Ionizing radiation is widely used in medical procedures, including cancer radiotherapy and the use in diagnosis.1 For over a century the biological effects of medium- and high-dose radiation (over 0.5 Gy) on human health have been investigated. Induction of cancer is one of the most severe longterm effects of radiotherapy. While the relationship between radiation effects and dose is well defined at higher doses, the effects of doses below 0.5 Gy are still unclear. Even though the use of radiotherapy resulted in a significant increase in cancer survivors2, it is important to investigate the potential negative long-term effects of radiation, considering that further advancements in therapy result in increased length of patients life after therapy. Table 1 presents the classification of radiation doses according to Kadhim
Dose ranges and sources of radiation exposureDose range Examples of exposure Very low doses < 0.05 Gy Mammography4, chest X-ray4 Low doses 0.05–0.5 Gy Cardaic CT angiogram4 Medium doses 0.5–5 Gy One fraction dose in standard fractionated radiotherapy5, dose absorbed by workers during Fukushima accident6 High doses 5–15 Gy Intraoperative radiotherapy (as boost)7 Very high doses > 15 Gy Intraoperative radiotherapy8, cumulative dose delivered during fractionated radiotherapy5
The damage to DNA is induced by radiation through two mechanisms: the direct and indirect effect.9 Directly induced damage results from the deposition of radiation energy in the DNA molecule creating a break. Damage induced indirectly is a result of an attack of the reactive species, which might be produced from ionization of water, on DNA molecule. These processes induced by radiation result in creation of single-strand breaks (SSB) and double-strand breaks (DSB) in DNA molecule.10 DSBs are prime lesions induced by ionizing radiation, and are responsible for its deleterious effect. Densely ionizing radiation has the ability to induce more damage per cell, making it possible for two or more lesions to be induced in proximity. These groups of breaks are called clustered DNA lesions, and are harder to repair than single DSBs and SSBs.11 Density of radiation correlates with reparability of DNA lesions, with densely ionizing radiation being harder to repair. Radiation stimulates DNA damage repair through non-homologous end-joining (NEHJ)12, an error prone pathway that can lead to induction of chromosome aberrations, which in turn may cause genomic instability.13 Genomic instability has been connected to induction of most of the human cancers, however currently the exact mechanism of radiation-induced carcinogenesis is not clear.
The dose-rate of radiation plays a major role in how much damage an exposed cell receives. High dose-rate irradiation, like radiation created by atomic bomb, results in deployment of energy in very short time to every irradiated cell. However if radiation is protracted over long periods of time, like the dose-rates considered in calculations of occupational and environmental radiation risks, the cell turnover influences how many ‘hits’ a cell will receive. Low-dose rates also influence how cell reacts regarding the repair of DNA damage, since low-dose rates allow more time for damage to be repaired which makes it more favorable for cells than high-dose rate radiation.14
This review contains a description of biological consequences of low-dose radiation and possible induction of cancer (Figure 1).
Damage to DNA is one of the most important factors in radiation induced cancer transformation. Even though ionizing radiation induces one DSB per 20 SSBs, research indicates that DSBs are much more impactful effect of irradiation (IR).15 This difference might be caused by the fact that SSBs are repaired by error-free mechanisms and are not sustained, while DSBs induced by radiation are repaired by mechanisms prone to mis-repair and repair failure, making DSBs a main cause of radiation induced cell death.16 Recently many authors pointed towards important differences between DNA damage response exerted by high and low doses of radiation.17,18 Research of low dose effect on normal tissues is especially significant considering side effects of radiotherapy treatment.
