Radiation induced bystander effects in the spleen of cranially-irradiated rats

Abstract

Objective:

To investigate the radiation-induced abscopal effect in terms of oxidative stress, apoptosis and DNA damage in the spleen cells following cranial X-rays irradiation of rats.

Methods:

Rats were cranially irradiated using 2 Gy X-rays. Another group was whole-body irradiated with 2 Gy X-rays and a third group was exposed to scattered radiation (measured to be 3 mGy). 24 hours following irradiation, sections from the spleen of the rats were dissected as well as plasma samples. The samples were examined for the desired endpoints.

Results:

The cranially irradiated animals showed a significant increase in the levels of glutathione, superoxide dismutase and catalase with no significant change in the lipid peroxidation product in the spleen cells with a significant increase in the C-reactive protein level the plasma. Apoptotic cell death in the spleen cells was demonstrated as indicated by the decrease of Bcl-2; the increase of p53, Bax, caspase-3 and caspase-8 and induction of DNA damage in the spleen in both of the cranially irradiated rats and whole body exposed rats. The exposure to 3 mGy scattered radiation increased the plasma level of C-RP and also induced apoptosis in the spleen cells.

Conclusion:

Cranial irradiation-induced abscopal effect in distant spleen cells. Very low doses of radiation can induce apoptosis in the spleen cells.

Advances in knowledge:

This paper provides an evidence on the incidence of radiation abscopal effect. Also, the results shed light of the effect very low doses of radiation as low as 3 mGy.

Introduction

The anticipation that the biological effects of ionizing radiation would be restricted to those cells directly traversed by radiation was challenged with an experiment performed by Nagasawa and Little¹ in 1992 defining a new paradigm in radiobiology which was named as “bystander effect”. Since then, bystander effect came into the spotlight and received attention from radiation biologists and radiation oncologists. The bystander effects of radiation can simply be defined as the response of cells to their neighbours being irradiated. Bystander effects can be induced by transmission of signals from irradiated to non-irradiated cells leading to biological changes in the recipient cells.² This definition has been broadly developed to include effects related to the production of clastogenic factors and the longer-range abscopal effects studied in whole organisms.³

Abscopal effects (in vivo bystander effect) pertain to the response of a tissue that is physically distant from the irradiated one, a concept that encompasses both distant tumour and distant normal tissue effects. This has often been described as “out-of-field or distant bystander” effects. These effects have been observed after the clinical use of radiation.^4,5 Hence, both “clastogenic factors” and “abscopal effects” are deemed as in vivo bystander response-like phenomena regardless of exposure to scatter radiation. In other words, scatter and out-of-field doses may deliver sufficient radiation dose to trigger bystander effects. So, most animal studies utilize either the shielded partially body irradiation method^6–9 or animals that received the estimated scatter dose as whole-body irradiation^10,11 to demonstrate the effects in non-irradiated areas.

The incidence of these effects is controlled by the transmission of stress signals from the irradiated cell population to distant cells, which can vary from a few millimeters up to several centimeters. The signals involved in the radiation-induced bystander effects have been reported to be cell signal mediated either through direct physical connection between cells via gap junction intercellular communication^12,13 or through secreted, diffusible signalling molecules into the surrounding media with its further reception by non-irradiated cells such as reactive oxygen species (ROS), reactive nitrogen species (RNS),^14–16 cytokines,¹⁷ peroxidase¹⁸ and more recently exosomes.^19,20

However, radiotherapy continues to be an important therapeutic modality for the treatment of cancer, it has been reported that local anticancer radiotherapy may have a risk for adverse consequences such as the formation of secondary tumours at the same organ, or at some distanced part of body.²¹ The appearance of these distant effects suggested the involvement of stress signals transmitted from the irradiated to the non-irradiated parts.²² On the other hand, Demaria et al observed that irradiation (2 or 6 Gy using a 60Co radiation source) of mammary carcinoma positioned on one flank of the mice as primary tumour resulted in inhibition of growth of secondary tumour of the same line implanted a few days later on the other flank. However, this effect was tumour type specific since the abscopal effect was absent when lymphoma was passaged as secondary tumour.²³ Moreover, Camphausen et al²⁴ observed a reduction in the growth of fully shielded tumours which were implanted into the midline dorsum region of mice upon the irradiation of the legs of mice in a manner dependent on the radiation dose and its fractionation.

