Since radiation-induced chromosome aberrations lead to calamitious consequences at cellular level, via genetic damage, chromatin organization, and chromosome function [1
], systematic study of differences in inter-chromosomal radio-sensitivities is imperative. The relationship between chromosome size or DNA content and its effect on radio-sensitivity is debatable [2
]. Our study was aimed to characterize the relative radio-sensitivities of human chromosome-1 and -2, both in interphase- and metaphase-spreads and thereby broadly understand differences in radiation-induced genetic damage, chromatin organization, and chromosome function. Chromosome-1 spans about 279 million nucleotide base pairs and chromosome-2 about 251 million base pairs [12
] but, the former seems to contain approximately 3-times more genes [13
]. Imperatively, differences in radio-sensitivity between these chromosomes will result in differences in genetic consequences.
The difference in radio-sensitivities between chromosome-1 and -2 is evident from the studies of Fernadez et al., in metaphase spreads [14
] and in interphase cells [15
]. Our laboratory earlier developed a simple and rapid signal transduction method to study radiation-induced specific chromosome damages directly in un-stimulated peripheral blood lymphocytes. This approach allowed comprehensive identification and quantification of radiation-induced damages, which represents breaks and/or translocations in interphase cells [8
]. In the present study, we further developed this method to be able to simultaneously measure damages in two chromosomes enabling studies on inter-chromosomal differences in radiation sensitivity directly in un-stimulated peripheral blood lymphocytes and compare these observations in metaphase spreads.
We observed a linear dose-response in radiation-induced aberration frequency and aberrant cells for both the chromosomes in interphase cells, confirming our previous results on chromosome 1 damage [8
]. The p
values of the linear dose-response curves for aberrant cells with chromosome-1 and -2 indicated statistically significant (p, 0.001
0.94 and 0.90, respectively) goodness of fit with this model. The observed significant difference (p
) between dose-response curves for chromosome-1 and -2 aberrant cells indicates a difference in radio-sensitivity. Further, chromosome-2 aberrant cells were higher indicating a higher radio-sensitivity, which is evident in the linear component of the model as seen by a significant difference in slope (Fig. ).
We further characterized the differences in radio-sensitivity between chromosome-1 and -2 by measuring aberration frequencies. Linear dose response curves as seen for aberrant cells reflected in aberration frequencies for chromosome-1 and -2. We plotted dose response curves with a linear equation by subtracting the background aberration frequency for specific chromosomes from radiation-induced aberration frequency. The p values of 0.0004 and 0.0003 as well as r2 values of 0.98 and 0.98, respectively, for chromosome-1 and -2 aberration frequencies reflect significant goodness of fit with the model. We noticed higher aberration frequency for chromosome-2 as evident from the significant difference (p <0.05) in dose response curves reflecting higher radio-sensitivity of chromosome-2.
Using whole chromosome DNA hybridization probes we measured fragments and/or translocations involving human chromosome-1 and -2 in metaphase spreads. Linear-quadratic dose-response model is routinely used by others to study radiation-induced aberrations involving specific chromosome(s) [8
]. Our observations that linear-quadratic increase in aberrant cell as well as total aberrations with radiation doses corroborates the observations of Fernandez et al. [14
] on translocations and dicentrics involving chromosome-1 and -2 after exposure to 100 kVp X-rays. Linear-quadratic dose-response curves were also seen for translocations involving chromosomes 2, 8, and 14 after exposure to 60
Co γ-rays [6
Our studies in metaphase spreads indicate a lower aberration frequency in chromosome-1 similar to observations in interphase cells. Comparison of two dose-response curves demonstrated a significant difference (p
). Previously, Wojcik and Streffer [7
] observed a lower frequency of acentric fragments involving chromosome-1 than -2 after irradiation with 1 Gy of X-rays. However, at a higher dose of 2 Gy the over-expression of acentric fragments (breaks) was only found in one experiment.
We observed dose-dependent increase in the frequency of different aberration types involving human chromosome-1 and/or -2 in metaphase cells. The stable translocation frequencies involving both the chromosomes were higher than breaks. Earlier, Grigorova et al. [16
] reported lower frequencies of acentric fragments (breaks) compared to translocations involving chromosome 2, 3, 8, X, and Y in Chinese hamster splenocytes exposed to X-rays and neutrons.
The frequencies of stable translocations were higher compared to dicentrics in our studies involving chromosome-1 and/or -2, similar to the observations of others [17
]. Translocation frequency was significantly higher than dicentrics in Chinese hamster splenocytes exposed to both low LET X-rays and high-LET neutron beams [16
]. Higher relative frequencies of stable translocations compared to dicentrics was also observed in interphase cell as measured by the PCC-FISH technique involving human chromosome 8 after X-irradiation [21
Our study revealed that human chromosome-2 is more prone to aberration induction compared to chromosome-1 both in interphase and metaphase cells though the DNA content of chromosome-2 is approximately 2.4% less than that of chromosome-1. The apparent reasons for differences in radio-sensitivities between these two chromosomes may be due to a difference in spatial organization in the nucleus. Recently Branco et al. showed a difference in spatial organization of human chromosome territories as indicated by the average radial positions. The average radial position of chromosome-2 is higher than chromosome-1 both in resting and PHA stimulated human lymphocytes [22
], which we strongly believe to be involved in bringing out the differential radio-sensitivity among chromosome 1 and 2.
In general interphase cells display more number of aberrations per unit dose compared to metaphases (Tables and ). However, this is influenced by: (i) a dose-dependent increase in aberration yield in both interphase- and metaphase- spreads, but the degree of difference is not dose-dependent because of a differential saturation of aberrations and elimination via cell death. This is evident from the apparent similar yield in aberrations, particularly at 5 Gy. (ii) In our studies we used whole chromosome DNA probes for both interphase- and metaphase-spread analyses; therefore, it is prudent to expect loss of some signal in interphase cells, since the DNA probes are region specific and bind along the length of whole chromosome, where DNA is not completely condensed akin to metaphases.
The differential radio-sensitivity between chromosome-1 and -2 may also be linked with the differences in the GC and AT base pairs among them and particularly in the regions of GC or AT richness [23
]. We are currently investigating such differences at the molecular and genomic level that contribute to the radio-sensitivity among chromosomes and the effect of ionizing radiation and repair mechanisms on the chromosomes.
Various factors such as inter individual variability, DNA content, genetic background etc., can influence the radio sensitivity of individual chromosome [6
]. In lieu
of potential inter-individual variability as well as genetic background, affecting the aberration yield involving specific chromosomes, we have deliberately chosen only one donor for our experiments, so that that profound difference among chosen chromosomes would surface. This would abet the design of more targeted studies keeping specificity and sensitivity and underlying variation among genetic population of interest in mind. It is difficult to generalize or draw any systematic and precise conclusion on genetic contribution in context of the entire genomic population by using one donor. Never-the-less, the work presented here should emphasize the importance and need for tailored assessment of health risks for individuals, based on relative individual chromosomal radio sensitivities, given differences among chromosomes may exist among individuals and in populations. Further while gene mapping for health risks for instance like cancer, is complex and evolving using chromosomal changes may be a simpler alternative approach with additional emphasis on sensitivity and specificity metrics. The differential radio-sensitivity with respect aberration induction will affect the chromosome function via change in the genetic organization especially translocations. Detection of chromosome translocations assists in diagnosis, treatment and prognosis of many blood related cancer and childhood sarcoma [26
]. For instance, a specific translocation between chromosome-1 and -13 results in alveolar rhabdomyosarcoma [27
]. Similar interesting studies can be designed and investigated using the differences we have proposed in our current paper.