Relative biological effect of alpha particle radiation on low dose phenomena: lethal mutation, hyper-radiosensitivity and increased radioresistance

At high doses, the current recommended radiation weighting factors advise a significantly higher effectiveness of alpha particles relative to gamma radiation. However, at lower doses, the ratio of effectiveness between radiations of varying linear energy transfer values is complicated due to the relative importance of low dose phenomena such as genomic instability, bystander effects, low dose hyper-radiosensitivity and increased radioresistance (HRS/IRR). Radium is the most common source of alpha radiation exposure to humans, but the dosimetry is complicated by the decay chain which involves gamma exposure due to radon daughters. This study aimed to isolate the relative biological effect of alpha particles after low doses of radium to cells and their progeny. This was done by subtracting the survival values of a human keratinocyte cell line (HaCaT) and an embryonic Chinook salmon cell line (CHSE-214) exposed to gamma irradiation, from survival of the same cell lines exposed to mixed alpha and gamma radiation through chronic exposure to Ra-226 and its decay products. The human cell line showed increased radioresistance when exposed to low doses of alpha particles. In contrast the fish cell line, which demonstrated radioresistance to low dose gamma energy, demonstrated increased lethality when exposed to low doses of alpha particles. The results confirm the need to consider the dose-response relationship when developing radiation weighting factors for low dose exposures, as well as the need to be aware of possible cell line and species differences.


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Linear energy transfer (LET), describes the amount of energy deposited to the interacting 43 material, per unit of distance. Photons such as gamma rays are able to traverse great distances 44 unchanged before being absorbed, however monoenergetic ions such as alpha particles cause 45 frequent direct ionizations within a smaller range. Due in part to the clustered nature of damage 46 caused, the relative biological effectiveness (RBE) of an alpha particle is often described to be 47 significantly higher than that of a gamma ray. This is a result of the concentration of damage 48 given the same amount of absorbed energy [1]. 49 While this may be true for high doses, it has been shown that if a single alpha particle 50 traverses a cell, it causes zero to small risk of oncogenic transformation [2]. Further, the work of 51 Nagasawa and Little has shown significantly higher frequencies of mutation than would be 52 expected through linear extrapolation from data for high doses, at doses where the mean number 53 of alpha particle traversals per nucleus was significantly less than one [3]. At low doses, both 54 alpha and gamma rays can cause non-targeted effects (NTE) like genomic instability where 55 damage does not cause direct mortality and cells appear completely normal but de novo effects 56 are seen in distant progeny and lethality occurs generations later (often referred to as lethal 57 mutations or delayed reproductive death) [4]. Cell survival at sub-lethal doses of gamma 58 irradiation has also been observed to differ from what is expected by the traditional linear-59 quadratic model, instead displaying a region of low-dose hyper-radiosensitivity (HRS) followed 60 by increased radioresistance (IRR) [5]. Currently accepted recommendations for the radiation 61 weighting factor (w_r) of alpha particles, which apply the concept of RBE to derive equivalent 4 62 dose, are dose independent [6]. However, research has shown instances for RBE to be dose 63 dependent when high dose biological effects are substantially different to low dose effects [7]. 64 Of particular interest to this study is whether NTE amplify low dose effects such that they 65 are higher than what would be expected from established linear no-threshold model (LNT) 66 related RBE values following exposure to low doses of an environmental alpha emitter: radium-67 226. Despite being an alpha emitter by itself, it is known that the uranium decay chain of which 68 radium is part of involves many gamma emissions, thereby making it difficult to measure pure 69 alpha effects. To approach this problem, observations from gamma irradiation (through acute 70 exposure to Cs-137) will be subtracted from mixed alpha and gamma irradiation (through 71 chronic exposure to Ra-226 and its progeny).

