Markers of oxidative stress and inflammation increase in the lung and liver of partially irradiated rats

ABSTRACT

This manuscript seeks to examine the induction of oxidative stress (OS) and the inflammatory response in the abscopal liver and lung of partially irradiated rats in comparison with the effects induced in the same organs of whole-body irradiated rats. For this purpose, 20 Sprague‒Dawley adult male rats were divided into four groups: 1) the control group, the nonirradiated group; 2) the 2 Gy whole-body γ-irradiated (WB) group; 3) the 2 Gy cranially γ-irradiated (Cr) group; and 4) the 2 Gy lower-limb γ-irradiated (LL) group. The rats were euthanized 24 h postirradiation, and lung and liver samples were collected for MDA, GSH, NF-κB, TGF-β1, caspase 3 and p53 analyses. Our data demonstrated increased levels of MDA in the liver and lung of the partially irradiated groups associated with GSH overexpression. Our data also revealed notable elevation in NF-κB and TGF-β1 levels in both organs associated with significant increase in p53 and caspase-3 levels in the partially irradiated groups. Our findings provide evidence of OS and inflammatory response in abscopal liver and lung tissues. Furthermore, the abscopal effect (AE) induced was comparable and, in some cases, greater than the effect of direct irradiation reported in the whole-body irradiated group.

KEYWORDS:

• Ionizing radiation (IR)
• abscopal effect (AE)
• malondialdehyde (MDA)
• transforming growth factor-β1 (TGF-β1)
• p53

Introduction

Reactive oxygen species (ROS) mediate the biological effects of ionizing radiation (IR). ROS are generated through ionization and the production of free electrons as a result of the interaction of IR with water molecules in biological systems [1,2]. ROS, such as superoxide anion (O₂⁻), hydroxyl radicals (^•OH), and hydrogen peroxide (H₂O₂), can act as signals in the intercellular signaling process between irradiated and nonirradiated cells [3–7] and induce oxidative damage to cells [4,8,9]. ROS attack cellular macromolecules such as DNA, RNA, proteins, and cell membranes, resulting in their dysfunction and destruction [10]. Numerous pathological states are associated with either a decrease in free radical scavenging capacity or an increase in free radical levels [11]. Oxidative stress (OS) refers to an imbalance between the production of ROS and the ability of the body to detoxify them or repair the resulting damage. OS can lead to damage to various cellular components, including DNA, proteins, and lipids [12,13]. Cellular damage can lead to various diseases and conditions, such as neurodegenerative diseases, cardiovascular diseases, cancer, and aging. Several mechanisms are involved in the cellular damage caused by OS, including lipid peroxidation, protein oxidation, DNA damage, and mitochondrial dysfunction [14].

In addition to the effects of direct irradiation, the abscopal effect (AE) of IR is an in vivo phenomenon, which refers to the implementation of the radiation effects in cells that were not directly irradiated but were nearby to the irradiated cells as a result of signaling between the irradiated and nonirradiated cells. Therefore, AE is thought to be responsible for the expansion of radiation effects to distant nonirradiated regions [15–20]. OS has been reported to play a pivotal role in the induction of AE [9,21,22]. Many of the proposed mechanisms contributing to abscopal radiation effects are based on tumor irradiation experiments [23–25]. Moreover, OS has been reported in abscopal tissues following partial irradiation of rats, which in some cases was equivalent to the effect of direct irradiation [18,26,27].

To counteract the harmful effects of IR-induced OS as a result of direct or abscopal irradiation, cells have several antioxidant defense mechanisms [28,29]. These mechanisms include enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, as well as nonenzymatic antioxidants such as vitamin C, vitamin E, and glutathione. These antioxidants help neutralize ROS and prevent them from causing further damage. Cellular antioxidant production following radiation is a complex process that involves the activation of various molecular pathways. One of the key players in cellular antioxidant production is the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) [30]. Upon activation, NRF2 translocates to the nucleus and binds to antioxidant response elements (AREs) in the promoter region of target genes. This induces the transcription of various antioxidant enzymes, including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1), among others [31].

In addition to NRF2-mediated transcriptional activation, posttranscriptional and posttranslational mechanisms also contribute to cellular antioxidant production following radiation. These include the stabilization of antioxidant enzymes, the modulation of ROS detoxification pathways, and the regulation of redox balance. Overall, cellular antioxidant production following radiation is a tightly regulated process that involves the coordinated activation of multiple antioxidant defense mechanisms. Understanding the intricacies of this process is crucial for developing strategies to enhance cellular resilience against radiation-induced oxidative damage.