In addition, similar effects have been observed in many experiments in vivo using animal models²⁵ with some of these abscopal effects were evidenced to be p53 dependent.²⁴ Moreover, other abscopal effects in vivo were also observed in the form of epigenetic changes when Koturbash et al²⁶ observed a significant reduction of DNA methylation in splenic cells of rats after localized cranial exposure to 20 Gy (X-rays, 90 kV, 5 mA) applied as two doses of 10 Gy while the rest of rat's body is shielded. Abscopal effects in vivo were also evidenced to initiate tumourigenesis in a dose dependent manner in non-irradiated tissues in vivo.⁸

The incidences of these abscopal effects draw attention to other possibilities of unexpected effects of ionizing radiation in distant bystander organs. So, further studies under conditions relevant to human radiation exposures are still needed to address the existence of molecular and cellular mechanisms of the bystander effects and other radiation-induced phenomena using animal models. There is also an insufficiency of studies investigating radiation-induced signaling effects from irradiated tumour to out-of-field normal healthy cells for clinically radiotherapy dose fraction of 2 Gy. Additionally, radiobiological effects may possibly be observed in normal healthy cells that can be subjected to out-of-field scattered dose only. However, there is a lack of published experimental evidence to support this proposal to date.

In this study, we investigate of an in vivo radiation-induced abscopal effect in the form of oxidative stress, apoptosis and DNA damage in spleen cells following cranial irradiation of rats using a dose of 2 Gy X-rays. Additionally, we investigated the effect of out-of-field dose from the scatter radiation, measured to be 3 mGy, on the spleen cells in animals positioned outside of the irradiation field.

methods and Materials

Animal model and irradiation of the animals

20 male Sprague-Dawley rats, 4 months old weighing 180 ± 20 g were divided randomly into 4 groups, 5 rats in each group. The animals were housed in individual cages with free access to food and drinking water. The animals were acclimated to laboratory condition of 22–24°C a 12-h light/dark cycle for 1 month before experimentation. Handling and care of the animals in addition to the experimental procedures were approved by the animal care and ethics committee at the National Research Centre, Cairo, Egypt (approval no:17–018).

The first group: sham-irradiated group (0 Gy). The animals were treated exactly the same way as the irradiated animals apart from being irradiated. The animals were anaesthetized and were taken to the irradiation centre, placed on the irradiation table for 30 s (equivalent to irradiation time) without turning the irradiator on. Afterwards, the animals were taken back to the animal house.

The second group: cranially irradiated group; animals received 2 Gy irradiation (dose rate 4 Gy min⁻¹) on head.

The third group: whole-body irradiated group; animals received 2 Gy whole-body irradiation.

The fourth group: animals exposed to scattered radiation only. The animals were placed outside of the irradiation filed (2 Gy) so as to be exposed to scattered radiation only. The radiation dose in this area was measured to be 3 mGy.

Irradiation

Rats were irradiated with 2 Gy of X-rays produced by a 6 MV linear accelerator (Precise Linear Accelerator; Elekta, Stockholm, Sweden) at the oncology and nuclear medicine centre at Mansoura University hospital, Mansoura, Egypt.

Dosimetry of the 6 MV photon beams generated by Elekta Precise linac was carried out using dual ionization chambers (detector and reference) of volume 0.125 cc connected to the dual channel PTW electrometer (TANDEM, PTW -Freiburg, Germany). The chambers are used with a 3D MP3-S water phantom connected to MP3-S therapy beam analyser system. The measurements were carried from 0 to 35 cm depth in 1 mm increments and the collected data was stored and analysed using the computer program, MEPHYSTO v. 7.3 (PTW -Freiburg, Germany). Dosimetry determined the beam configuration and symmetry as well as the percentage depth doses for each field size.

