|Year : 2017 | Volume
| Issue : 1 | Page : 51-55
Radiation-induced non-targeted effect in vivo: Evaluation of cyclooygenase-2 and endothelin-1 gene expression in rat heart tissues
Reza Fardid1, Masoud Najafi2, Ashkan Salajegheh1, Elahe Kazemi1, Abolhasan Rezaeyan3
1 Department of Radiology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Medical Physics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Medical Physics, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran
|Date of Web Publication||16-May-2017|
Department of Medical Physics, School of Medicine, Tehran University of Medical Sciences, Tehran
Source of Support: None, Conflict of Interest: None
Aim: In this study, we investigated expression levels of cyclooxygenase-2 (COX-2) and endothelin-1 (ET-1) genes after pelvis and heart irradiation in a rat model. These factors are involved in heart diseases (HDs).
Materials and Methods: We used seven groups, including two groups of pelvic irradiation, two groups of whole body irradiation, two groups of heart irradiation, and one control nonirradiated group. Pelvis irradiations were conducted at a 2 cm × 2 cm in the pelvis area. Irradiation condition conducted using 1.25 MeV cobalt-60 gamma-rays (30 cGy/min). The changes at ET-1 and COX-2 gene expressions in heart tissue after pelvis and heart irradiation were measured and compared to the control and whole body irradiation groups at 24 h and 72 h after the exposure.
Results: In heart irradiation groups, 3-fold up-regulation of both ET-1 and COX-2 was observed. In pelvis irradiation groups, 3-fold up-regulation of ET-1 was seen, but not significant changes in COX-2 gene expression have observed at distant heart tissues after pelvis irradiation.
Conclusion: This study reveals that nontargeted effect induced by radiation may be considered as an important phenomenon for induction of HD after radiotherapy.
Keywords: Cyclooxygenase-2, endothelin-1, heart, nontargeted, radiation
|How to cite this article:|
Fardid R, Najafi M, Salajegheh A, Kazemi E, Rezaeyan A. Radiation-induced non-targeted effect in vivo: Evaluation of cyclooygenase-2 and endothelin-1 gene expression in rat heart tissues. J Can Res Ther 2017;13:51-5
|How to cite this URL:|
Fardid R, Najafi M, Salajegheh A, Kazemi E, Rezaeyan A. Radiation-induced non-targeted effect in vivo: Evaluation of cyclooygenase-2 and endothelin-1 gene expression in rat heart tissues. J Can Res Ther [serial online] 2017 [cited 2020 Jul 10];13:51-5. Available from: http://www.cancerjournal.net/text.asp?2017/13/1/51/203601
| > Introduction|| |
There is accumulating evidence which show radiation induces heart diseases (HDs) in lung and breast cancer patients which undergoing radiotherapy (RT). The occurrence of HDs in these patients is high and it has been considered as a major cause of death during years after the treatment. The main HD due to irradiation are cardiac fibrosis, coronary vascular injury, atherosclerosis, heart valves disease which are responsible to heart attack.
Several studies have shown immune responses have a key role in HDs among RT patients. Cyclooxygenase-2 (COX-2) and endothelin-1 (ET-1) are two key factors that play important roles in regulating cardiovascular function in inflammation condition. Several studies have shown that over-expression of some inflammatory genes such as COX-2 are associated with an increase in the probability of many HDs, such as inflammation, ischemia, and myocardial infarction., Furthermore, abnormal up-regulation of COX-2 increases the production of prostaglandins such as PGI2 and PGE2, and some signaling pathways that can result to increase of matrix metalloproteinase enzymes activity that may lead to fibrosis and atherosclerosis.