In radiotherapy, much care and many safety measures are applied to avoid the direct irradiation of the organs at risk in close vicinity of the tumor to be treated. The development of the AE concept sheds light on the potential risk of IR toxicity in normal tissue distant from the irradiated sites. Therefore, studies are needed to evaluate the potential damage induced in abscopal tissues following irradiation. These studies may lead to modulating the safety measures applied to radiotherapy patients to minimize the side effects of radiotherapy.

In the present study, we aim at investigating the damage and OS induced in organs distant from the irradiated regions in an effort to get a glimpse of the possible impact of the AE on normal tissue toxicity using a radiotherapy-relevant radiation dose. For this purpose, we investigated radiation-induced AE in liver and lung tissues resulting from cranial and lower-limb irradiation with 2 Gy γ-radiation, which represents the most common therapeutic dose/fraction used, in comparison with direct irradiation in the whole-body irradiated group and the nonirradiated control group.

Materials and methods

Experimental animals

Twenty Sprague‒Dawley (SD) adult male rats weighing 160–200 g randomly divided into four cohorts, each consisting five rats, were used in this study. The first group, the control group, in which the rats were not γ-irradiated. The second group, the whole-body irradiation (WB) group, in which the rats were subjected to 2 Gy whole-body γ-irradiation with. The third group, the cranially irradiated group (Cr), in which the rats were γ-irradiated at the head only. The fourth group, the lower limb irradiated (LL) group, in which only the thigh of the rat’s right leg was γ-irradiated.

The animals were maintained on plastic chests with bedding at a temperature of 20–24°C with a 12/12 h light/dark cycle under a balanced diet with free access to food and water. The rats were euthanized 24 h post-irradiation, and lung and liver samples were collected from the rats in all groups.

All the experimental procedures described in this study complied with the ethical guidelines and principles for the care and use of laboratory animals adopted by the ethics committee at the National Research Centre, Cairo, Egypt (approval no: 17–018).

Irradiation

The experimental animals were irradiated with 2 Gy of 1.25 MeV γ-radiation using the⁶⁰Co γ-irradiator Theratron Gammabeam 100–80 (Best Theratronics Ltd., Canada) at a dose rate of 0.5 Gy/min. The 2 Gy dose was chosen to represent the most common therapeutic dose/fraction used. For each experimental group, different radiation fields were used to accommodate the designated target region, with the five rats being irradiated at once (Supplementary data).

Tissue homogenate preparation

The liver and lung samples were first perfused with ice-cold 1X PBS containing 0.16 mg/ml heparin to remove any remaining red blood cells and clots. Then, the samples were rinsed with 1X PBS. The samples were then homogenized in 20 ml of 1X PBS (containing protease inhibitors) and stored overnight at −20°C. Two freeze‒thaw cycles were performed to disrupt the cell membranes. Furthermore, the samples were homogenized using a tissue homogenizer (Omni International, USA) and centrifuged for 5 min at 5000 × g. The supernatant was then removed.

Biochemical assays

The concentrations of the lipid peroxidation product malondialdehyde (MDA) and the reduced form of glutathione (GSH) in the liver and lung tissue homogenates were quantified by a colorimetric method using an MDA detection kit (MD 2529, Biodiagnostic, Egypt) and a glutathione detection kit (GR 2511, Biodiagnostic, Egypt), following the manufacturer’s protocol.

Enzyme-linked immunosorbent assay (ELISA)

First, the protein concentrations in the tissue samples were determined using a protein estimation kit (2624800021730, Genei, India) according to the method of Bradford et al. [32], where Coomassie blue G250 dye binds to proteins. The quantity of protein could then be estimated by measuring the absorbance at 595 nm using a spectrophotometer (Labomed UVD-2950, USA).

The concentrations of NF-κB, active TGF-β1, cleaved caspase-3 and p53 in liver and lung tissue homogenates were estimated by ELISA kits (CSB-E13148r, CUSABIO, USA), SEA124Ra, Cloud-Clone Corp., USA), HEA626Ra, Cloud-Clone Corp., USA) and NBP2–75359, Novus Biologicals, USA), respectively, following the manufacturers’ protocols. (Further details are provided in the supplementary data). In all of the tests, the concentration of the marker was quantified by measuring the optical density (OD) spectrophotometrically at a wavelength of 450 nm and comparing the obtained OD with the standard curve.