In our study, two field sizes were used 3 × 35 and 35 × 35 cm for cranial irradiation and whole-body irradiation, respectively. For each field size, the percentage depth dose was calculated using the device's operating program in order to be able to give the desired dose to the target that was assumed to be 1 cm deep under the skin. For irradiation, in each irradiated group, one rat was put at the central axis and four rats were put off axis (two at each side) but adjacent to the central one with the maximum off axis distance at each side not exceeding 15 cm. Thus, all the rats were in the plateau region of the field where the dose distribution is uniform and the penumbra region is avoided. For the 6 MV linear accelerator used, there was a build up region of 1.5 cm which was dealt with by putting 1.5 cm thick perspex sheet in direct contact above the rats during all irradiations.

Before irradiation, the animals were anaesthetized by intraperitoneal injection of ketamine and xylazine cocktail (75 and 5 mg kg⁻¹, respectively). In average, the animals were under anaesthesia for 60 min during which the animals were kept at room temperature (20–25°C). During irradiation, the animals were restrained in the sternal recumbent position on the irradiation table. For the cranially irradiated animals, the radiation field was adjusted to fit only the head of the animal and it was centred in the radiation field. The average dose of scattered radiation at the irradiation room was 3 mGy as measured by thermoluminescent dosimeters (TLDs) placed on the irradiation table just out of the radiation field in a position equivalent to where the rest of the rats’ bodies will be.

At the end of the experimental period, all animals were anaesthetized with halothane and sacrificed 24 h post-irradiation/sham-irradiation. For biochemical examinations, blood was collected by cardiac puncture into heparinized tubes and plasma was separated by centrifugation at 500g for 5 min then kept at −80°C until analysis. Also, sections from the spleen were collected and kept frozen at −80°C until analysis. Samples of spleen tissues were homogenized in 9 volumes (9:1 w/w) of cold Tris-HCl buffer (0.1 M) pH 7.4 for preparation of 10% homogenate. The homogenate was then centrifuged at 500 g at 4°C cooling centrifuge. The clear supernatant was used for the biochemical assay in the spleen.

Biochemical investigations

Lipid peroxidation was investigated by quantification of the lipid peroxidation product malondialdehyde (MDA) using MDA kits MD2529 (Bio-diagnostic, Giza, Egypt) following the manufacturer’s protocol. The antioxidant levels were estimated using colourimetric method by determining superoxide dismutase (SOD, SD 2521), catalase (CAT, CA 2517) activities and the glutathione (GSH, GH 2511) concentration using kits provided by Bio-diagnostic, Egypt. The calcium ion concentration was determined according to the instruction of the cornley AFT-500 apparatus. The C-reactive protein (C-RP) level was measured in plasma samples with an immunoturbidimetric assay (Archem-diagnostic, Egypt) according to the manufacturer’s protocol.

Flow cytometric study

Spleen tissue samples were disaggregated by passing through syringe and stainless sieves followed by filtering on nylon strainer and finally centrifuged. Before staining, cells were fixed using 2% paraformaldehyed and permeabilized with saponin, (0.5% v/v in PBS, pH 7.4). The cells were washed and suspended in PBS (pH 7.4) with 0.2% BSA, divided into aliquots in round-bottom tubes (Becton Dickinson) and stored at 4°C for flow cytometric analyses.

Detection of p53

A cell suspension was prepared with a PBS/BSA buffer and the antibody (mouse anti-p53 “aa20–25” FITC, Clone: DO-1) was added. After the addition of the antibody, they were mixed well and incubated for 30 min at room temperature. The cells were washed with PBA/BSA, centrifuged at 400 g for 5 min and the supernatant was discarded. After centrifugation and aspiration of the supernatant, cells finally were resuspended in 0.5% paraformaldehyde in PBS/BSA and analysed using flow cytometry.

