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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 8  |  Page : 135-139

Expression of transforming growth factor-beta and interferon gamma biomarkers after whole body gamma irradiation


1 Department of Radiology; Ionizing and Nonionizing Radiation Protection Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Radiology, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Radiotherapy and Oncology; Ionizing and Nonionizing Radiation Protection Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
4 Department of Laboratory Medicine, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Web Publication22-Mar-2019

Correspondence Address:
Dr. Reza Fardid
No. 14, Namazee Complex, Khalil St., Shiraz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1324_16

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 > Abstract 


Context: There are plenty of evidence that suggest that the potential high doses of radiation result in severe health effects to exposed individuals, although there is no consensus about the health impact of low dose of ionizing radiation (IR).
Aims: This study aimed to discuss the effect a range of IR doses on the changes of gene expression and serum protein levels of two immune factors transforming growth factor-β (TGF-β) and interferon gamma (IFN-γ) in rats. Findings from this study can be useful to develop a suitable biomarker for biological dosimetry applications.
Subjects and Methods: After 24 h of irradiation of rats with the doses of 1000, 500, 100, 50, and 20 mGy, the gene expression of TGF-β and IFN-γ in lymphocytes was assessed using quantitative polymerase chain reaction. Besides, the protein level of these two factors in blood plasma was determined by enzyme-linked immunosorbent assay (ELISA) kits.
Statistical Analysis Used: One-way analysis of variance Tukey–Kramer Multiple Comparisons Test was used. P <0.05 was considered statistically significant.
Results: Significant increases in the expression levels of TGF-β and IFN-γ genes were observed by increasing the dose from 100 to 500 mGy and then 1000 mGy compared to the control (P < 0.05). The ELISA tests showed significant differences in the serum level of TGF-β cytokine in the dose of 1000 mGy, while the serum level of IFN-γ cytokine showed significant differences in doses of 20 mGy and 1000 mGy compared to the control (P < 0.05).
Conclusions: The results of this study showed the changes in the expression of TGF-β and IFN-γ genes after irradiation more than 100 mGy in lymphocytes compared to the control group; the changes in the serum levels of these cytokines only occurred in the specific doses compared to the control group.

Keywords: Enzyme-linked immunosorbent assay, gene expression, interferon gamma, ionizing radiation, quantitative polymerase chain reaction, transforming growth factor-beta


How to cite this article:
Fardid R, Ghahramani P, Mosleh-Shirazi MA, Kalantari T, Behzad-Behbahani A, Kazemi E, Okhovat MA. Expression of transforming growth factor-beta and interferon gamma biomarkers after whole body gamma irradiation. J Can Res Ther 2019;15, Suppl S1:135-9

How to cite this URL:
Fardid R, Ghahramani P, Mosleh-Shirazi MA, Kalantari T, Behzad-Behbahani A, Kazemi E, Okhovat MA. Expression of transforming growth factor-beta and interferon gamma biomarkers after whole body gamma irradiation. J Can Res Ther [serial online] 2019 [cited 2019 Nov 19];15:135-9. Available from: http://www.cancerjournal.net/text.asp?2019/15/8/135/243482




 > Introduction Top


There are plenty of evidence that high doses of ionizing radiation (IR) reported to have severe health effects on the biological systems, due to direct and indirect action of radiation. In direct interaction, IR causes chromosomal breaks, leading to lethal damage in cell while the production of free radicals happens in an indirect way which generates reactive oxygen species (ROS) (e.g., NO, OH, and H2O2).[1] These species are very unstable but reactive and tend to react with other molecules, imposing damages on lipids, proteins, or nucleic acids. When the immunity of the cells is significantly lower than the production of ROS, oxidative stress occurs, leading to other consequences like infection. Hence, IR can affect the immune system which protects the body against foreign microorganisms.[1],[2],[3] When the lymphocytes and macrophages in the immune system are affected by IR, the level of most cytokines such as interleukin-1 beta (IL-1 β), IL-1, tumor necrosis factor-alpha (TNF-α), IL-2, IL-10, interferon gamma (IFN-γ), and transforming growth factor-β (TGF-β) is elevated, giving rise to a risk of second malignancy after radiotherapy or increasing the level of proliferation and differentiation of the stem cell. Activating macrophages by increasing cytokine production leads to an increase in chromosomal damage, change in DNA bases, mutagenesis and apoptosis. In addition, monocytes play a key role in the induction of immune response. Monocytes are important factor in antigen, the release of cytokines that regulate immune T-cell stimulation, and differentiation.[4] Cytokines are soluble proteins secreted by the immune cells which mediate many actions of these cells. The main cytokines involved in the immune response are IFN-γ, IL-4, IL-17, and TGF-β.[3],[5]