Furthermore, ET-1 is the most potent and long-lasting vasoconstrictor in heart tissue, and has been proposed as an important factor in HDs after exposure to radiation. ET-1 has a key role in pro-inflammatory and profibrotic effects in the heart tissues. In addition, ET-1 contributes to endothelial dysfunction, increasing oxidative stress, and also promoting hypertension and atherosclerosis.,, It seems, ET-1 increases ROS production and inflammatory markers through activation of NADPH oxidase.,
Nontargeted (or out-of-field) effects induced by ionizing radiation (IR) can lead to damages to tissues which not directly irradiated. The mechanisms of this phenomenon have been not completely recognized.In vivo and in vitro studies have shown that cytokines such as transforming growth factor (TGF)-β, tumor necrosis factor-α, interleukin (IL)-1, IL-2, IL-8, and IL-33 are involved in the nontargeted damages after irradiation. The levels of these cytokines rises after RT for a long time and exerts their effects on nonirradiated tissues. They also elevate the expression of reactive oxygen species (ROS) generating enzymes such as COX-2, inducible nitric oxide synthase and NADPH oxidase. Besides mutation and carcinogenesis, changed immune responses after exposure to IR is a serious concern that is involved in several disorders include HDs. COX-2 production in nonirradiated tissues is tissue dependent and can be induced by some cytokines, including TGF-β. Chronic inflammation associated with long-term up-regulation of COX-2 is regarded as a potential risk for cancer and noncancerous diseases like heart failure. Chai et al. have investigated COX-2 over-expression in nontargeted lung and bronchial epithelial cells after lower abdominal irradiation. Up-regulation of COX-2 has resulted in the increase of ROS production and inflammatory responses in nonirradiated tissues. With respect to the fact that the levels of immune mediators such as TGF-β remain high for a long time after RT, we hypothesized that local pelvis irradiation may result in changes in COX-2 and ET-1 gene expression in the heart. This may be a threat to cause HDs after RT.
| > Materials and Methods|| |
In this experimental study, we used seven groups, including irradiation to two groups of pelvic, two groups of whole body, two groups of heart, and one control nonirradiated group. Animal in two groups were exposed to the heart (3 Gy, 60CO gamma rays, 30 cGy/min). For evaluation of effect of pelvis irradiation, animal irradiated at a 2 cm × 2 cm in the pelvis area (3 Gy, 60CO gamma rays, 30 cGy/min) and the other tissues were protected by lead shield. For detection of effects of scatter radiation on expression of COX-2 and ET-1 in pelvis irradiation groups, two groups (five rats) were exposed to the whole body (7.5 mGy, 60CO gamma rays, 30 cGy/min). The control group was included five sham-treated rats as non–irradiated control rats. Before irradiation, the rats received anesthesia using ketamine (50 mg/kg) and xylazine (10 mg/kg) via an intramuscular injection. Animals were anesthetized and sacrificed 24 and 72 h after the exposure. Heart tissues were extracted and frozen at −80°C. The expressions of COX-2 and ET-1 genes in lung tissue were evaluated 24 h and 72 h after heart and pelvis irradiation.
Irradiation and measurement of scattering radiation dose
All of the local heart and pelvis irradiations groups were exposed to 3 Gy cobalt 60 gamma rays (1.25 MeV) at a dose rate of 30 cGy/min. Local irradiation was done at a 2 cm × 2 cm area of the animal's pelvis. For detection of scattering dose received by lung in local pelvis irradiation, we used a rat phantom. The phantom was prepared from Plexiglas and cork as soft tissue and lung equivalent. The semiflex ionization chamber was used to measure the scattering dose received by equivalent lung tissue in rat phantom. The measured scattered radiation dose in the lung tissue equivalent, after irradiation of the pelvis in rat phantom with 3 Gy of gamma ray dose was 7/5 mGy. This radiation dose was received by the scatter groups.
Quantitative real-time polymerase chain reaction analysis
Total RNA was extracted from the heart tissues using the RNX-Plus extraction kit (CinnaGene, Iran). The total RNA concentration was assessed by spectrophotometer (WPA BIOWAVE II, UK(. Total RNA was reverse transcribed to cDNA using RevertAid ™ First Strand cDNA Synthesis Kit (Fermentase, Germany). mRNA expression was quantified using an ABI step-one real-time polymerase chain reaction (PCR) system (Applied Biosystem ™, ABI, USA) and the amplifications were performed with Ampliqon RealQ Plus 2x Master Mix Green (Ampliqon, Denmark). Expression of target genes was quantified relative to the reference gene. Gene expression results were normalized with GAPDH as housekeeping gene and targets are evaluated using a relative standard curve method. The quantitative real-time PCR was then performed at 94°C for 12 min, 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with the last three steps repeated for forty cycles. Data were analyzed by the relative standard curve method using StepOne™ Software version 2.3. Primer sequences for GAPDH, COX-2, and ET-1 genes (Applied Biosystems) are listed in [Table 1].