Induction of DSBs is considered to be one of the main mechanisms by which radiation exerts its deleterious effect. One of the earliest events appearing in response to DSB induction is phosphorylation of histone H2AX by protein kinases: DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia and Rad3-related (ATR) and ataxia telangiectasia mutated (ATM).19 Phosphorylated H2AX (γH2AX) foci appears around the DNA break as a part of DSB repair mechanism20 and it corresponds directly with the number of DSBs.21 The correlation between the radiation dose and the initial number of γH2AX foci it induces has already been thoroughly described.21,22 Rothkamm
HRS is described as an increase in radiosensitivity in cells exposed to low-dose radiation, usually below 0.2–0.3 Gy for low LET radiation. When cells are exposed to doses higher than 0.3 Gy an increase in radioresistance is observed and the transition towards it on dose response curve is described as increased radioresistance (IRR).29Figure 2 presents the dose-response curves with and without the evidence of HRS and IRR. Occurrence of HRS and IRR in cells was first described using an
In order to repair the damaged DNA, irradiated cells utilize cell cycle arrest to allow sufficient time for the DNA repair. The most important checkpoint pathways that initiate the radiation-induced arrest inhibit the cell cycle progression from G2 phase to mitosis. Two checkpoints are utilized for the repair of DNA damage induced by radiation. The classic G2/M checkpoint is activated after high doses of radiation and its role is to arrest the cells damaged in S or G1 cell cycle phase.34 The early G2/M checkpoint is activated shortly after exposure to radiation and it exclusively protects cells in G2 phase of cell cycle from radiation effects.35 This checkpoint is especially relevant to assessment of low-dose radiation risks because induction of this checkpoint occurs after irradiation with doses up to 10 Gy of low LET radiation35 and a threshold for its activation is observed at radiation doses of 0.3 Gy.36 This threshold dose coincides with radiation doses at which a transition between HRS and IRR is observed, suggesting a correlation between the two phenomenon, which was first suggested by Marples
While even very low doses of radiation are capable of inducing DSBs, there is evidence suggesting a threshold number of 10–20 DSBs for the triggering of ATM dependent G2/M checkpoint.38 This can mean that low doses of radiation inducing few DSBs may not trigger G2/M arrest in irradiated cells, making it possible for cells with unrepaired DSBs to enter mitosis, which in turn might result in loss of genetic material.39 Deckbar
Mutation in ATM gene observed in Ataxia telangiectasia, has been connected with increased chromosomal radiosensitivity and increased susceptibility to cancer.42 Since both DNA damage repair and G2 checkpoint induction depend on ATM kinase, Ataxia telangiectasia (AT) cells are often used to investigate the mechanisms of these processes. Using AT cells researchers were able to show for the first time that G2 checkpoint facilitates not only the repair of DSBs, but also the repair of chromosomal breaks, after irradiation with 1 Gy ionizing radiation.43 Failure to activate the G2 checkpoint might be one of the reasons for increased cancer rates in patients with Ataxia telangiectasia, since unrepaired damage to DNA might alter cells genome leading to malignant transformation.42
Radiation induced DNA damage, specifically DSBs, are very important in occurrence of chromosomal instability, which may involve aneuploidy, deletions, and aberrations, which in turn might contribute to carcinogenesis.44 The non-targeted effects, including bystander effects and genomic instability, contribute to the induction of cancer in a less clear way.45 The progeny of exposed cell might exhibit phenotypes such as chromosomal aberrations and rearrangements, gene mutations and enhanced cell death rate.46 Acquisition of alterations in the genome of progeny of irradiated cells is described as genomic instability (GI) and it has been accepted as the hallmark of cancer cells and one of the most important factors involved in the development of some cancers.44 RIGI was firstly observed in one-cell mouse embryos irradiated with X-rays and with neutrons.47 The results suggested that chromosomal aberrations might appear de novo two or three mitoses after initial exposure and also that radiation of a different quality (different LET) induces aberrations at different frequencies.
In the context of RIGI the effect on genome is mediated via the accumulation of genetic changes in the progeny of surviving irradiated cells through many generations.48 The capacity to induce genomic instability depends on both dose and quality of radiation, with high LET radiation generally inducing chromosome- and chromatid-type aberrations more effectively.49 This effect was observed in murine bone marrow cells irradiated with 0.25 Gy, 0.5 Gy and 1 Gy of densely ionizing α-radiation.50 Colonies arising from irradiated cells exhibited high frequency of non-clonally induced chromatid aberrations consistent with them arising de novo and not directly from irradiation. Early studies using human lymphocytes irradiated
TGF-β signaling can be induced in nontransformed cells irradiated with very low doses of radiation. It has been shown that TGF-β signaling selectively induces reactive oxygen species (ROS) production in transformed cells leading to their apoptosis.53 This signaling pathway could be a part of surveillance network protecting cells from malignant transformation. It is important to note, that radiation induced EMT phenotype can be mediated by TGF-β, making it a “two-edged sword” in need of further investigation.54 Authors suggest that while TGF-β might play a role in elimination of irradiated, genomically unstable cells, the chronic exposition to radiation and TGF-β signaling can induce EMT in remaining cells.53
Dicentric chromosomes are an established marker used to assess IR induced chromosomal aberrations.55 Using the measurement of dicentric frequency in human fibroblasts after irradiation, researchers were able to investigate the role of dose-rate effect in induction of chromosome aberrations. [13] In cells irradiated with a single X-ray dose the frequency of dicentrics correlated exponentially with doses higher than 0.2 Gy. Irradiation of cells with a single 1 Gy dose of X-rays resulted in similar rise of frequency of dicentrics as irradiation with fractionated 1 Gy dose (fractionation: 10 × 0.1 Gy; 5 × 0.2 Gy; 2 × 0.5 Gy) given with intervals of 1 min between fractions. When intervals between fractions were increased above 5 min, the frequency of dicentric chromosomes was significantly lower, which suggests that the maximum of DNA damage repair, in relation to chromosome dicentrics, is reached after 5 min. These results show that high dose rate irradiation is more harmful to the genome than low dose rate. The measurement of frequency of dicentrics in patients lymphocytes could be utilized in examination of radiosensitivity. Linear dose response of dicentric frequency was observed in human lymphocytes after irradiation with X-ray doses above 20 mGy.51 Frequency of dicentrics was also used to evaluate human lymphocytes response to low LET γ-rays. While for doses above 20 mGy a linear increase in dicentric frequency was observed, authors were not able to detect statistically significant changes in dicentric frequencies below 20 mGy doses, despite counting over 5000 metaphases.56 This means that dicentric frequency count might not be a precise enough method for assessment of response to very low doses.