In contrast to high doses of radiation, there is no consensus about the health effects of low-dose radiation.[6],[7] The increasing concerns regarding the health risks arise from exposure to low doses of IR, particularly in the radiation staff involved in occupational exposures or public exposed. Mechanisms behind the cell and tissue response to low doses are not yet clearly defined.[8] Evidence suggests that low doses of IR induce activation of the immune response, leading to molecular and cellular responses, the name of this conceptual process which is known radiation Hormesis. However, this process has not been studied sufficiently yet.[8] In the present study, we investigated the effects of doses of gamma irradiation (20, 50, 100, 500, and 1000 mGy) on the expression of immune factors (TGF-β and IFN-γ) in rat's lymphocytes and blood serums. We applied quantitative reverse transcription-polymerase chain reaction (PCR) to monitor the molecular changes followed by different doses of radiation. In addition, we examined the levels of these two cytokines in the plasma using enzyme-linked immunosorbent assay (ELISA) kits after irradiation with the mentioned identical doses. Findings from this study can be useful to find a suitable biomarker for biological dosimetry applications.


 > Subjects and Methods Top


The study was approved by the ethics committee of SUMS before commencing.

We used 48 male rats (Rattus Norvegicus), 8 in each group, with average body weight of 220 ± 10 g. The animals were housed in animal laboratory of SUMS and fed under standardized controlled condition of temperature and food.

The whole-body gamma-ray irradiation of the animals was performed through the use of Cobalt60 Telegamma unit (Theratron 780, Atomic energy of Canada Limited, Canada), belonging to the Radiotherapy ward of Namazi hospital. The corrected dose rate was determined to be 36/32 cGy/min and irradiation with a distance of 80 cm on the field size of 30 × 30. The animals were randomly divided into five groups for irradiation to different doses of 20, 50, 100, 500, and 1000 mGy. The control group was kept unexposed to radiation.

Twenty-four hours after irradiation, 5cc of the peripheral blood samples was collected using cardiac puncture. It was divided into two parts; 1cc for ELISA tests and the rest of them for PCR tests.

Quantitative polymerase chain reaction methods

RNA preparation and quantification

After isolation of the lymphocytes from the peripheral blood samples, total RNA was extracted from blood samples with the RNX-Plus extraction kit (CinnaGene, Iran), according to the manufacturer's protocol and stored at −20°C. The concentration and quality of RNA were determined by measuring the absorbance by spectrophotometer system (Beckman, USA) at 260 nm (A260) and A260/A280 ratio, respectively.

cDNA synthesis and real-time polymerase chain reaction (quantitative polymerase chain reaction)

cDNA synthesis from RNA was performed with RevertAid™First Strand cDNA Synthesis Kit (Thermo Scientific, k1622). Quantitative measurements of specific transcripts were acquired using a Corbett real-time quantitative PCR (QPCR) system (Applied Corbett, USA) and the amplifications were performed with YTA SYBR Green QPCR masterMix reagents (YT2551, Iran). Verification of amplification of a single PCR product was performed by generating a melting curve. Comparisons of the expression levels for the irradiated and control samples were performed using Gapdh gene as an internal reference for normalization of the results. The primers for Gapdh, TGF-β, and IFN-γ were designed and are shown in [Table 1].
Table 1: The primers and their sequences

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Verification polymerase chain reaction and primer design

To verify IFN-γ, TGF-β and Gapdh primers with 140, 156, and 158 base pair (bp) band lengths, PCR was performed with a cDNA sample. Five microliters of PCR product was loaded on 2% agarose gel. The distinct mentioned band lengths of each gene were visible after ethidium bromide staining. [Figure 1] represents the electrophoresis image of the two primers IFN-γ and TGF-β (IFN-γ, 156 bp, and TGF-β, 140 bp). No template control (NTC) was used to assess the reagent contamination.
Figure 1: Electrophoresis image of the designed interferon gamma, and transforming growth factor-β primers (interferon gamma, 156 base pair transforming growth factor-β, 140 base pair) and the no template control, which is visible in the image

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Enzyme-linked immunosorbent assay method

After collection of the whole blood, the blood was allowed to clot by leaving it undisturbed at room temperature. This took 15–30 min. The clot was removed by centrifuging at 1000–2000×g for 10 min in a refrigerated centrifuge and stored at −20°C until it was used for further investigation/test.

The serum samples were removed from the −20°C freezer and allowed to thaw on ice. The ELISA test was performed with an ELISA kit (eBioscience, USA) according to the manufacturer's protocol. After performing the steps listed in the protocol, the entire plate was placed into a plate reader, and the optical density (i.e., the amount of colored product) was determined for each well. The amount of color produced was proportional to the amount of primary antibody bound to the proteins on the bottom of the wells.