Statistical analysis

The values are expressed as mean ± standard deviation (S.D.), for five rats in each group. Statistical analyses were performed using two-way analysis of variance (ANOVA), where degrees of freedom (df) between columns = 9, df between rows = 3, df_residual = 27, and df_total = 39, followed by a Tukey – multiple comparisons post-hoc test to recognize which groups differ from the rest. Differences were considered significant at p < 0.05, n = 5 in each of the four groups. Statistical analyses were carried out using GraphPad Prism software (GraphPad Software Inc. V8.01, San Diego, CA, USA). Shapiro–Wilk test for normality and Pearson r correlation of effect size analysis were used to check assumptions of ANOVA and magnitude of differences observed, respectively.

Results

In this study, we investigated the OS and inflammatory response induced in the liver and lung of partially (Cr and LL groups) and whole-body (WB group) γ-irradiated rats in comparison with the control group that was mock irradiated. Furthermore, the data of the Cr and LL groups were compared with the WB group. In the case of Cr and LL groups, the liver and lung were not irradiated and considered abscopal to the irradiated region. Therefore, the effect detected in both Cr and LL groups represents the abscopal effect induced as a result of cranial or lower limb irradiation. On the other hand, the liver and lung of the WB group were directly irradiated. Therefore, the effect induced in the WB group represent the effect of direct irradiation.

The lipid peroxidation, as indicated by MDA expression level, and variation of endogenous GSH expression were examined as indicators of the OS. Lipid peroxidation was assessed by measuring lipid peroxidation product (MDA) levels in the different groups. Figure 1 shows that the MDA levels in both the liver and lung were significantly greater in all of the irradiated (WB, Cr and LL) groups than in the control group (p˂0.05). Moreover, the MDA level in liver of the LL group was significantly greater than that of the WB and Cr groups (p˂0.05), while no significant difference was detected between the WB and Cr groups. On the other hand, the lung samples from all the irradiated groups showed comparable levels of MDA, indicating equality in the effect of both direct irradiation and abscopal irradiation.

Figure 1. MDA levels (mmol/g tissue) in the liver and lung of the control, WB, Cr and LL groups. The values represent the means ± S.D. (n = 5). *p < 0.05 versus control, and @p < 0.05 versus Cr. group.

The antioxidant activity was assessed by measuring the level of GSH. Figure 2 shows a significant (p < 0.05) increase in the GSH level in the liver of the Cr and LL groups compared with that in the control group. In contrary, no significant increase was reported in the WB group in comparison with the control group. Furthermore, the GSH level in the liver of the LL group was significantly (p < 0.05) greater than that in the lung of the WB and Cr groups. The GSH level in the lung of the LL group was significantly (p < 0.05) greater than that of the control, WB and Cr groups, while no significant change was recorded in the WB and Cr groups in comparison with the control group.

Figure 2. GSH levels (mmol/g tissue) in the liver and lung of the control, WB, Cr and LL groups. The values represent the means ± S.D. (n = 5). *p < 0.05 versus control, $p < 0.05 versus WB group, and @p < 0.05 versus Cr. group.

Furthermore, we investigated TGF-β1 expression due to its direct correlation with radiation-induced oxidative stress. TGF-β1 signaling has been shown to be activated in response to radiation-induced oxidative stress and TGF-β1 signaling leads to the activation of downstream signaling pathways, such as SMAD proteins, that play a role in the regulation of oxidative stress and antioxidant defense mechanisms. In the context of radiation-induced inflammation, TGF-β1 plays a complex role. On the one hand, it can promote tissue repair and fibrosis by stimulating extracellular matrix deposition and myofibroblast differentiation. However, excessive TGF-β1 signaling, as a result of the activation of TGF-β1 by radiation-induced ROS, can also exacerbate inflammation and contribute to fibrotic tissue remodeling, leading to long-term complications post-radiation therapy. Figure 3 shows significant elevation (5–7-fold) (p < 0.05) in TGF-β1 levels in the liver and lung in all of the irradiated groups as compared with the control group. No significant difference between the abscopal (Cr and LL) and the WB groups was observed for either organs, indicating that the abscopal effect was comparable to the effect of direct irradiation.

Figure 3. TGF-β1 levels (pg/mg protein) in the liver and lungs of the control, WB, Cr and LL groups. The values represent the means ± S.D. (n = 5). *p < 0.05 versus control.

As a key transcription factor that regulates the expression of genes involved in inflammation and immunity, NF-κB was investigated. Activation of NF-κB signaling is a hallmark of radiation-induced inflammation, driving the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. These cytokines recruit immune cells to the irradiated site and amplify the inflammatory response, leading to tissue damage and dysfunction. Similar to the TGF-β1 data, Figure 4 shows that the NF-κB levels significantly (p < 0.05) increased (2.5–3-fold) in the liver and lung of all of the irradiated groups compared with the control group. No significant difference between the abscopal (Cr and LL) and the WB groups was observed for either organs

Figure 4. NFκB levels (pg/mg protein) in the liver and lung of the control, WB, Cr and LL groups. The values represent the means ± S.D. (n = 5). *p < 0.05 versus control, and @p < 0.05 versus Cr. group.

