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Dexmedetomidine acts as an oxidative damage prophylactic in rats exposed to ionizing radiation
Journal of Clinical Anesthesia (2016) 34, 577–585
Dexmedetomidine acts as an oxidative damage prophylactic in rats exposed to ionizing radiation☆ Dilek Kutanis (Assistant Professor)a,⁎, Engin Erturk a , Ahmet Besir a , Yucel Demirci b , Selcuk Kayir c , Ali Akdogan a , Birgul Vanizor Kural d , Zumrut Bahat e , Emine Canyilmaz e , Hanife Kara d a
Karadeniz Technical University, Medical Faculty, Department of Anaesthesiology and Intensive Care, Trabzon, Turkey Giresun University, Medical Faculty, Department of Anaesthesiology and Intensive Care, Giresun, Turkey c Hitit University, Corum Education and Research Hospital, Corum, Turkey d Karadeniz Technical University, Medical Faculty, Department of Biochemistry, Trabzon, Turkey e Karadeniz Technical University, Medical Faculty, Department of Radiation Oncology, Trabzon, Turkey b
Received 4 March 2016; revised 16 May 2016; accepted 7 June 2016
Keywords: Ionizing radiation; Oxidative damage; Dexmedetomidine
Abstract Study objective: To investigate the effects of dexmedetomidine on oxidative injury caused by ionizing radiation. Design: Randomized controlled experimental study. Setting: Department of radiation oncology and research laboratory of an academic hospital. Interventions: Twenty-eight rats were randomized to 4 groups (n = 7 per group). Group S rats were administered physiologic serum; group SR rats were administered physiologic serum and 10 Gy external ionizing radiation. Groups D100 and D200 were administered 100 and 200 μg/kg dexmedetomidine intraperitoneally, respectively, 45 minutes before ionizing radiation. Measurements: Liver, kidney, lung, and thyroid tissue and serum levels of antioxidant enzymes (glutathione peroxidase [GPX], superoxide dismutase, and catalase) and oxidative metabolites (advanced oxidation protein products, malondialdehyde, and nitrate/nitrite, and serum ischemia-modified albumin) were measured 6 hours postprocedure. Main results: In group SR, IR decreased antioxidant enzyme levels and increased oxidative metabolite levels (P b .05). In plasma, antioxidant enzyme levels were higher and oxidative metabolite levels were lower in groups D100 and D200 than in group SR (P b .01). In tissues, hepatic and lung GPX levels were higher in groups D100 and D200 than in group SR (P b .001). Renal and thyroid GPX levels were higher in D200 than in group SR (P b .01). Thyroid superoxide dismutase levels were higher in groups D100 and D200 than in group SR (P b .01). Renal, lung, and thyroid catalase levels were higher in group D200 than in group SR (P b .01). Hepatic, renal, and lung advanced oxidation protein products and malondialdehyde levels were lower in groups D100 and D200 than in group SR (P b .01). Hepatic, renal, and lung nitrate/nitrite levels were lower in group D200 than in group SR (P b .05).
☆
We declare that there is no conflict of interest. ⁎ Corresponding author at: Karadeniz Technical University, Faculty of Medicine, Department of Anaesthesiology and Intensive Care, 61080 Trabzon, Turkey. Tel.: +90 462 3775898. E-mail address: [email protected] (D. Kutanis). http://dx.doi.org/10.1016/j.jclinane.2016.06.031 0952-8180/© 2016 Elsevier Inc. All rights reserved.
