1. Introduction
In 1993, Marples and Joiner discovered a new, low-dose phenomenon using the survival response of V79 hamster fibroblasts to single X-ray doses [
1], termed later HRS/IRR. “Low dose hyper-radiosensitivity (HRS) is characterised by an increased sensitivity to radiation doses less than 0.3 Gy, which is followed by a more radioresistant response per unit dose between 0.3 and 0.6 Gy termed increased radioresistance (IRR)” [
2] (p. 98). This phenomenon has been later demonstrated in numerous studies on mammalian cell survival, chromosomal aberration, and micronucleus induction in mammalian and plant cells, and also using DNA double-strand break (DSB) test. It was shown that not all types of cells were capable of exhibiting the effects. Only 75% of the 50 mammalian normal and malignant cell lines tested by 2010 using a clonogenic assay in vitro exhibited the HRS/IRR response [
2]. Some high-LET radiations did not trigger this effect.
Many hypotheses exist on mechanisms of HRS and IRR, starting from the assumption that HRS/IRR, or rather the specific pattern of the initial part of dose curves, is exhibited in the cell population, which was a mixture of cells differing in radiation sensitivity (e.g., [
3]). The main problem was the mechanism of HRS, while as far as IRR was concerned, it was agreed that this cellular response resulted from radiation damage repair induction. It was hypothesised that HRS was due to the presence of G2-phase cells subpopulation in the cell culture, or due to apoptotic death of damaged cells to prevent mutation perseverance in survived cells. The latter was later invalidated by observing the HRS response in mammalian cells using chromosomal and gene mutation assays [
4]. Other possible mechanisms of low-dose HRS that have been investigated include impairment of DNA damage repair, the impact of cell cycle checkpoints, DNA DSB repair pathways and their regulation, as well as NO-mediated cell death. Following a series of studies [
5] using clonogenic, micronucleus induction, γH2AX, and pATM foci assays, researchers have concluded that HRS was observed in primary normal fibroblasts in both asynchronous and G2-phase cells of HRS-positive patients, while it was absent in cells from HRS-negative patients. Enrichment of the population with G2-phase cells has been shown to have no effect on eliciting HRS, though the “HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumour and other cells” [
6] (p. 45). Wang et al. [
7] suggested that early G2-phase checkpoints play important roles in the induction of the DNA damage repair and IRR after threshold doses of 0.2–0.3 Gy.
According to a current radiobiological paradigm, the DNA double-strand breaks are the major cellular damage that results in cell death, mutations, and chromosomal aberrations (CA). In this regard, radiations with different LET values are a helpful tool to investigate the influence of DSB complexity on various radiobiological effects manifestation using the above endpoints because the DSB complexity increases with LET. It should be also noted that the typical dose range of HRS is up to 0.1–0.2 Gy, i.e., it is within the small dose range considered in radiation protection [
8], where stochastic rather than deterministic effects prevail. At these doses, the mean specific energy z in a 8 μm cell nucleus becomes constant at doses <2.5 and <73 mGy for
60Co γ-rays and 14 MeV neutrons, respectively. After this kind of threshold, the mean elemental dose begins to rise proportionally to the absorbed dose [
9]. However, the fractions of cells hit increase with a dose below those thresholds and reach 100% above them. The above micro-dosimetric considerations raise the question of an HRS threshold for radiations differing in LET levels.
The HRS survival response has been observed after exposure to some high-LET radiation: pi-meson and proton [
10,
11,
12,
13], as well as 14 MeV neutrons given at a low dose rate [
14]. On the basis of experiments with pi-mesons in the Bragg peak and d(4)-Be fast neutrons, the authors of [
11,
12,
13] suggested that the IRR response was only evident after low and intermediate LET radiation exposures. Experiments with 59 and 79 keV/μm α-particles allegedly confirmed this suggestion, but those with 102 keV/μm α-particles did not [
15]. Results of experiments with carbon ions in which LET levels were 45.2 keV/μm [
16], 70 keV/μm [
17,
18], and 252 keV/μm [
19] also disagreed with this suggestion. Our cytogenetic studies with CHO-K1 cells exposed to low-dose rate 14.5 MeV neutrons have also shown HRS/IRR response [
20], as well as in B14–150 cells irradiated with carbon ions at the plateau and “tail” of the Bragg curve [
21]. Both cell cultures were in the stationary (plateau) growth phase.
Most of the results on HRS/IRR in vitro were obtained using asynchronous cell populations and clonogenic assay, with an emphasis on G2-phase cells where the effect was most readily expressed. However, G1-phase cells also showed the effect [
22]. It is known that cells irradiated in G0 or G1 phases die predominantly due to the visible Giemsa-stained chromosome-type CA dicentrics, centric and acentric rings, and terminal deletions. Cornforth and Bedford established a one-to-one relationship between the average number of these aberrations per cell and −ln
S, where
S is the fraction of surviving cells [
23]. The fact allows for comparing results of HRS/IRR response cytogenetic studies with those obtained using survival assays.