Aneuploidy is a phenotype very often observed in tumors and it arises from incorrect chromosome segregation during mitosis.57 Delayed appearance of aneuploid cells induced by low-dose radiation has been observed
One of the radiation induced phenotypes used to investigate genetic effects of low-dose radiation is loss of heterozygosity (LOH). LOH is conventionally associated with cancer as a mechanism inactivating tumor suppressor genes, however it is also found in regions of genome responsible for cancer induction.61 For research on induction of mutations
Another non-targeted effect playing a role in carcinogenesis is radiation induced bystander effect (RIBE). This effect together with other non-targeted effects, is often described as most relevant to low-dose radiation63 and is mediated through two mechanisms: secretion of soluble factors by irradiated cells64 and also by signaling through cell-to-cell junctions.65 One of the early works in which this effect was described used Chinese hamster ovary (CHO) cells irradiated with low doses of α-radiation.66 The authors chose very low radiation dose (0.31 mGy), so that less than 1% of cells were traversed through by the radiation. After irradiation close to 30% cells exhibited presence of sister chromatid exchanges which suggests, that the genetic damage was induced even in the cells whose nuclei was not traversed through by radiation. Authors speculated that this effect might have been mediated through the production of ROS by the cells which were irradiated directly. The occurrence of RIBE was also confirmed for very low doses of X-ray radiation in human fibroblasts.67 After irradiation with doses ranging from 1.2 to 200 mGy the induction of DSBs, measured by ATM phosphorylation, followed a supralinear relationship. While cells were treated with an inhibitor of gap junction intercellular communication, the number of DSBs induced by radiation was smaller than in untreated cells. The largest differences between treated and untreated cells were observed at doses between 1.2 to 5 mGy, meaning that RIBE has the most influence on DSB induction at doses up to 10 mGy. RIBE has also been shown to induce mutations in bystander cells and the mechanism of these mutations is different than in the cells irradiated directly.68 In bystander CHO cells, the frequency of deletions was higher after using 10 cGy α-radiation and after using 0.5 cGy α-radiation the frequency of point mutations was higher. Very low doses of α-radiation also have the ability to induce ROS production in irradiated cells69 and ROS has been shown to induce point mutations70 making it the likely mechanism for induction of mutations in bystander cells. However it is not clear whether the effect is mediated through ROS produced by irradiated cells and transferred to unirradiated cells, or the irradiated cells produce factors inducing ROS production in uniradiated cells.
The role of factors secreted by irradiated cells has also been confirmed for low-dose induced RIBE. Seymour and colleagues71 irradiated human keratinocytes with doses ranging from 0.01 to 0.5 Gy of γ-radiation. Then the medium from irradiated cells was collected and transferred to unirradiated cell culture. The results of clonogenic assays measuring clonogenic death of cells show that RIBE mediated by the secreted substances is most predominant in doses below 0.5 Gy. RIBE has also been confirmed to occur
Evidence has also been presented for oncogenic effect of RIBE in
Radiation to which humans are exposed comprises mainly of low-dose and low-dose rate radiation from both natural and man-made sources. In recent years the biological effects of low-dose radiation became a point of interest due to the increase in popularity of radiation therapy and diagnostic radiology. Even though many studies point toward a link between carcinogenesis and exposure to radiation, the exact mechanism is still not clear. Induction of genomic instability is suspected to play a major role in malignant transformation after high-dose irradiation, and it might be responsible for carcinogenesis after exposure to lower doses. Latest research suggests that phenomena characteristic for low-dose exposures like HRS and RIBE might be the factors contributing to induction of genomic instability after exposure. Better understanding of these processes is crucial for the proper estimation of low-dose exposure risks for radiation workers, patients and people exposed to high background radiation.