The mean ± standard deviation was calculated and statistical analysis was done using SPSS 21. Data were statistically evaluated using one-way ANOVA Tukey-Krammer test to determine the significance of the mean differences. P< 0.05 was considered statistically significant.
| > Results|| |
Cyclooxygenase-2 gene expression
Direct irradiation (3 Gy 60CO) leads to a significant increase in COX-2 gene expression at both 24 h (P < 0.001) and 72 h (P < 0.05) after irradiation in comparison to the normal control group. Low dose whole body scatter radiation (7.5 mGy) does not cause a significant change in COX-2 gene expression in heart tissue at both 24 h and 72 h compared to the control group. In addition, the differences between whole body and pelvis irradiation were not significant at both 24 h and 72 h after irradiation [Figure 1].
|Figure 1: Level of cyclooxygenase-2 gene expression in the heart tissues followed by pelvis, heart and whole body irradiation. Heart and whole body irradiation were compared to the control group and pelvis irradiation was compared to the whole body irradiation. Error bars indicate the standard deviation of the mean, n = 5 (one-way ANOVA Tukey-Krammer test *= P< 0.05)|
Click here to view
Endothelin-1 gene expression
Direct irradiation causes a significant increase in ET-1 gene expression at both 24 h (P < 0.001) and 72 h (P < 0.001) after irradiation in comparison to the normal control group. The scatter radiation dose could increase ET-1 gene expression 72 h after irradiation (P < 0.001) but not after 24 h. Comparisons between the whole body and pelvis irradiation at 24 h showed a significant increase in ET-1 gene expression (P < 0.001). In addition, difference between the whole body and pelvis irradiation 72 h after irradiation was significant (P < 0.05) [Figure 2].
|Figure 2: Level of endothelin-1 gene expression in the heart tissues followed by pelvis, heart and whole body irradiation. Heart and whole body irradiation were compared to the control groups and pelvis irradiation were compared to the whole body. Error bars indicate the standard deviation of the mean, n = 5 (one-way ANOVA Tukey-Krammer test*P< 0.05, **P < 0.001)|
Click here to view
| > Discussion|| |
Nontargeted effect induced by radiation is a very important phenomenon, especially in acute localized irradiation situation like RT. Although different studies have been done, the comprehensive mechanisms involved in this phenomenon remain unknown. However, some immune mediators have been proposed. Inflammatory cytokines in irradiated tissues could immigrate to distant regions; stimulate the expression of genes such as mitogen-activated protein kinases, nuclear factor (NF)-κB, and COX-2 and then lead to the production of free radicals and inflammatory responses.,
In this study, we hypothesized that targeted and nontargeted effects of radiation may cause changes in ET-1 and COX-2 gene expression in heart tissues. Up-regulation of these genes is important in some HDs such as inflammation, atherosclerosis, ischemia, and stroke. We showed that heart irradiation to a single 3 Gy gamma radiation cause up-regulation of both COX-2 and ET-1 at 24 h and 72 h after exposure. We found that pelvis irradiation can increase the expression of ET-1 in heart both 24 h and 72 h after exposure compared to whole body irradiation. By contrast, the increased level of COX-2 in both 24 h and 72 h was not statically significant compared to whole body irradiation. These result show local pelvis irradiation result in biological signaling pathways that can regulate ET-1 in heart for at least 3 days after exposure. Up-regulation of ET-1 in heart tissue after pelvis irradiation was very similar to direct heart irradiation, especially at 24 h after exposure. This may indicate that ET-1 overexpression in the heart tissue is after distant local radiation treatment may be very important similar to direct exposure of heart to radiation. We showed that direct irradiation with 3 Gy can cause up-regulation of COX-2 gene expression. However, pelvis irradiation and low dose whole body irradiation did not show any significant change in COX-2 gene expression. These may indicate up-regulation of ET-1 after exposure to radiation is independent to COX-2 and has more critical role in response to IR. Moreover, we revealed that low radiation dose (7.5 mGy) can up-regulate ET-1 gene expression 72 h after exposure but not at 24 h.