In this paper, we present the results of cytogenetic studies with Chinese hamster ovary cells exposed to protons and 14.5 MeV neutrons and compare them with previous data obtained using neutrons, carbon ions, and γ-rays. Taken together, the data show a certain dependence of HRS/IRR response on the LET and in general agree with the assumption that it is induced by low and intermediate LET radiations.
3. Discussion
In the present study, we demonstrated a low-dose hypersensitivity and induced radioresistance response using CA induction in stationary-phase Chinese hamster CHO-K1 cells exposed to a scanning proton beam at the Bragg curve plateau and to 14.5 MeV neutrons. The data normalisation per the absorbed radiation dose provided another line of evidence of the above response. Although the radiations had very different dose-averaged LET values, ca. 1 and 100 keV/μm, the phenomena patterns were rather close, viz., a distinct region of HRS response with a steep increase in CA frequency, compared with linear-quadratic extrapolation from higher doses. Another specific region in the dose curve then followed—that of induced radioresistance—where CA yield came to plateau (neutrons) or slowed down (protons) (
Figure 1 and
Figure 3). It is of importance to note that radiosensitivity, i.e., CA frequency per unit radiation dose, was persistently increased in both dose curves regions until the transition to regular dose dependence, linear–quadratic or linear ones. Therefore, the CA yield also increased when compared with the linear–quadratic or linear prediction (
Figure 1 and
Figure 3).
Results of the present cytogenetic study, together with those previously published on the observation of HRS/IRR response in plateau-phase CHO-K1 cells exposed to photon and particle radiations with different LET values (
Figure 5), enable us to draw further conclusions about LET dependence of the effect. The most prominent one is that the CA frequency level in the IRR region rises in general with radiation LET. We may note that this is in line with a well-known increase in the biological efficiency of radiation with LET. The next two conclusions coincide with those inferred by Marples et al. from HRS/IRR studies on cell survival exposed to low-LET radiation—X-rays, and γ-rays—and medium- and high-LET ones—negative pi-mesons, protons, and neutrons. They stated that “HRS is a ubiquitous response for all radiation qualities” and added, “The transition point (i.e., differential effectiveness of radiation killing per unit dose) between HRS and IRR differs for differing LET radiations” [
4] (p. 1311). In our studies, HRS upper border shifted with LET from 0.08 Gy for
60Co γ-rays to 0.15 Gy for 14.5 MeV neutrons. The authors also concluded, on the basis of results published up to 2008, that the IRR response is only evident after low and intermediate LET radiation exposures [
4] (p. 1311) and that the dose range of IRR response decreases with the increase in LET, up to fully diminishing at high LET [
11]. We observed decreases in that dose range, 0.08–0.6 Gy (
60Co γ-rays), 0.1–0.5 Gy (protons), 0.12–0.35 (carbon ions), and 0.15–0.35 Gy (14.5 MeV neutrons). Data for 14.5 MeV neutrons appear contradictory to some of the above conclusions. Neutron LET
d value of ≈100 keV/μm is too high to induce an IRR response according to suggestion in [
4]. However, the IRR response do exist and quantitatively is close to that of carbon ions at the Bragg curve “tail” where the LET
d value was 25–27 keV/μm.
The reason for the discrepancy between the neutrons’ high LET
d value and an intermediate-LET-like biological effect may lie in the spectrum of secondary charged particles produced in tissue by 14.5 MeV neutrons. Partial doses of protons, α-particles, and heavy recoils C, N, and O are 72.9%, 12.4%, and 10.9%, respectively [
28]. Corresponding particles LET ranges are approximately 3.5–93 keV/μm, 50–240 keV/μm, and 200–1000 keV/μm. It is the heavy recoils with their LET that give the LET
d value of 90–100 keV/μm. However, if we consider the number of particles crossing the cell nucleus rather than deposited energy, we obtain a spectrum-averaged LET value of 12 keV/μm, the so-called track average LET
t. The numerous protons determine low-neutron LET
t. We may further suggest that the observed cytogenetic effects at low doses < 0.5 Gy were mainly due to protons, the dose-average LET of which was between 20 and 25 keV/μm [
29,
30,
31]. Cells damaged heavily with high LET α-particles and C, N, and O recoils may escape from analysis due to cell division delay or apoptotic death. Another piece of evidence revealing the proton role in the effect follows from our assessment of 14.7 neutrons effective LET
d for CA induction in Chinese hamster cells, which was found to be 25–30 keV/μm. Our putative explanation agrees with the notion that LET, and especially averaged values, is not a good and comprehensive characteristic of radiation quality [
32,
33,
34].