Detection of Bax

A cell suspension was prepared with PBS/BSA buffer, mixed well and incubated with antiBax [6A7] antibody (ab5714, Abcam, UK). After 30 min of incubation at room temperature, cells were washed with PBA/BSA, centrifuged at 400g for 5 min and the supernatant was discarded. After all, the resultant cell pellet was re-suspended in 0.5% paraformaldehyde in PBS/BSA and analysed via flow cytometry.

Detection of Bcl-2

A cell suspension was prepared with PBS/BSA buffer and was mixed well and incubated with antiBcl-2 [100/D5] antibody (ab692, Abcam, UK) for 30 min at room temperature. The cells were washed with PBA/BSA, centrifuged at 400g for 5 min and the supernatant was discarded. The cells were finally resuspended in 0.5% paraformaldehyde in PBS/BSA and analysed using flow cytometry.

Detection of caspase-3 and caspase-8

For caspase-3 and caspase-8 assays, the following antibodies, FITC rabbit antiactive caspase-3 antibody (CPP32; Yama; Apopain, BD Bioscience, USA) and anticaspase-8 [E6] antibody (ab32125; Abcam, UK) were used according to the manufacturer's instructions.

Detection of apoptosis using annexin V/PI staining

Samples from spleen were stained with fluorescein isothiocyanate-conjugated annexin V/PI using the annexin-V-FITC/PI apoptosis detection kit (ab14085; Abcam, UK),according to the manufacturer's instructions.

Single cell gel electrophoresis (comet assay)

DNA damage in spleen samples was assessed using the single cell electrophoresis (comet assay) method, a sensitive and rapid technique for quantifying and analysing DNA strand breaks in individual cells as described previously.^27,28 The DNA strand breaks were quantified in the obtained images using CASP software (CaspLap, Poland) to directly obtain the percent of DNA in the tail, the tail length and the tail moment.

Results

Biochemical observations

Oxidative stress and antioxidant

Figure 1 illustrates lipid peroxidation and antioxidants in the spleen samples of the different groups. Cranial irradiation of the animals lead to an insignificant change in the level of MDA in the spleen tissue compared with sham-irradiated animals (Figure 1a). Compared with the sham-irradiated animals, the level of MDA was significantly increased (p < 0.05) in the spleen of both the whole-body irradiated animals and the animals exposed to scattered radiation only (Figure 1a). Additionally, the GSH content was significantly (p < 0.05) increased in the spleen of cranially irradiated animals (Figure 1d) compared with the sham-irradiated animals. On other hand, GSH content was significantly (p < 0.05) decreased in the spleen of whole-body irradiated animals and insignificantly changed in the spleen of animals exposed to scattered radiation only compared with sham-irradiated animals (Figure 1d).

Figure 1.

Changes in the levels of the lipid peroxidation product MDA (nmol/g tissue) (a), superoxide dismutase (SOD; U/g tissue) (b), and catalase (CAT; U/g tissue) (c) and glutathione (GSH; ng/g tissue) (d) in the spleens of sham-irradiated, 2 Gy whole-body irradiated, 2 Gy cranially irradiated animals and animals exposed to scattered radiation only (measured to be 3 mGy). The values are expressed as the means ± SEM (n = 5). *Significant at p < 0.05, **significant at p < 0.01 and ***significant at p < 0.001 with respect to the sham-irradiated group. CAT, catalase; GSH, glutathione; MDA, malondialdehyde; SOD, superoxide dismutase.