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D. Kutanis et al. Conclusions: Dexmedetomidine preserves the antioxidant enzyme levels and reduces toxic oxidant metabolites. Therefore, it can provide protection from oxidative injury caused by ionizing radiation. © 2016 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
People are often exposed to radiation during medical or paramedical procedures. Ionizing radiation is used for surgery, angiography, and several imaging protocols. It is also frequently used as a cancer treatment. However, these procedures can produce unwanted adverse effects. The basic mechanism of action for ionizing radiation is DNA damage that induces cell death [1]. Ionizing radiation creates reactive oxygen species (ROSs) through water ionization and subsequent reactions with chemicals in the immediate cellular environment. ROSs react with membrane lipids, proteins, and DNA, which is lethal to cells [2,3]. In addition, ROSs negatively impact antioxidant defense mechanisms in cells by reducing the activity of antioxidant enzymes, especially glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) [3,4]. Moreover, some destructive oxidative species, such as advanced oxidation protein products (AOPP), malondialdehyde (MDA), nitrite/nitrate (N/N), and ischemia-modified albumin (IMA), are produced during ionizing radiation reactions. Several recent studies have investigated methods to minimize oxidative damage [2] induced by ionizing radiation [2,5-8]. Reducing the amount of ionizing radiation the patient is exposed to is one method of minimizing damage; however, this technique is not a viable damage-reduction method for cancer radiotherapy due to negative effects on tumor response. Antioxidant drugs are another damage-reduction method because they inhibit ROS formation or inactivate ROSs after ionization has occurred. Anesthetics [9] and anesthesia methods are frequently used during procedures involving radiation, such as radiotherapy [10], invasive imaging applications, or surgery under anesthesia [11]. Several urologic and orthopedic surgical procedures and invasive interventional procedures are performed with radiation imaging. Sedoanalgesia, which combines sedation and analgesia, is commonly used for these types of procedures [11]. For example, sedoanalgesia is frequently used when implanting catheters for high-dose radiation brachytherapy [12]. Dexmedetomidine, an α2 adrenergic receptor agonist, has sedative, anxiolytic, and analgesic effects. Thus, it is ideal for sedoanalgesia procedures [10]. Dexmedetomidine has been shown to have antioxidant properties in experimental studies [13], and 1 study showed that dexmedetomidine enhanced the SOD activity of human blood [14]. In this randomized prospective study, we investigated the effects of dexmedetomidine on ionizing radiation damage. To this end, we measured the plasma and tissue levels of GPX, SOD, CAT, AOPP, MDA, N/N, and IMA in rats treated with dexmedetomidine before exposure to ionizing radiation.
2.1. Animals and groups All methods were approved by the Ethical Committee of Laboratory Animal Research of Karadeniz Technical University (protocol no. 4-2013). Ten- to 12-week-old SpragueDawley male rats weighing 250-300 g were used in this study. The rats were divided into 4 groups. Group S was administered physiologic serum and were not irradiated (n = 7). Group SR was administered physiologic serum and radiation (n = 7). Group D100 was administered 100 μg/kg dexmedetomidine and radiation (n = 7). Group D200 was administered 200 μg/kg dexmedetomidine and radiation (n = 7). Rats were housed in transparent polycarbonate cages, maintained on a 12-hour light-dark cycle, and kept at 20°C-22°C ± 2°C with 45%-65% humidity. Rats were fed ad libitum with standard food and fresh tap water.
2.2. Anesthesia and radiation Rats were initially anesthetized by injecting 10 mg/kg xylazine hydrochloride (Rompun 23, 32 mg/mL, Bayer) and 50 mg/kg ketamine (Ketalar 50 mg/mL, Pfizer) intraperitoneally (IP) before irradiation and blood and tissue extraction. For groups S and SR, physiologic serum was injected IP. For groups D100 and D200, 100 and 200 μg/kg dexmedetomidine (Precedex 100 μg/mL, Abbott Park, IL), respectively, was injected IP. At 45 minutes postinjection, all rats were transported in metal-free polycarbonate cages to the Radiotherapy Department at Karadeniz Technical University. Groups SR, D100, and D200 were removed from their cages and exposed to 10 Gy of external ionizing radiation using a cobalt-60 teletherapy machine (80-cm fixed source to surface distance, 2.5cm depth). Group S underwent a sham irradiation where the rats went through the same handling steps as the other groups but were not irradiated. After the procedure, rats were returned to their cages.
2.3. Plasma and tissue extraction At 6-hours postprocedure, all rats were sacrificed using cardiac puncture blood collection. Blood was collected and stored in tubes containing no anticoagulant. Subsequently, plasma was collected after centrifugation at 1800g for 10 minutes. Aliquots of plasma were prepared and stored in Eppendorf tubes at −80°C for downstream tests. Liver, right kidney, right lung, and thyroid tissue samples were extracted and stored in Eppendorf tubes at −80°C for downstream tests.