The analysis of the number of CA per one aberrant cell and chromosome-type exchanges fraction in chromosome spectra suggested that lesion repair occurred in the HRS/IRR dose range. It follows from comparing ≈1 CA per aberrant cell with 1 to 10–20 DSB produced in a mammalian cell in the dose range of HRS/IRR response. An increase in chromosome-type exchange fractions resulting from DSB misrepair from ≈20% for carbon ions to 45–50% for γ-rays and protons agrees with the suggestion of simple DSB production in the latter case. Fractions of aberrant cells in the HRS/IRR dose range up to 10% are close to killed cells fractions in survival assays, of 10–20%, i.e., survival levels of 80–90%.
A peculiar shape of the dose dependence of the HRS/IRR phenomenon observed in mammalian cell survival was earlier seen in cytogenetic studies on human lymphocytes. The description of the effect for both tests is actually the same: “The yield [of dicentrics] is very small at 5 rad, then shows a rapid rise between 7.5 and 10 rad, followed by a plateau between 10 and 30 rad and a new rise from 30 rad on” [
35] (p. 372). Prior to this publication, Luchnik had proposed a hypothesis about the existence of two types of repair. We continue to cite: ”The “regular” repair occurs during each mitotic cycle and ensures the maintenance of genetic stability. The “emergency” repair is induced by an elevated level of genetic damage. One can speculate that the threshold is produced at doses of radiation where the “regular” repair is already not sufficient but the “emergency” repair is still not induced” [
35] (p. 375). Unfortunately, cytogeneticists had paid major attention to the plateau region of the dose curve while overlooking the existence of the HRS region, though they had pointed out “the rapid rise” in dicentrics before the plateau, and that “the number of dicentrics appears to be higher than both theoretical predictions”. However, the similar shape of dose curves for both endpoints is not surprising since the so-called asymmetric types of chromosome aberrations contributed mainly to cell death assessed with clonogenic assay (at least for cells in the G1 or plateau phase). This emphasises the importance of considering the results of the two assays in parallel or concomitantly because the information gained independently might be complementary.
As an example of such a complementary approach, let us consider the possible existence of the low-dose threshold of X-rays for HRS response. All cell survival dose–response data fitted well to the induced-repair model [
1] in which HRS response, if existing, started from zero doses, without any threshold. However, Luchnik and Sevan’kaev pointed to it directly in the citation above, placing it between 0.05 and 0.075 Gy. Nearly the same figures follow from the results of multi-lab collaborative studies with human lymphocytes, which have shown a lack of dose–response data in the dose range below 0.02–0.05 Gy [
36] and no evidence of HRS up to 0.05 Gy [
36,
37]. The next dose points in those studies were ≈0.3 Gy, according to Luchnik’s data ([
35],
Figure 4a). the HRS–IRR response occurred at doses between 0.075 and 0.3 Gy, which were not included in these studies. Therefore, the question of a threshold remained open. It should be noted that Lloyd et al. [
37] claimed that linear coefficients in the low dose range studied were consistent with extrapolation from high doses, unlike Luchnik’s finding. This claim agrees with a micro-dosimetric calculation of the mean specific energy z in a cell nucleus of 8 μm diameter which began to linearly increase at doses ≥ 0.0025 Gy after it was constant at lower doses of
60Co γ-rays ([
9],
Figure 1). In a more recent study on dicentric yield in human lymphocytes, the authors did not observe the dependence of chromosome frequency on doses up to 100 mGy [
38]. They also did not observe any HRS/IRR response because there were only two dose points. In general, the dose curve for five individuals was linear–quadratic. By contrast, HRS response started already at the dose of 0.05 Gy in cytogenetic studies using micronuclei induction in human fibroblasts, keratinocytes [
39], and G2-phase lymphoblastoid cells [
40]. However, dose–response curve for total aberrations in G2-phase lymphocytes from donor 1 indicated the apparent threshold somewhere between 0.1 and 0.2 Gy ([
40],
Figure 1a). Thus, to date, there is no clear evidence for a low border of HRS response in cells exposed to X-rays to be disregarded. It may lie, according to cytogenetic data discussed [
35,
36,
37,
41], between 0.05–0.075 Gy and 0.1 Gy (or somewhat higher).