A significant increase (p < 0.05) was demonstrated in the SOD activity in the spleen of cranially irradiated animals compared with the sham-irradiated animals (Figure 1b). On the other hand, compared with the sham-irradiated animals, a significant decrease in the SOD activity was observed in the spleen of the animals exposed to scattered radiation only and an insignificant change was demonstrated in the SOD activity in spleen of the whole-body irradiated animals (Figure 1b). In addition, there was a significant increase (p < 0.05) in CAT activity in the spleen of the cranially irradiated animals (Figure 1c). Also, CAT activity appeared to be significantly decreased in the spleen of whole-body irradiated animals and insignificantly changed in the animals exposed to scattered radiation only (Figure 1c).

C-reactive protein (C-RP)

Table 1 illustrates changes in the levels of the C-RP in the plasma of the different animal groups expressed as mg l⁻¹. In the cranially irradiated group, whole-body irradiated group, animals exposed to scattered radiation only group, a significant increase (p < 0.05) of the C-RP level in the plasma was detected compared with sham-irradiated animals.

Table 1.

Changes in the levels of the Ca ++ (mmol l⁻¹) in the spleen and plasma of sham-irradiated, 2 Gy whole-body irradiated, 2 Gy cranially irradiated animals and animals exposed to scattered radiation only (measured to be 3 mGy)

	Sham-irradiated	Cranially irradiated	Whole-body irradiated	Scattered radiation irradiated
C-RP (mg l−1)	2.19 ± 0.29	5.420 ± 0.9238*	4.890 ± 0.2974***	4.23 ± 0.59*
Ca++ (mg dl−1) (spleen)	0.52 ± 0.0070	0.52 ± 0.019	0.51 ± 0.014	0.51 ± 0.0050
Ca++ (mg dl−1) (plasma)	0.24 ± 0.0055	0.23 ± 0.0085	0.21 ± 0.019	0.24 ± 0.0088

The values are expressed as the means ± SEM (n = 5).

*Significant at p > 0.05 and

***significant at p > 0.001 with respect to the sham-irradiated group

Calcium ion

The calcium ion content was measured in the plasma and the spleen tissue in all groups and expressed as (mg dl⁻¹). As evident from the obtained data in the Table 1, insignificant changes were detected in the calcium ion concentration neither in the plasma nor the spleen cells of any of the groups compared with the sham-irradiated animals.

Molecular analysis

p53

The analysis of the flow cytometry data demonstrated that p53 expression was significantly increased (p < 0.05) in the spleen of both cranially irradiated and whole-body irradiated animals compared with sham-irradiated animals (Figure 2). Additionally, in the group in which animals were exposed to scattered radiation only, p53 was significantly upregulated in the spleen compared with the sham-irradiated group (Figure 2).

Figure 2.

Changes in the BCL-2, Bax, caspase-8, caspase-3 and p53 levels in spleen cells of the sham-irradiated, 2 Gy whole-body irradiated, 2 Gy cranially irradiated animals and animals exposed to scattered radiation only (measured to be 3 mGy). The values are expressed as the means ± SEM (n = 5). *Significant at p < 0.05 and ***significant at p < 0.001 with respect to the sham-irradiated group.

Bax

The Bax level significantly increased (p < 0.05) in the spleen of both cranially irradiated and whole-body irradiated animals compared with sham-irradiated animals (Figure 2). Furthermore, a significant increase was observed in the Bax level in the spleen of the group that was exposed to scattered radiation only compared with the sham-irradiated group (Figure 2).

Bcl-2

Both cranial-irradiation of the animals with 2 Gy of X-rays and whole-body exposure to 2 Gy of X-rays lead to a significant reduction in Bcl-2 level in the spleen (p < 0.05) compared with that measured for the sham-irradiated animals (Figure 2). When the animals were exposed to scattered radiation only, a significant (p < 0.05) decrease was observed in the level of Bcl-2 in the spleen compared with that measured for sham-irradiated animals (Figure 2).

Caspase-8

In Figure 2b, the expression level of caspase-8 was significantly (p < 0.05) increased in the spleen of both a cranially irradiated and whole-body irradiated animals compared with sham-irradiated animals. When animals were exposed to scattered radiation only, a significant (p < 0.05) increase was observed in the caspase-8 level compared with the sham-irradiated group.