We further investigated the p53 expression. The data in Figure 5 show, for both liver and lung, p53 expression is increased significantly (1–2 fold) in all of the irradiated groups, whether partially or whole body irradiated, as compared with the control. The levels of p53 expression in the Cr and LL groups were comparable to that of the WB groups for both organs too. In analogous to p53 data, caspase 3 data (Figure 6) show significant (p < 0.05) increase in the liver of all irradiated groups compared with that in the control group, with 1-fold and 2-fold increases in the whole body, cranially irradiated and lower limb-irradiated groups, respectively. The lung data also show significant (p < 0.05) increase in the caspase 3 expression level in all of the irradiated groups above the control level with no significant difference between the p53 levels in the Cr and LL groups, on one side, and the WB group on the other side. The data of TGF-β1, NF-Κb, p53 and caspase 3 refer in general to the induction of abscopal effect in both organs, which is comparable to the effect of direct irradiation and lead to p53-dependent apoptosis.

Figure 5. P53 levels (pg/mg protein) in the liver and lung of the control, WB, Cr and LL groups. The values represent the means ± S.D. (n = 5). *p < 0.05 versus control.

Figure 6. Caspase-3 levels (pg/mg protein) in the liver and lung of the control, WB, Cr and LL groups. The values represent the means ± S.D. (n = 5). *p < 0.05 versus control.

Discussion

Radiation-induced AEs have long been the focus of research due to their influence on normal distant tissue toxicity. Therefore, it is fundamental to consider the influence of localized radiotherapy on distant regions. In this study, we investigated radiation-induced AEs in the liver and lungs of partially irradiated rats and compared the effects of direct irradiation on the same organs in whole-body irradiated and control groups by examining lipid peroxidation, antioxidant expression, and inflammatory and apoptosis markers.

One of the key markers of oxidative damage to lipids is malondialdehyde (MDA), an end product of lipid peroxidation. The products of lipid peroxidation, such as malondialdehyde (MDA), are believed to be secondary messengers of OS, enhancing OS-stimulated injury by increasing the damage caused by other biological molecules, such as proteins. Consequently, injury to lipids can lead to injury to the cell membrane and influence the various tasks exerted by the cell membrane, which may lead to cell death [33]. In addition, MDA has mutagenic effects by destroying DNA either by interacting with nucleic acid bases, which produce numerous adducts [34,35], or activating DNAase via caspase signaling, resulting in DNA decay [36].

In the present study, MDA levels are significantly greater in the liver and lungs of the WB, Cr and LL groups than in those of the control group. These results are consistent with data obtained by our group [18] and others [37,38], which show increase in the level of lipid peroxidation in abscopal tissues. Our data also show that the levels of MDA in both the abscopal (Cr and LL groups) and directly irradiated (WB group) organs are comparable. Therefore, this study provides evidence of damaging AE, which is equivalent to the effect of direct irradiation.

Cells acquire protection against OS through both enzymatic and nonenzymatic antioxidant defense systems [39]. These antioxidant defense systems consist of antioxidant enzymes such as catalase and low molecular weight antioxidants such as glutathione (GSH). The stimulation of endogenous GSH after exposure to IR can be useful in protecting cells from OS [29]. Our data reveal that GSH expression is significantly elevated in the liver of the Cr group compared with that in the control group. In addition, the GSH levels in the liver and lung of the LL group are notably greater than those in the control and WB groups. These findings are consistent with previous finding that GSH levels are significantly elevated in the spleens of cranially irradiated rats [18].

Interestingly, after tissue exposure to radiation, the levels of chemokines, cytokines, and growth factors increase, resulting in an inflammatory response [40]. A fluent mechanism of how radiation might stimulate an AE through IR-stimulated changes during cell death, which excites numerous inflammatory mediators that are liberated from the demise cell, with a resultant attraction and/or stimulation of immune cells inside the cellular microenvironment. These cells can produce further cytokines, which act both topically and systemically over the general and lymphatic circulation, influencing distant normal nontargeting tissues [41].