As for other radiations, cytogenetic data give no indication of a low border of HRS response in mammalian cells and dose curves starting from zero doses, or more accurately, from the least doses. In our investigations with CHO-K1 cells, they were 0.05 Gy (protons, this paper); 0.03, 0.05 Gy (neutrons [
20]); 0.07 and 0.1 Gy (
12 C, [
24]); and 0.02, 0.03 Gy (protons, fibrosarcoma B14–150 cells [
21]). The first dose points were 0.075 Gy (14 MeV neutrons, human melanoma cells) in [
14]) and 0.1 Gy (
12 C ions, HPRT mutation frequency) in [
17]. In another multi-lab collaborative study with 14.83 MeV neutrons, in which the studied doses were 3.55, 8.4, 16.4, 24.5, 40.8, 81.1, and 244 mGy, total aberration frequencies increased linearly, starting from 3.55 mGy [
31], without any indication of HRS response. In this regard, we should remind the existence of HRS positive/negative patients with cancer, as revealed by Slonina et al. [
6], and we could also presume that feature in healthy people. This presumption may partly explain the absence of HRS/IRR response in cells of human origin including lymphocytes, as was observed in several multi-lab studies [
31,
36,
37,
42].
The existence of a low-dose threshold—at least for X- or γ-ray exposure—ensues from the Luchnik’s hypothesis about “regular” and “emergency” types of cellular repair (see citation above, this paragraph). To compare quantitatively cytogenetic patterns of HRS/IRR response, it is helpful to apply a piecewise linear fit model of the kind proposed by Geras’kin [
43] or separate linear fitting of data in HRS and IRR dose ranges [
41].
The results of our investigations with different radiations agree with the assumption made by Marples that HRS/IRR response is seen only after exposure to low/intermediate-LET radiations (with a reservation of about 14 MeV neutrons). Marples had further assumed that there is an upper LET threshold for increased radioresistance detection, using survival assay. The basis for the assumption was the lack of the IRR response in the survival of V-79 cells exposed to d(4)-Be neutrons (LET = 60–70 keV/μm) and Bragg-peak-negative pi mesons (LET = 35–55 keV/μm). However, some articles appeared later in which authors reported HRS/IRR response for 102 keV/μm α-particles [
15], and carbon ions with LET values of 45.2 keV/μm [
16], 70 keV/μm [
17,
18], 252 keV/μm [
19]. A survival assay was used in all studies, whereas Xue et al. [
17] additionally used an HPRT mutation assay. As for the latter study, we may speculate that the effect was due to appreciable low-LET components in high-LET beams with remarkable inhomogeneity for high-LET fractions and homogeneity for low-LET ones, in line with our consideration of 14 MeV neutrons. After X-ray exposure, the IRR response dose range was 0.1–0.5 Gy and 0.12–0.5 Gy for survival and mutation assays, respectively. After exposure to carbon ions (70 keV/μm), range origins shifted to larger doses (0.17 and 0.2 Gy), while range endings did not. Thus, the dose ranges for high-LET ions were shorter than for X-rays, just as in our studies with 14 MeV neutrons.
Identification of DNA DSB as a main cellular radiation lesion that results in radiobiological effects accentuates the role of radiation with various LET values differing in DSB complexity to test conceptions/mechanisms of DNA damage repair. In this regard, mechanistic biophysical modelling of low-dose effects of radiations with different LET values is a perspective tool in the investigation of HRS/IRR phenomena. The Monte Carlo simulation code PARTRAC is a standard of such modelling which considers all stages of radiation effects on subcellular and cellular scales: physical, physiochemical, chemical, biochemical, and biological [
44]. In addition to modelling DNA lesions induced by different radiations, it models DSB repair by NHEJ, production of chromosomal aberrations, and cell death. The PARTRAC code is currently being developed to involve radiation-induced effects of protons and light ions at radiotherapy-relevant energies [
45]. On the basis of another simulation code Geant4-DNA_2019 similar to PARTRAC, a new version of Geant4-DNA is being developed which additionally takes into account all known DSB repair pathways NHEJ, HR, SSA, alt-NHEJ [
46]. However, from the HRS/IRR point of view, the latest proposed mechanisms of low-dose effects should be considered [
47]. Another example of less sophisticated modelling is the DNA damage-repair dynamic model for HRS/IRR effects of C. elegans induced by neutron irradiation, developed by Feng et al. [
48].
There are suggestions to use the HRS/IRR phenomena in radiation therapy, and reports of such trials in schemes of hyperfractionation with right daily fractions [
49]. The goal is either to decrease the total dose to tumours, due to the HRS effect, and consequently to healthy tissues, or to expose healthy tissue at doses in the range of IRR effect, thus sparing it. The latter may be implemented, for example, in multi-directional proton therapy. A certain obstacle in the way of clinical application is the existence of HRS-positive and -negative patients [
6], so we need marker(s) of patient HRS/IRR status.
The HRS/IRR phenomenon appears to be many aspects in its manifestations. It was documented in the induction of DNA DSBs, mutations, chromosomal aberrations, micronuclei, and some form of cellular death (reproductive, apoptotic, etc.), using radiation with different LET values. We speculate that its mechanism might involve many aspects as well.