Caspase-3

In Figure 2, caspase-3 was significantly (p < 0.05) increased not only in the spleen of whole-body irradiated animals, but also in the spleen of cranially irradiated animals compared with sham-irradiated animals. Additionally, a significant increase (p < 0.05) was observed in the caspase-3 level in the spleen of the group that was exposed to scattered radiation only compared with the sham-irradiated group.

Apoptosis

Flow cytometry was used to determine the apoptosis levels in the spleen of different animal groups using annexin V-FITC staining. The analysis of the flow cytometry data demonstrated that the percentages of necrotic cells and the late apoptotic cells were significantly (p < 0.05) higher in the spleen of both the cranially irradiated and whole-body irradiated animals than in those of the sham-irradiated animals (Figure 3). Results also demonstrated that the percentage of early apoptotic cells was significantly (p < 0.05) increased in the spleen of cranially irradiated animals and significantly (p < 0.05) decreased in the spleen of whole-body irradiated animals compared with those of sham-irradiated animals (Figure 3). Moreover, when animals were exposed to scattered radiation only, a significant (p < 0.05) increase in the percentages of early and late apoptotic cells and necrotic cells was observed in the spleen compared with sham-irradiated animals (Figure 3).

Figure 3.

Changes in the apoptosis levels in spleen cells of the sham-irradiated, 2 Gy whole-body irradiated, 2 Gy cranially irradiated animals and animals exposed to scattered radiation only (measured to be 3 mGy). The values are expressed as the means ± SEM (n = 5). *Significant at p > 0.05, **significant at p > 0.01, and ***significant at p > 0.001 with respect to the sham-irradiated group.

Comet assay observations

The changes in the levels of the comet parameters are displayed in Figure 4 and summarized in Table 2. Cranial irradiation of the animals lead to a significant increase in the levels of all comet attributes, including percentage of DNA in tail, tail length and tail moment in the spleen of animals compared with that measured for the sham-irradiated ones. Also, 2 Gy X-ray whole-body irradiation showed a significant (p < 0.05) increase in the comet parameters in the spleen of animals, suggesting that DNA damage occurred in the spleen of both cranially irradiated and whole-body irradiated animals. Furthermore, a slight increase was observed in the comet parameters in the spleen of the group that was exposed to scattered radiation only, compared with the sham-irradiated group.

Figure 4.

Changes in the percentage of DNA damage in the spleen cells of the sham irradiated, 2 Gy whole-body irradiated, 2 Gy cranially irradiated animals and animals exposed to scattered radiation only (measured to be ~3 mGy) using the alkali comet assay that detects DNA single-strand breaks. (a) Percent of tail DNA, (b) tail length and (c) tail moment. The appearance of the microscopic images of representative comets for the different groups is shown at the bottom of histograms. The values are expressed as the means ± SEM (n = 2). *Significant at p > 0.05, ** significant at p > 0.01, and ***significant at p > 0.001 with respect to the sham-irradiated group.

Table 2.

Changes in the percentage of DNA damage in the spleen cells of the sham irradiated, 2 Gy whole-body irradiated, 2 Gy cranially irradiated animals and animals exposed to scattered radiation only (measured to be 3 mGy)

	Sham-irradiated	Cranially irradiated	Whole-body irradiated	Scattered radiation irradiated
% of tail DNA	1.5 ± 0.16 n = 4	2.1 ± 0.069*	2.6 ± 0.16**	1.9 ± 0.17
Tail length	1.7 ± 0.055	2.5 ± 0.092***	2.7 ± 0.11***	2.2 ± 0.072**
Tail moment units	2.6 ± 0.35	5.2 ± 0.36**	6.9 ± 0.14***	4.2 ± 0.51*

The values are expressed as the means ± SEM (n = 2).

