Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures

Abstract

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

FIG. 1.

Linear energy transfer (LET) effects of pure neutrons, electrons, or photons measured by the high-throughput dicentric assay: Panel A: The RABiT-II DCA dose-response curves for dicentric chromosome aberrations produced by five radiation qualities: IND-spectrum neutrons (LET of ~70 keV/μm), photons (320-kVp X rays, LET of 0.4 keV/μm; 10-MV X rays, LET of 0.2 keV/μm; ¹³⁷Cs γ rays, LET of 0.91 keV/μm), and electrons (LET of 0.2 keV/μm). Each value represents an average of the dicentrics per total number of chromosomes scored automatically by the chromosome identification software (Table 1). Error bars indicate standard errors (n = 3). Panel B: RBE as a function of LET for human lymphocytes exposed to various radiation types: a comparison between RBE values extracted by the RABiT-II DCA and the classical DCA (, –14, 16). All RBE values were calculated at a damage level equivalent to 2 Gy of γ rays. The black-dashed line shows the general trend of the data.

FIG. 2.

Mixed vs. pure radiation field effects measured by the high-throughput dicentric assay: Panel A: A bubble chart shows relationships between three variables: the neutron dose (y-axis), the photon dose (x-axis), and dicentric yields (Table 3, column 8) induced by pure or mixed (photons + IND-neutrons) fields as a function of photon and neutron mixed doses (the third dimension, encoded by the radius of the circles, n = 4). The yield percentages (%) are encoded by the color matrix depicted in the chart on the right. Black-dashed arrows show each percentage of neutrons (%). Panel B: Dicentric dose-response curves for mixed (10%, 19%, or 35% neutrons) and pure (0% or 83% neutrons) radiation fields as a function of total dose. Actual dicentric yields (Table 3, column 8) represent an average of dicentrics per normal chromosomes scored by the software and are indicated by white (mixed field) or black (pure field) symbols. Error bars indicate standard errors. Predicted dicentric yields (Table 9, column 9) are estimated using best-fit parameters for the fixed effects regression model and are shown by colored dashed (mixed fields), black straight (pure photon field), or black dotted (IND-neutron field with parasitic photons) trendlines.

FIG. 3.

Dose rate effects measured by the high-throughput dicentric assay: Panel A: Dose-response curves for dicentric aberration yields induced by photon exposures. Dashed lines with black markers correspond to high-energy X rays (10 MV); solid lines with white markers correspond to highly filtered low-energy X rays (320 kVp). Panel B: Dose-response curves for dicentric aberration yields induced by 9-MeV electrons. Each value represents an average of the dicentrics per total number of chromosomes scored automatically by the chromosome identification software (Table 1) and plotted against the dose. Error bars indicate standard errors (n = 3). Panel C: The effects of the dose rate on dicentric yields as a function of absorbed energy per unit of time (Gy/s). Dicentric yields caused by 320-kVp X rays are shown by straight lines with black markers, 10-MV X rays by dotted lines with gray markers, and 9-MeV electrons by dashed lines with white markers. For all radiations, 3 Gy data is shown in triangle-shaped markers while 8 Gy data – is in circle-shaped markers. Each data point indicates the mean value (a total of 10 and 8 donors in photon or electron study, respectively) ± standard error (n = 3).

FIG. 4.

Effects of radiomitigators measured by the high-throughput dicentric assay. Dicentric yields from human lymphocytes precultured for 24 or 48 h with 0.1 μg/mL Neupogen (NEP, green lines) or 1.7 μg/mL Neulasta (NEU, orange lines) after exposure to ¹³⁷Cs γ rays (Table 5). Twenty-four-hour data is shown by squared markers, 48-h data – by triangle markers. Each data point indicates the mean value (4 donors) ± standard error (n = 3).