In this study, we used a single dose for assessment of nontargeted effect in the heart tissue. Previous studies have shown that fractionated irradiation leads to more obvious damages in distant nonirradiated tissues. In fractionated RT or brachytherapy that will continue for several weeks, these changes may be more prominent compared to single dose irradiation. Also, the comparison of micronucleus formation with different radiation qualities includes X-ray, and carbon, neon, and argon ions showed numbers of micronuclei in bystander cells was greater for higher LET. Micronucleus formation occurred in a dose-dependent manner for all radiation qualities.
Expression of ET-1 increases in response to several pathways include COX-2, NF-κB, hypoxia inducible factor-1, activator protein-1, GATA-2, Smad proteins, ROS, and epigenetic regulation such as acetylation of histone H3 and H4. Probably, other stimulating mediators such as NF-κB, ROS and epigenetic regulators that have been seen associated with nontargeted effect are involved in up-regulation of ET-1 in heart tissue. However, the exact signaling pathways remain unknown. COX-2 could be stimulated with cytokines such as IL-1 and TGF-β and promote the expression matrix metalloproteinase enzymes and enhance the cell death, tissue destruction and collagen production in cardiac tissues.
COX-2 and ET-1 induce hypertrophic growth and collagen deposition that leads to myocardial fibrosis and ischemia that increase risk of myocardial fibrosis and ischemia.,, Moreover, up-regulation of ET-1 influence the renin-angiotensin-aldosterone system and the sympathetic nervous system, impair NO production and is associated with dilated cardiomyopathy and pulmonary hypertension.,,,, Recently, a clinical study has revealed an association between increased ET-1 levels with subsequent heart failure, death rate, and heart failure. This study proposed ET-1 as a marker for prediction of heart failure.
| > Conclusion|| |
Finally, we concluded that overexpression of COX-2 and ET-1 after direct exposure to IR or induced by nontargeted effect induces detrimental effects on the heart function. Both COX-2 and ET-1 are involved in free radical production and chronic inflammation. Overproduction of these enzymes during RT may lead to long-term HDs. Long-term overexpression of ET-1 in the heart after distant tissue radiation treatment may cause inflammation, oxidative damage, and hypertrophy in heart tissue. However, further studies are needed to confirm this hypothesis and identify potential biological mechanisms. Radiation-induced heart changes are a long time process, and there is a need to study it after many years of exposure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Rutqvist LE, Lax I, Fornander T, Johansson H. Cardiovascular mortality in a randomized trial of adjuvant radiation therapy versus surgery alone in primary breast cancer. Int J Radiat Oncol Biol Phys 1992;22:887-96.
Jaworski C, Mariani JA, Wheeler G, Kaye DM. Cardiac complications of thoracic irradiation. J Am Coll Cardiol 2013;61:2319-28.
Wong SC, Fukuchi M, Melnyk P, Rodger I, Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation 1998;98:100-3.
Saito T, Giaid A. Cyclooxygenase-2 and nuclear factor-kappaB in myocardium of end stage human heart failure. Congest Heart Fail 1999;5:222-7.
Walton LJ, Franklin IJ, Bayston T, Brown LC, Greenhalgh RM, Taylor GW, et al
. Inhibition of prostaglandin E2 synthesis in abdominal aortic aneurysms: Implications for smooth muscle cell viability, inflammatory processes, and the expansion of abdominal aortic aneurysms. Circulation 1999;100:48-54.
Ruef J, Moser M, Kübler W, Bode C. Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells. Cardiovasc Pathol 2001;10:311-5.
Kähler J, Mendel S, Weckmüller J, Orzechowski HD, Mittmann C, Köster R, et al
. Oxidative stress increases synthesis of big endothelin-1 by activation of the endothelin-1 promoter. J Mol Cell Cardiol 2000;32:1429-37.
Kähler J, Ewert A, Weckmüller J, Stobbe S, Mittmann C, Köster R, et al
. Oxidative stress increases endothelin-1 synthesis in human coronary artery smooth muscle cells. J Cardiovasc Pharmacol 2001;38:49-57.