*Significant at p < 0.05,

**significant at p < 0.01, and

***significant at p < 0.001 with respect to the sham-irradiated group.

Discussion

The abscopal effect is considered one of the most controversial aspects in radiobiology and drew the attention of many researchers. The controversy of the abscopal effect comes from the contradictory findings in the literature with respect to the beneficiary of the harmfulness of the radiation-induced abscopal effect. From one point of view, the abscopal effect has been shown to be harmful. These effects were mostly detrimental effects and the abscopal effect was considered to be an extension of the detrimental effects of radiation.²⁹ However, other data show how abscopal effect could be beneficial in the form of reduced growth of tumours distant from irradiated areas.^23,24

Hence, comprehensive examination of the bystander effects under conditions relevant to human radiation exposures are needed to assess their potential significance in evaluating the harmful health effects of radiation. This could influence our understanding to the overall result of radiotherapy.

The current study investigated abscopal effect on the spleen of rats upon cranial-irradiation with 2 Gy of X-rays. The spleen has been previously reported to be an important radiation target organ for elucidation of the bystander effects.³⁰ Upon cranial-irradiation, the spleens of the rats were out of the primary radiation beam.

The induction of ROS and RNS following irradiation can perturb the normal redox balance in the cells leading to chronic inflammatory responses.^31,32 Lipid peroxidation is one of the major effects of oxidative damage following irradiation³³ and can affect function of cellular membranes. The products of lipid peroxidation can activate caspases, which subsequently activate DNase that degrades DNA leading to apoptosis.³⁴ As demonstrated in the present study, both whole-body irradiation with 2 Gy of X-rays and out-of-field scattered dose alone cause an increase in the MDA level MDA, with decreased levels of SOD, CAT and GSH 24 h post-irradiation establishing excessive oxidative stress in the spleen. The increase in oxidative stress is a consequence of higher ROS generation, which are mainly produced by the electron transport chain of the mitochondria and by the cytoplasmic NADPH oxidases³⁵ that can cause DNA damage, as well as cellular responses such as apoptosis, senescence, cell cycle arrest and possibly cancer.³⁶ However, cranial irradiation of the animals cause a comparable level of MDA in the spleen cells to that measured for the sham-irradiated animals, with increased levels of SOD, CAT and GSH 24 h post-irradiation. Some reports showed that MDA is less toxic than other lipid peroxidation products.^37,38 In support with that, our results suggest that the MDA is not implicated in the induction of damage in the distance spleen cells.

Moreover, C-RP is used as a specific marker of tissue damage and inflammation³⁹ induced by radiation.^40,41 The present study demonstrated that the C-RP levels significantly increased in the plasma of whole-body, cranially irradiated rats as well as rats exposed to scattered radiation. Thus, even exposing only part of the body to ionizing radiation or exposing the whole body to a radiation dose as low as 3 mGy has a significant inflammatory effect. This is in agreement with the reported data of the increased levels of C-RP in patients receiving radiotherapy.⁴² This also suggests a role of the innate immune system in the response, as C-RP is known to be a part of the innate immunity system.⁴³

Since an increased calcium ions (Ca2+) concentration have been shown to stimulate the mitochondrial ROS formation,⁴⁴ it is believed it can influence various cellular activities such as enzyme activation, cycle regulation and apoptosis.^45,46 Previous report suggested that in vitro, a sharp transient calcium flux triggered the response in cells receiving bystander signals.^47,48 Investigation of various stress pathways suggested a role for the MAPK pathway leading to induction of apoptosis.⁴⁹ Therefore, in the current study calcium ion concentration was measured to see if the Ca2+ was associated with apoptosis. By analysing Ca2+ concentration in the spleen and the plasma of whole-body irradiated animals, cranially irradiated animals and animals which received dose from the scattered radiation only, the data in Table 1 don't show any change in the calcium ion concentration neither in the spleen cells nor in the plasma compared with sham-irradiated animals. Although, the results here suggest that the abscopal effects seen in the spleen cells are not related to the calcium level or transport, it would not be appropriate to conclude the independence of the observed abscopal effect on the calcium level as it could be that the 24-h time point used in our study was late enough not to detect any increase in the calcium level. Mothersill et al⁵⁰ demonstrated that irradiated C57BL/6 mice but not CBA/Ca mice produce bystander signals that induce calcium fluxes, loss of mitochondrial potential and apoptosis in reporter HPV-G keratinocytes, indicating in vivo induction of bystander signals that are strongly influenced by genetic factors.