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This study will measure the acute survival and the lethal mutation phenotype assayed as 73 reduced cloning efficiency in culture of a human keratinocyte cell line (HaCaT). In addition, due 74 to the increasing relevance of protecting non-human biota from radium in hydrogeologic 75 contaminations from mining, etc., this study will also investigate relative alpha exposure effects

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Reduction in cloning efficiency was observed using the clonogenic assay technique 93 developed by Puck and Marcus [9]. Cell stocks were maintained in T75 flasks with 30ml 94 medium. Upon reaching 80-90% confluence, flasks were subcultured. Here cells were gently 95 rinsed with calcium and magnesium-free DPBS in a biosafety level 2 laminar flow cabinet.

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HaCaT cells were detached using a 0.25% (v/v) trypsin-1 mM EDTA solution (Gibco) at 37°C 97 for 8 minutes, while CHSE-214 cells were detached using a 0.125% (v/v) trypsin-1 mM EDTA 98 solution (Gibco) at 19°C for 8 minutes. Trypsin was neutralized using fresh culture media, and 99 the cell solution was centrifuged at 125g for 4 minutes. The pellet was resuspended, and cells 100 were counted using an automated cell counter (Bio-Rad TC20). The cells were then seeded into 101 fresh flasks with fresh culture media at the required cell density such that at least 100 viable 102 colonies could be expected to form in control flasks.

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Reporter T25 flasks were maintained in the incubator for 9 days. Following this 104 incubation period, colonies in sham irradiated (control) flasks were visible to the naked eye.

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Flasks were stained using a 1:4 (v/v) dilution of Fuchsin-Carbol (Ricca Chemical Co., Arlington, 6 106 TX) in water, and macroscopically visible colonies (confirmed to have more than 50 cells when 107 observed under a microscope) were scored as survivors. days before being stained as described above (initial). The remaining fourth flask of each dose 141 was incubated until cells became 80-90% confluent, after which they were subcultured as 142 described above with fresh culture medium. This process was also repeated twice as above (P2 143 and P3).   Through subtracting the functions of fitted curves for cells exposed to Cs-137 from those 234 exposed to Ra-226 at each interval, the relative effect of alpha exposure to the residual survival 235 of HaCaT cells was isolated (see Table 1). Figure 3 describes the functions of the isolated effect 236 of alpha exposure to residual survival graphically at each observation.   In contrast to the studied human cell line, there was no significant cell death observed in 256 the directly exposed cells of the CHSE-214 fish cell line and their progeny to acute Cs-137 257 exposure (Fig 4). This shows an existing radioresistance when compared to human cell culture Using the same methodology as was done for the human cell line, the relative effect of 275 alpha exposure to the residual survival of CHSE-214 cells was isolated (see Table 2). As 276 exposure to gamma irradiation caused little to no effect in residual survival, the isolated relative   Considering the potential for sub-lethal doses from chronic exposure to radium and its 311 daughters found in waste products, to remnants of historic commercial and medical usage of 312 radium (ranging from self-luminous paints to cancer treatment), the unconventional behaviors 313 observed in both cell lines of this study have potential importance in radiological protection.

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Further, the presence of radium in waste reaching the ecosystem from mining and nuclear 315 applications is important given the currently growing interest for non-human radiological 316 protection.

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The radioprotective quality of sub-lethal doses of alpha radiation that was observed in At sub-lethal doses, survival greatly depends on repair mechanisms. Since the HaCaT cell 352 line demonstrates hyper-radiosensitivity to gamma energy at low doses, high-LET alpha particle 353 radiation may be able to produce sufficient genomic instability to induce radioresistance. In such 354 instances, the ratio of relative biological damage caused by alpha exposure is significantly lower 19 355 than an equivalent dose of gamma energy alone, and as such a lower radiation weighting factor 356 might be considered. However, while the CHSE-214 cell line demonstrates increased 357 radioresistance to gamma energy, the concentrated nature of energy deposited causes increased 358 lethality when exposed to alpha particles. These cases would suggest a higher radiation