Li L, Chu Y, Fink GD, Engelhardt JF, Heistad DD, Chen AF. Endothelin-1 stimulates arterial VCAM-1 expression via NADPH oxidase-derived superoxide in mineralocorticoid hypertension. Hypertension 2003;42:997-1003.
Pu Q, Neves MF, Virdis A, Touyz RM, Schiffrin EL. Endothelin antagonism on aldosterone-induced oxidative stress and vascular remodeling. Hypertension 2003;42:49-55.
Koyama S, Kodama S, Suzuki K, Matsumoto T, Miyazaki T, Watanabe M. Radiation-induced long-lived radicals which cause mutation and transformation. Mutat Res 1998;421:45-54.
Yang H, Asaad N, Held KD. Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts. Oncogene 2005;24:2096-103.
Marozik P, Mothersill C, Seymour CB, Mosse I, Melnov S. Bystander effects induced by serum from survivors of the Chernobyl accident. Exp Hematol 2007;35 4 Suppl 1:55-63.
Najafi M, Fardid R, Hadadi G, Fardid M. The mechanisms of radiation-induced bystander effect. J Biomed Phys Eng 2014;4:163-72.
Chai Y, Calaf GM, Zhou H, Ghandhi SA, Elliston CD, Wen G, et al
. Radiation induced COX-2 expression and mutagenesis at non-targeted lung tissues of gpt delta transgenic mice. Br J Cancer 2013;108:91-8.
Najafi M, Fardid R, Takhshid MA, Mosleh-Shirazi MA, Rezaeyan AH, Salajegheh A. Radiation-Induced Oxidative Stress at Out-of-Field Lung Tissues after Pelvis Irradiation in Rats. Cell J 2016;18:340-5.
Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor. Circulation 2004;109 21 Suppl 1:II2-10.
Mothersill C, Seymour CB. Bystander and delayed effects after fractionated radiation exposure. Radiat Res 2002;158:626-33.
Autsavapromporn N, Suzuki M, Funayama T, Usami N, Plante I, Yokota Y, et al
. Gap junction communication and the propagation of bystander effects induced by microbeam irradiation in human fibroblast cultures: The impact of radiation quality. Radiat Res 2013;180:367-75.
Stow LR, Jacobs ME, Wingo CS, Cain BD. Endothelin-1 gene regulation. FASEB J 2011;25:16-28.
Bishop-Bailey D, Mitchell JA, Warner TD. COX-2 in cardiovascular disease. Arterioscler Thromb Vasc Biol 2006;26:956-8.
Ammarguellat FZ, Gannon PO, Amiri F, Schiffrin EL. Fibrosis, matrix metalloproteinases, and inflammation in the heart of DOCA-salt hypertensive rats: Role of ET(A) receptors. Hypertension 2002;39(2 Pt 2):679-84.
Mendez M, LaPointe MC. PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation. Am J Physiol Heart Circ Physiol 2005;288:H2111-7.
LaPointe MC, Mendez M, Leung A, Tao Z, Yang XP. Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse. Am J Physiol Heart Circ Physiol 2004;286:H1416-24.
Agapitov AV, Haynes WG. Role of endothelin in cardiovascular disease. J Renin Angiotensin Aldosterone Syst 2002;3:1-15.
Cody RJ, Haas GJ, Binkley PF, Capers Q, Kelley R. Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 1992;85:504-9.
Ramzy D, Rao V, Tumiati LC, Xu N, Sheshgiri R, Miriuka S, et al
. Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway. Circulation 2006;114 1 Suppl: I319-26.
Yang LL, Gros R, Kabir MG, Sadi A, Gotlieb AI, Husain M, et al
. Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice. Circulation 2004;109:255-61.
Yang LL, Arab S, Liu P, Stewart DJ, Husain M. The role of endothelin-1 in myocarditis and inflammatory cardiomyopathy: Old lessons and new insights. Can J Physiol Pharmacol 2005;83:47-62.
Perez AL, Grodin JL, Wu Y, Hernandez AF, Butler J, Metra M, et al
. Increased mortality with elevated plasma endothelin-1 in acute heart failure: An ASCEND-HF biomarker substudy. Eur J Heart Fail 2016;18:290-7.
[Figure 1], [Figure 2]