A significant increase in number of apoptotic cells in the spleen of both whole-body and cranially irradiated rats was demonstrated with significant reduction in the number of the viable spleen cells in both of the whole-body and cranially irradiated rats. These data are in good agreement with previously reported increase in apoptosis in bystander cells and 3D tissue models.^51,52 Moreover, this increase in apoptosis was also associated with an increase in the expression of the p53 protein. The p53-dependent apoptosis measured here is in consistence with the reported p53-dependence of the abscopal effect measured in the partially irradiated mice.²⁴ The results also demonstrated an increase in the expression of Bax, caspase-3 and caspase-8 associated with a decrease in the level of the anti-apoptotic protein Bcl-2.

Apoptosis is developed through mitochondrial and death receptor pathways as caspase-8 is an initiator protein via two parallel pathways: it can directly cleave and activate caspase-3, or alternatively, it can cleave Bid, a pro-apoptotic Bcl-2 family protein. Truncated Bid translocates to mitochondria, inducing cytochrome c release, which sequentially activates caspase-9 and caspase-3.⁵³

To test whether the radiation have any effect on the DNA damage in the distant organ, an investigation of the DNA damage in the spleen of the cranially irradiated rats was performed using comet assay. Our data show an increase in the DNA damage measured in the spleen of cranially irradiated rats, which is concomitant with the increase in the expression of p53 and caspase-3. It is reported that active caspase-3 has the ability to mobilize the caspase-activated DNase into the nucleus leading to DNA fragmentation.^34,54 These results are in agreement with previously reported data that the radiation-induced apoptosis in the directly hit and bystander cells occurred from the DNA damage.^55,56 Although, neither the nature of the bystander signals nor the mechanisms by which these signals are transmitted to the distant organ is still unidentified, soluble clastogenic factors transmitted through the blood could be one possibility.⁵⁷

In the case, animals that were exposed to scattered radiation only, although, the radiation dose was as low as ~3 mGy, this dose seemed to be sufficient to trigger an effect in the spleen marked by: (1) an increase in the apoptotic cells, (2) a decrease in the viable cells, (3) an increase in the expression of the pro-apoptotic proteins p53, Bax, caspase-3 and caspase-8 and (4) a decrease in the anti-apoptotic protein Bcl-2. Although, the changes in the levels of these factors were much less than those measured for both of the whole-body and cranially irradiated groups, they were significantly different from that measured for the sham-control group. This indicates that low doses of ionizing can have measurable molecular effects on the cells and tissue. Our findings are in agreement with the previous data showing that out-of-field radiation dose alone can have a damaging effect on the proliferation of PNT1A cells when a clinically relevant dose of 2 Gy is delivered in in-field⁵⁸ and the induction of the bystander effect existed when the cells were irradiated with γ-ray at a dose as low as 3 mGy.^59,60

In conclusion, an abscopal effect in the form of DNA damage and induction of apoptosis in spleen of cranially irradiated rats that was demonstrated. It is equal to or sometimes higher than the apoptosis induced in the whole-body irradiated rats with an observed innate immune system response. The mitochondrial and death-receptor apoptosis pathways are involved in the apoptosis induction. A low dose of 3 mGy can induce apoptosis in the spleen cells. This highlights the possible occurrence of unexpected effects of low-dose radiation and its implication on human health.

Therefore, it is essential to perform more studies to explain the mechanism of the bystander effect to more precisely evaluate radiation risks and for better management of radiotherapy protocols.