Protective effects of dexmedetomidine on oxidative stress
2.4. Biochemical assessments 2.4.1. GPX activity The GPX activity was determined using the Cayman Chemical Glutathione Peroxidase Assay Kit (catalog no. 703102; Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, this kit is a glutathione reductase––coupled assay that uses H2O2 as the substrate. The activity was measured by tracking the NADPH oxidation rate spectrophotometrically at 340 nm. 2.4.2. SOD activity The SOD activity was determined using the Cayman Chemical Superoxide Dismutase Assay kit (catalog no. 706002) according to the manufacturer's instructions. Briefly, the kit uses a tetrazolium salt to detect superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the amount of enzyme needed to produce 50% dismutation of the superoxide radicals. 2.4.3. CAT activity The CAT activity was measured using the Cayman Chemical Catalase Assay Kit (catalog no. 707002) according to the manufacturer's instructions. Briefly, CAT can use alcohols as electron donors to decompose H2O2, forming water and an aldehyde. CAT activity can be measured spectrophotometrically at 540 nm using a chromagen to determine the aldehyde concentration in solution. 2.4.4. Serum and tissue AOPP The AOPP levels were measured using the Rat AOPP ELISA (enzyme-linked immune sorbent assay) kit (catalog no. CSB-EQ027429RA; Cusabio Biotech, Wuhan, Hubei Province, China) according to the manufacturer's instructions. 2.4.5. N/N levels The N/N levels were measured using the Cayman Chemical Nitrate/Nitrite Colorimetric Assay Kit (catalog no. 780001) according to the manufacturer's instructions. Briefly, total nitrite reacts with Griess reagent (sulfanilamide and naphthalene–ethylenediamined hydrochloride), and the reaction is measured spectrophotometrically at 540 nm. 2.4.6. Plasma IMA levels The IMA levels in plasma were assessed using the Rat IMA ELISA kit (catalog no: CSB-E13620 r; Cusabio Biotech). The results are reported in units per milliliter. 2.4.7. Plasma MDA activity assay The MDA concentration in rat plasma samples was determined based on lipid peroxidation using the method described by Yagi [15]. Tetramethoxypropane was used as a standard, and MDA levels were presented as nanomoles per milliliter. 2.4.8. Tissue MDA activity assay Tissues were weighed and homogenized in ice-cold 1.15% w/v KCl. The homogenate was centrifuged at 2000g for 10
579 minutes. The MDA levels in tissue samples were determined using the method described by Mihara and Uchiyama [16]. Tetramethoxypropane was used as a standard, and tissue MDA levels were presented as nanomoles per gram wet tissue.
2.5. Statistical analysis Statistical analysis was performed using SPSS version 13.0 (International Business Machines Corp, Armonk, NY). Tests on individual samples were performed in triplicate, and the mean value of individual samples was used to calculate group values. Compatibility of the variables to normal distribution was evaluated using analytical methods (1-sample KolmogorovSmirnov tests). All group values are presented as the mean ± SD. Kruskal-Wallis test was used as a nonparametric analysis of variance (ANOVA) which was used to compare 4 different samples. One-way ANOVA was used as a parametric ANOVA which was used to compare 4 different samples. For intergroup comparisons with ANOVA, we clarified this discrepancy by using the Tukey honestly significant difference (HSD) and Tamhane T2 tests. Tukey HSD test was performed on tissue SOD levels; renal AOPP levels; lung MDA levels; and plasma GPX, SOD, CAT, and MDA levels. Post hoc Tamhane T2 tests were performed on tissue GPX, CAT, and AOPP levels; hepatic MDA levels; hepatic and lung N/N levels; and plasma AOPP levels. Statistical significance was defined as a P b .05 for both tests. For intergroup comparisons with Kruskal-Wallis, we clarified this discrepancy by using the Mann-Whitney U test with Bonferroni correction. This test was performed on plasma and renal N/N levels. In this case, a P b .008 was considered as statistically significant.
