Ex vivo analysis of irradiated fingernails: chemical yields and properties of radiation-induced and mechanically-induced radicals

Abstract

A qualitative and quantitative analysis of the radicals underlying the radiation-induced signal (RIS) in fingernails was conducted in an attempt to identify properties of these radicals that could be used for biodosimetry purposes. A qualitative analysis of RIS showed the presence of at least three components, two of which were observed at low doses (<50 Gy) and the third required higher doses (>500 Gy). The low dose signal, obtained by reconstruction, consists of a 10 gauss singlet at g = 2.0053 and an 18 gauss doublet centered at g = 2.0044. Based on the initial slope of the dose-response curve, the chemical (radical) yields of the radicals giving rise to the singlet and doublet were 327 (+/-113) and 122 (+/-9) nmol J-1 (standard error, SE), respectively. At doses below 50 Gy, the singlet signal is the dominant component. Above this dose range, the signal intensity of the singlet rapidly dose-saturates. At doses <50 Gy, there is a small contribution of the doublet signal that increases in its proportion of the RIS as dose increases. A third component was revealed at high dose with a spectral extent of approximately 100 gauss and displayed peaks due to g anisotropy at g = 2.056, 2.026, and 1.996. The total radical yield calculated from the initial slope of the dose-response curve averaged 458 +/- (116) nmol J-1 (SE) in irradiated nail clippings obtained from six volunteers. Such high yields indicate that nails are a strong candidate for biodosimetry at low doses. In a comparison of relative stabilities of the radicals underlying the singlet and doublet signals, the stability of the doublet signal is more sensitive to the moisture content of the nail than the singlet. This differential in radical stabilities could provide a method for removing the doublet signal under controlled exposures to high humidities (>70% relative humidity). The decay of the singlet signal in RIS varies with exposure of a nail clipping to differing ambient humidities. However, long exposures (>6 h) to relative humidities of 72-94% results in singlet intensities that approach 7.0 +/- (3.2)% (standard deviation) of the original intensities in an irradiated nail. This result suggests the existence of a subpopulation of radicals underlying the singlet signal that is relatively insensitive to decay under exposure of nails even to high humidities. Therefore, exposures of an irradiated nail clipping under controlled humidities may provide a method for estimating the exposure dose of the nail that is based on the intensity of the signal of the humidity insensitive radical population underlying the singlet signal.

Figure 1

Spectra from an x-ray irradiated single nail (8.9 mg) given additive doses of 0, 10, 30, 50, 100, 200, 500, and 1,000 Gy showing the qualitative changes in the spectra as a function of increasing dose. At a 10 Gy dose a singlet with a 10 G linewidth is the predominant feature. However as the dose increases beyond 10 Gy there are other, broader features that build into the spectrum.

Figure 2

Qualitative changes in the MIS spectra as a function of time and humidity in the same sample. The analysis was conducted on 6 nails (same volunteer) cut into 2×2 mm pieces (46 mg) to maximize the MIS signal for the purposes of spectral deconvolution analysis. The spectrum A was taken within 20 min of the sample preparation under ambient temperature (294-296 K) and humidity (55%). Spectra B, C and D were obtained after 4, 12 and 24 hr, respectively, of exposure to 72% relative humidity. The spectral trace at the bottom of the figure is from Fremy salt which is used as a field reference (g = 2.0056, aN = 13.09 G).

Figure 3

RIS spectrum from a single nail clipping (8.9 mg) x-ray irradiated to 1,000 Gy (Spectrum A). The basis spectrum (B) for the singlet was subtracted from spectrum A, resulting in the doublet shown in spectrum C.

Figure 4

MIS spectrum from 6 nails (same volunteer) cut into 2×2 mm pieces (46 mg) to maximize the MIS signal for the purposes of spectral deconvolution analysis. The basis spectrum (B) for the singlet was subtracted from spectrum A, resulting in the doublet shown in spectrum C.

Figure 5

Decay in the RIS signal in nails gamma-irradiated to 10 Gy as a function of contact time in water at 296 K. The points represent the mean peak-to-peak intensity ratios for the spectral line at g = 2.005 in clippings at each time point (RIS_X)relative to the initial intensity (RIS₀) post-radiation. Each time point represents a separate nail clipping obtained from the same individual and matched for mass (10 mg ± 1 mg). The error bars represent the standard deviation in the intensity ratios between five individuals. The nails were presoaked in water for 10 min, followed by 20 air dry before receiving the 10 Gy dose to remove the MIS.

Figure 6

Structural representations of a perthiyl (A) and sulfuranyl (B) radical. Either radical structural assignment would give rise to an anisotropic EPR spectrum with g-values at 2.056, 2.021, and 1.999, consistent with the downfield line positions seen in the high dose RIS and the MIS spectra in Figs. 4 and 5.

Figure 7

Dose response curves for a finger nail (8.9 mg) given x-ray doses from 10 to 1,000 Gy. Spectra for each data point were taken within 10 min of the cessation of the radiation dose. The number of spins in the irradiated nail were determined against a CuCl₂•2H₂O standard (1.27×10¹⁷ spins). The r² value of the exponential fit to the data was 0.9977. The chemical yield calculated from this data for the total spectrum (square) is 403 nmol J^-1. The total spectra for each of the dose points was deconvoluted into the singlet and doublet spectra (i.e. as is shown in Fig. 4). From these deconvolutions, separate plots of the dose response behavior for the singlet (circle) and double (triangle) were obtained, with chemical yields of 327 and 122 nmol J^-1 calculated for the singlet and doublet, respectively.

Figure 8

Chemical yields calculated from the six different dose response studies performed on x-ray irradiated nail clippings from six volunteers. The error bars represent the standard error of the estimate. Nail masses used in these studies are as follows: Vol. 1 (8.9 mg), Vol. 10 (10.2 mg), Vol 12 (12.0 mg), Vol. 13 (10.4 mg), Vol. 14 (10.1 mg) and Vol. 15 (10.1 mg).

Figure 9

Power saturation of the singlet at g = 2.0053 in RIS (A) and MIS (B) spectra. The data is plotted as the peak-to-peak (g = 2.0053) as a function of the square-root of the incident microwave power. The RIS spectrum was obtained from a nail (10 mg) x-ray irradiated to 100 Gy and then exposed to 74% relative humidity for 2 hr to remove the underlying doublet signal. The MIS spectrum was obtained from 10 2×2 nail clippings (same volunteer) that were exposed to 74% relative humidity for 12 hr to remove the underlying doublet and sulfuranyl radical signals.

Figure 10

Structural representation of an alpha-carbon radical on the peptide backbone.

Figure 11

Decay in the RIS signal in a single nail (8.9 mg) exposed to a humidity of 72% following a 1,000 Gy dose of x-ray radiation. The points represent the double integrals of the central singlet of the spectra taken at time points from 10 – 300 min post-irradiation. The number of spins in each time point spectrum was determined in reference to a CuCl₂•2H₂O standard. The data for the total spectrum (square) was fit to a two-phase exponential decay model. The r² value for this particular fit was 0.987. For each time point, spectral deconvolutions were conducted to extract the singlet and doublet signals. These data are plotted for the singlet (circle) and doublet (triangle) in the figure and fit to a one-phase exponential model.