation of dose–effect response
The correlation coefficient, r (y, d) = 0.514 ± 0.004, illustrates that there was a average linear correlation between doses and dicentric frequencies, but diagram [Figure 1] of correlation between dose and frequencies of dicentric shows a parabolic form for all of the 15 combinations. The r (y, d) reflected exactly the effect of low LET radiation. The general experimental equation had the form of quadratic equation: y = α D + βD ^{2} + C [Table 2].  Figure 1: The dose–effect calibration curve for dicentric chromosomes in human blood lymphocytes (red  experimental data, blue  graph of calibration equation)
Click here to view 
 Table 2: The results of linear correlative coefficients for 15 independent curves
Click here to view 
Counting experimental recurrent coefficients of model equation y = aD + bD ^{2} + C
Finding the experimental recurrent coefficients a, b, and C of quadratic equation: y = aD + bD ^{2} + C of the independent curves was conducted by solving of the set of three equations:
In these equations, D_{i} indicates the absorbed doses that used for exposing blood samples (eight doses) and y_{i} is induced dicentric frequencies to D_{i}. Using experimental data of dose–effect and using replaced method for solving of the set of three equations above produced, the recurrent values of a, b, and C are shown in [Table 3].  Table 3: The results of the experimental recurrent coefficients of 15 independent curves
Click here to view 
[Table 3] presents the values α, β, and C of 15 independent curves, the averages of these values were α = 1.01 ± 0.93; β = 4.43 ± 0.30; C = 0.56 ± 0.39.
The general experimental recurrent coefficients were α = 1.01 ± 0.93; β = 4.43 ± 0.30; C = 0.56 ± 0.39 (α = 10^{−2} × Gy ^{−1}; β = 10^{−2} Gy ^{−2}). The general experimental recurrent equation was y = 1.01D + 4.43D ^{2} + 0.56.
The dose–effect calibration curve is shown in [Figure 1] (red  experimental data, blue  graph of the general calibration equation).
The data from our study indicated that the samples were exposed in the uniform radiation field. Poisson distribution (Utest) of dicentric chromosomes among metaphases was a parameter that used to check the uniformity of the radiation field.^{[2],[6],[7],[9]} Consulting Chisquare table with k = 5, α = 0.05 had χ^{2}_{k−1} (α) = 9.19. The data presented Σχ_{i}^{2}< χ^{2}_{k−1} (α), it means that induced dicentric distribution in the irradiation cells fitted well with Poisson distribution. This result ensured the reliability of the samples exposed in the uniform radiation field.
Dose–effect relationship followed to the model equation with linear correlative coefficient. The correlation coefficient r (y, d) = 0.514 ± 0.004 did not fit linear correlation between doses and observed dicentric frequencies, but fitted reasonably well with an exponent correlation. A weak correlation coefficient r (y, d) = 0.514 ± 0.004 fitted with a dose–effect distribution y = αD + βD ^{2} + C that was in accordance with low LET radiation in observed source. Our result showed the suitability of the basic principles of radiation effects, such as dependence on LET in line with earlier reports.^{[2],[4],[6],[11],[12],[13],[14],[15],[16]}
The dependence of model equation on the LET as well as the dependence of the coefficient's rate α/β of the calibration equation of dose–effect on dose rate of radiation sources have been observed in earlier publications.^{[10],[11],[12],[13],[14],[15],[16]} Solving of experimental recurrent equation y = αD + βD ^{2} + C showed the experimental recurrent coefficients α = 1.01 ± 0.93 (10^{−2} Gy ^{−1}), β = 4.43 ± 0.30 (10^{−2} Gy ^{−2}), C = 0.56 ± 0.39 (10^{−2}), α/β = 0.228 and calibration dose–effect y = 1.01D + 4.43D ^{2} + 0.56. Our result showed the suitability of the basic principles of radiation effects, such as dependence on energy, LET, and dose rate.
Conclusion   
Calibration of doseeffect was conducted for ^{60} Co gamma rays with dose rate of 12.5 mGy/s. The investigated data of 15 indipendent calibration curves presented that distribution of dicentric chromosome among metaphases of exposed cells was fitted a Poisson distribution with p = 95%, this evidence showed irradiation field was uniform radiation field.