Statistical analysis

Statistical analysis of data was performed using the SPSS version 16 software (Armonk, New York). One-way Analysis of Variance Tukey–Kramer Multiple Comparisons Test was used. P < 0.05 was considered statistically significant.


 > Results Top


Transforming growth factor-beta and interferon gamma gene expression

Mean gene expression values for the control and irradiated groups are shown in [Table 2] and [Table 3]. The mean gene expressions levels of both TGF-β and IFN-γ genes showed a significant difference between the control and irradiated groups by increasing dose from 100 to 1000 mGy (P < 0.05).
Table 2: Results of mean difference of IFN-α gene expression using reverse transcription polymerase chain reaction technique

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Table 3: Results of mean difference of TGF-β gene expression using reverse transcription polymerase chain reaction technique

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[Figure 2] shows the changes in the TGF-β gene expression following irradiation with different doses of gamma-irradiation. The error bar in all graphs represents ± 1 standard deviation. This figure shows that the expression of this gene in all cases increased almost linearly by increasing the dose and at the dose of 1000 mGy this increase was doubled.
Figure 2: Transforming growth factor-β gene expression changes at different doses obtained using polymerase chain reaction

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[Figure 3] shows the corresponding changes in IFN-γ gene expression following irradiation with gamma rays. This figure shows that the expression of this gene in all cases increased with increasing dose.
Figure 3: Interferon gamma gene expression changes at different doses obtained using polymerase chain reaction

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Changes in transforming growth factor-beta and interferon gamma protein product

Mean gene expression values for the control and irradiated groups are shown in [Table 4] and [Table 5]. [Figure 4] and [Figure 5] show the changes in TGF-β and IFN-γ protein product following irradiation with gamma-irradiation in different doses. The expression of the TGF-β protein product can be seen in [Figure 4]; it increased with increasing dose in all cases.
Table 4: Results of mean difference of IFN-α protein expression using ELISA

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Table 5: Results of mean difference of TGF-β protein expression using ELISA

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Figure 4: Changes in the transforming growth factor-β protein product following different doses of gamma rays

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Figure 5: Changes in the interferon gamma protein product following different doses of gamma rays

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The ELISA tests showed significant differences in the serum level of TGF-β cytokine in the dose of 1000 mGy, while the serum level of IFN-γ cytokine showed significant differences in doses of 20 mGy and 1000 mGy compared to the controls (P < 0.05).


 > Discussion Top


The growing use of IR in the range of low dose radiation arose a lot of attention about the impact of low dose radiation on biological systems.[7],[9]

Cytokines are made by many cell populations, but they are mostly produced by helper T cells and macrophages; in other words, they are involved in biological immunity system. They are divided into two groups, pro- and anti-inflammatory cytokines. The latter regulates the pro-inflammatory cytokines receptors.[5] There is evidence which shows the effect of low-dose radiation on the release of cytokines. It is reported that low-doses of ionizing irradiation in mice lead to increased accumulation of the lymphocytes in tumor cells; the antitumor proteins such as IFN-γ and TNF-α are secreted by these lymphocytes.[10]

Safwat in a study showed that doses of 1.5–2 Gy of gamma rays cause changes in the cytokine secretion pattern and the secretion of cytokines IFN-γ and IL-2 increases. It also causes an increase in IL-2 receptors on the lymphocytes.[11] Zakeri et al. reported an increase in the concentration of cytokines IFN-γ in blood samples of radiographers compared to nonradiation workers.[12] The results of another study published by Hayashi et al. in 2003 showed that the level of TNF-α and IFN-γ cytokines in the blood plasma of survivors of nuclear explosions was more than the control group.[13] Spary et al. reported that proliferation and activity of blood mononuclear cells and IFN-γ cytokine production increased in 0.6–2.4 Gy of gamma-ray doses.[14] Li et al. reported that serum levels of cytokines IFN-γ and TNF-α in the blood samples of miners of uranium mines slightly increased.[15] Our results also indicated a significant increase in the concentration of IFN-γ and TGF-β.

In the present study, to investigate the effect of low doses of gamma radiation, we exposed 5 groups of 8 rats to different doses 20, 50, 100, 500, and 1000 mGy, and one group of 8 rats was also used as control group. We examined the changes in gene expression of 2 cytokines after irradiation with gamma-ray using QPCR in the gene expression level in the rat's lymphocyte cells and also by ELISA at the protein level in the rat's blood serum.