The TGF-β1 data show that the liver and lung TGF-β1 levels are significantly elevated in the Cr and LL groups compared with those in the control group and were comparable to those in the directly irradiated group (WB). These data suggest excess TGF-β1 was activated as a result of OS, which is supported by previous findings showing increase in TGF-β1 in response to oxidizing conditions following IR exposure [42–45] in addition to stimulating the elevation of intracellular ROS in nonirradiated cells [46]. Our data also suggest a role of TGF-β1 in mediating the AE in the liver and lung, which is concomitant with the findings of other groups [9,47,48] that TGF-β1 plays a significant role as a mediator of AE. Furthermore, the significant increase in TGF-β1 levels 24 h following irradiation, as shown in our study, is analogous to the findings of Jiang et al. [49] showing that TGF-β1 induced by ROS is clearly involved in early but not late abscopal responses.

To further study the inflammatory response in the abscopal liver and lung in comparison with the control and directly irradiated organs, we investigated the expression of NF-κB. NF-κB is a transcriptional regulatory protein that modulates the expression of immunoregulatory genes related to inflammatory diseases, cancer, and apoptosis. Compared with those in the control group, NF-κB levels in the liver and lung significantly increased in the Cr and LL groups, which can be attributed to the induction of OS [50]. These results are consistent with previous data showing that hepatic and pulmonary NF-κB levels are significantly increased following cranial irradiation in rats [38]. The interaction between TGF-β1 and NF-κB is complex and bidirectional, contributing to the fine-tuning of inflammatory responses post-radiation. TGF-β1 can activate NF-κB signaling through various mechanisms, including the upregulation of NF-κB subunits, promotion of NF-κB nuclear translocation, and induction of NF-κB target genes. Conversely, NF-κB activation can also enhance TGF-β1 expression and signaling, forming a positive feedback loop that sustains the inflammatory processes. NF-κB expression in the abscopal organs is found to be comparable to that in the directly irradiated organs in the WB group, yet again providing more evidence of the induction of AE due to partial irradiation of the rats.

We further investigated the expression of p53 following cranial and lower limb irradiation. Our data show that liver and lung p53 levels are significantly greater in the Cr and LL groups than in the control group and are comparable to those in the WB group, where the organs were directly irradiated. Our results are consistent with the data previously obtained by Mohye El-Din et al. and Koturbash et al., who reported that cranial irradiation of rats stimulated apoptotic cell death with elevated p53 in the abscopal spleen using similar radiation doses [18,26]. Our data are also in agreement with the data of Mukherjee et al., who showed an increase in the intrinsic apoptotic pathway mediator p53 in abscopal cells in comparison to that in nonirradiated controls [51]. These data, in combination with the MDA data, suggest the induction of p53-dependent apoptosis in the abscopal liver and lung tissues following cranial or lower limb irradiation. To test this hypothesis, we further investigated the expression of caspase-3, a hallmark of apoptosis and key executioner caspase involved in the final stages of apoptosis [52,53]. Therefore, caspase-3 data can indicate the induction of apoptosis in liver and lung cells.

Our data show that the caspase-3 concentration is notably greater in the Cr and LL groups than in the control group and is comparable to that in the WB group. The p53 and caspase 3 data, together with the MDA data, suggest the induction of OS in the abscopal liver and lung tissues, which resulted in p53-dependent apoptosis as a result of lipid peroxidation in the cells. This assumption is further supported by the increased inflammatory response indicated by increased TGF-β1 and NF-κB expression.

This study provides evidence of AE induced by IR in the liver and lung of cranially and lower-limb-irradiated rats 24 h post-exposure. The induced AE is comparable to the effect of direct irradiation, which means that normal tissue toxicity risk needs to be taken into account when planning radiation treatments. More time points and clinically related endpoints can provide more information on the kinetics of the induced AE. However, due to funding limitations, we were not able to expand the study further at this time, but this topic is well considered in future plans.

Conclusions

Cranial and lower-limb irradiation of experimental animals with 2 Gy γ-radiation leads to an increase in the lipid peroxidation in abscopal liver and lung as indicated by the increase in MDA levels. In addition, increases in the inflammatory markers TGF-β1 and NF-κB associated with increase in apoptosis markers p53 and caspase 3 were also reported in the abscopal liver and lung. The increases in the MDA levels as well as the inflammatory and apoptosis markers in the abscopal liver and lung were comparable to the levels in their directly irradiated counterparts in the WB group. These data indicate the induction of OS and an inflammatory response in the abscopal liver and lung and that AE is comparable and in some cases greater than the effect of direct irradiation demonstrated in the whole-body irradiation group. These findings highlight the potential jeopardy of normal tissue toxicity associated with localized radiotherapy. Further investigations using different time points to represent the late effects of IR are still needed for better understanding of the long-term effect of radiotherapy.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Research Centre, Cairo, under Grant number 13020305.