The linear related coefficient r (y, d) = 0.514 ± 0.004, equation model fitted to y = αD + βD ^{2} + C. The experimental recurrent coefficients were determined α = 1.01 ± 0.93 (10^{2} Gy ^{1}); β = 4.43 ± 0.30 (102 Gy ^{2}) and C = 0.56 ± 0.39 and calibration doseeffect was presented Y = 1.01D + 4.43D2 + 0.56.
Acknowledgements
We would like to thank IAEA for support (IAEA CRPE3.5008).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References   
1.  Savage JR. Classification and relationships of induced chromosomal structual changes. J Med Genet 1976;13:10322. 
2.  IAEA 1986: Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment. Technical Reports Series No. 260. IAEA, Vienna; 1986. 
3.  Bender MA, Awa AA, Brooks AL, Evans HJ, Groer PG, Littlefield LG, et al. Current status of cytogenetic procedures to detect and quantify previous exposures to radiation. Mutat Res 1988;196:10359. 
4.  IAEA 2001: Biological Dosimetry: Cytogenetic Analysis for Radiation Dose Assessment, A Manual. Technical Reports Series No. 405. IAEA, Vienna; 2001. 
5.  Romm H, Oestreicher U, Kulka U. Cytogenetic damage analysed by the dicentric assay. Ann Ist Super Sanita 2009;45:2519. 
6.  IAEA 2011: Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies. Cosponsored by IAEA and WHO, EPRbiodosimetry 2011, IAEA, Vienna; 2011. 
7.  Szluinska M, Edwards AA, Lloyd DC. Statistic Methods for Biological Dosimetry, Health Protection Agency, HPARPD011. Chilton, Didcot, Oxfordshire, UK; 2005. 
8.  Edwards AA: Dosimetric and statistical aspects of cytogenetics. I reunion internacional sobre dosimetria biologica. Empresa Nacional de Residuos Radioactivos, Madrid; 1990. p. 7585. 
9.  Hilali A, Léonard ED, Decat G, Léonard A. An appraisal of the value of the contaminated Poisson method to estimate the dose inhomogeneity in simulated partialbody exposure. Radiat Res 1991;128:10811. 
10.  Wilkins RC, Romm H, Oestreicher U, Marro L, Yoshida MA, Suto Y, et al. Biological dosimetry by the triage dicentric chromosome assay – Further validation of International Networking. Radiat Meas 2011;46:9238. 
11.  Fabry L, Leonard A, Wambersie A. Induction of chromosome aberrations in G0 human lymphocytes by low doses of ionizing radiations of different quality. Radiat Res 1985;103:12234. 
12.  Lloyd DC, Edwards AA. Chromosome aberrations in human lymphocytes: Effect of radiation quality, dose, and dose rate In:Radiationinduced Chromosome Damage in Man. New York, USA: Alan R. Liss, Inc.; 1983. p. 2350. 
13.  Sasaki MS. Cytogenetic biomonitoring of human radiation exposures: Possibilities, problems and pitfalls. J Radiat Res 1992;33:4453. 
14.  Bauchinger M, Schmid E, Streng S, Dresp J. Quantitative analysis of the chromosome damage at first division of human lymphocytes after 60Co gamma irradiation. Radiat Environ Biophys 1983;22:2259. 
15.  Stricklin D, Wilkinson D, Arvidsson E, Prud”hommeLalonde L, Thorrleifson E, Mullins D, et al. Biodosimetry Intercomparison: FOI and DRDC Ottawa. FOIR1929SE. Scientific Report, NBC Defence; 2006. 
16.  Prasanna PG, Loats H, Gerstenberg HM, Torres BN, Shehata CW, Duffy KL, et al. AFRRI's GammaRay, XRay, and FissionNeutron Calibration Curves for the Lymphocyte Dicentric Assay: Application of a Metaphase Finder System. 8901 Wisconsin Avenue, Bethesda, Maryland 208895603: Armed Forces Radiobiology Research Institute, Special Publication; 2002. p. 21. 
[Figure 1]
[Table 1], [Table 2], [Table 3]