Low-dose responses showed that the expression of TGF-β increased with increasing the radiation dose and this increase was more pronounced at higher doses. Comparison between different groups of radiation showed the difference in the expression of TGF-β at doses of 100, 500, and 1000 mGy compared with the control group was statistically significant; in addition, double expression of TGF-β occurred at 1 Gy. At doses of 20 and 50 mGy, we observed no statistically significant difference with the control group. The level of TGF-β gene product was also increased in doses more than 100 mGy.

It was also found that IFN-γ gene expression increases by increasing the radiation dose except in two doses of 20 and 50 mGy and the changes were not statistically significant. IFN-γ gene protein product level in the plasma was not significant after 20 mGy irradiation although by increasing the dose more than 50 mGy, this level increased as well.

The results obtained in this study showed that increased level of expression and protein product of these two genes mostly occurred by increasing the dose of radiation. In the second part of this study, we evaluated the protein levels in the serum after irradiation. Results showed that changes in the serum protein of IFN-γ and TGF-β gene showed a significant increase only in dose of 1000 mGy.

Li et al. reported that the cytokine TGF-β concentrations in the blood serum of miners of uranium mines compared with other miners showed no changes.[15] The findings of this study correspond with the results of the ELISA test.

Our results showed the maximum increasing level of gene expression of 2 cytokines and their protein products after irradiation of 1000 mGy. However, increasing the doses leads to an increase in the levels of these two markers. In addition, it was concluded that low doses of radiation can cause some changes in the biological systems.


 > Conclusions Top


The results of this study showed the changes in the expression of TGF-β and IFN-γ genes after irradiation more than 100 mGy in the lymphocytes compared to the control group; however, the changes in the serum levels of these cytokines only occurred in the specific doses compared to the control group.

Acknowledgment

The present article was extracted from the M. Sc thesis of Parisa Ghahramani and financially supported by Shiraz University of Medical Sciences, Shiraz, Iran (Grant No.: 90037). The authors would like to thank Centre for Development of Clinical Research of the Nemazee Hospital for statically analysis assistance and editing the manuscript.

Financial support and sponsorship

This study was supported by Shiraz University of Medical Sciences, Shiraz, Iran.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Fardid R, Bahreyni-Toossi MT, Rezaee A, Sadr-Nabavi A, Rafatpanah H. Expression of IFNg and TGFb1 genes can distinguish radiation workers from the normal population. Int J Low Radiat 2014;9:396-408.  Back to cited text no. 3
    
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United Nations Scientific Committee on the Effects of Atomic Radiation. Biological Mechanisms of Radiation Actions at Low Doses: A White Paper to Guide the Scientific Committee's Future Programme of Work; 2012.  Back to cited text no. 6
    
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Bahreyni-Toossi MT, Fardid R, Rezaee A, Sadr-Nabavi A, Rafatpanah H, Bolbolian M. Expression of apoptotic genes can distinguish radiation workers from normal population. Int J Low Radiat 2011;8:388-99.  Back to cited text no. 7
    
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El-Saghire H, Michaux A, Thierens H, Baatout S. Low doses of ionizing radiation induce immune-stimulatory responses in isolated human primary monocytes. Int J Mol Med 2013;32:1407-14.  Back to cited text no. 8
    
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Azimian H, Bahreyni-Toossi MT, Rezaei AR, Rafatpanah H, Hamzehloei T, Fardid R, et al. Up-regulation of bcl-2 expression in cultured human lymphocytes after exposure to low doses of gamma radiation. J Med Phys 2015;40:38-44.  Back to cited text no. 9
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Lugade AA, Moran JP, Gerber SA, Rose RC, Frelinger JG, Lord EM, et al. Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. J Immunol 2005;174:7516-23.  Back to cited text no. 10
    
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Safwat A. The immunobiology of low-dose total-body irradiation: More questions than answers. Radiat Res 2000;153:599-604.  Back to cited text no. 11
    
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Zakeri F, Hirobe T, Akbari Noghabi K. Biological effects of low-dose ionizing radiation exposure on interventional cardiologists. Occup Med (Lond) 2010;60:464-9.  Back to cited text no. 12
    
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Hayashi T, Kusunoki Y, Hakoda M, Morishita Y, Kubo Y, Maki M, et al. Radiation dose-dependent increases in inflammatory response markers in A-bomb survivors. Int J Radiat Biol 2003;79:129-36.  Back to cited text no. 13
    
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Spary LK, Al-Taei S, Salimu J, Cook AD, Ager A, Watson HA, et al. Enhancement of T cell responses as a result of synergy between lower doses of radiation and T cell stimulation. J Immunol 2014;192:3101-10.  Back to cited text no. 14
    
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Li K, Chen Y, Li X, Lei S, Chen Q, Liu J, et al. Alteration of cytokine profiles in uranium miners exposed to long-term low dose ionizing radiation. Sci World J 2014;2014:Article ID: 216408, 5.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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