3. Results 3.1. Alterations in GPX levels The GPX levels in plasma and hepatic, renal, lung, and thyroid tissues were significantly different between the 4 groups (all P b .005; Table). Changes in plasma and tissue GPX levels among various groups are shown in Figure 1A. In plasma, GPX levels dropped when rats were exposed to radiation, and these levels recovered in the presence of dexmedetomidine. The GPX level was significantly lower in group SR than in groups S (P = .038), D100 (P = .003), and D200 (P = .004). In hepatic tissue, GPX levels were significantly higher in groups D100 and D200 than in group SR (P = .020 and P b .001, respectively). The GPX level was also higher in group D200 than in group S (P = .002). Similar to plasma, in renal tissue, the GPX level was lower in group SR than in group S (P = .023), and the level was restored in group D200 (P = .041 compared with group SR). In lung tissue, dexmedetomidine administration (groups D100 and D200) produced significantly higher GPX levels than those of group S (P = .002 and P = .009, respectively) and group SR (P =
580 Table SD)
D. Kutanis et al. Plasma and tissue levels of various antioxidant enzymes and destructive oxidative species 6 hours post–radiation treatment (mean ± Group S
Plasma
Hepatic
Renal
Lung
Thyroid
GPX SOD CAT AOPP MDA N/N IMA GPX SOD CAT AOPP MDA N/N GPX SOD CAT AOPP MDA N/N GPX SOD CAT AOPP MDA N/N GPX SOD CAT AOPP MDA N/N
502.83 0.784 1737.16 10.52 0.007 1.87 0.52 2274.48 0.243 5557.77 433.69 1.0312 14.95 1080.25 0.361 5946.76 664.48 0.7232 14.80 633.44 0.381 5897.49 118.79 0.4151 7.96 510.302 0.637 6152.72 56.16 0.8777 13.53
Group SR ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
177.97 0.086 597.20 3.78 0.0019 0.11 0.20 526.90 0.073 978.03 141.23 0.1412 1.70 284.00 0.045 1228.47 88.48 0.1566 4.11 172.74 0.179 908.75 38.50 0.1094 1.19 216.65 0.235 1440.14 27.12 0.1709 8.24
233.51 0.637 813.44 31.74 0.015 1.72 1.53 1887.75 0.220 4950.64 525.33 1.2093 18.73 614.39 0.289 1798.56 819.77 0.7905 22.41 470.27 0.392 4602.41 177.99 0.6224 11.18 466.25 0.538 5157.75 91.99 0.9749 10.37
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
47.36 0.109 242.77 9.25 0.0038 0.14 2.34 503.81 0.083 456.97 66.87 0.1516 4.3 156.50 0.029 806.69 132.01 0.0890 11.14 249.42 0.085 865.93 25.16 0.1654 2.74 171.03 0.230 1529.89 50.82 0.1795 3.00
GroupD100
GroupD200
P values
483.99 0.822 1967.72 18.01 0.007 1.28 0.51 2946.08 0.205 4964.59 399.43 0.6411 13.15 708.42 0.320 7957.09 664.69 0.6792 11.26 2170.45 0.420 6738.91 116.66 0.4515 6.92 701.63 0.915 7804.87 60.48 0.8050 8.16
629.48 0.811 1881.49 19.88 0.0081 1.20 0.48 3629.56 0.208 5336.00 328.82 0.7760 12.43 1039.59 0.346 8722.54 683.93 0.6702 12.39 2141.47 0.336 7597.96 101.57 0.4286 6.79 2156.94 0.876 8240.34 58.21 0.8467 10.76
P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
109.01 0.100 477.37 2.78 0.0027 0.16 0.08 574.19 0.059 557.59 59.55 0.0896 1.59 174.60 0.055 1550.47 103.36 0.1247 3.16 610.25 0.116 1832.53 20.69 0.1043 1.06 268.86 0.159 2052.22 27.37 0.1299 2.61
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
175.26 0.098 425.41 1.16 0.0033 0.11 0.09 493.09 0.053 985.39 89.91 0.1462 1.66 285.776 0.053 1481.43 47.91 0.2437 2.05 749.76 0.091 1687.15 33.30 0.1046 1.49 655.41 0.208 1375.04 45.25 0.1242 6.81
b .001 = .006 b .001 b .001 b .001 = .004 = .283 b .001 = .693 = .440 = .007 b .001 = .001 = .001 = .042 b .001 = .017 = .514 = .015 b .001 = .648 = .004 b .001 = .015 b .001 b .001 = .009 = .005 = .283 = .225 = .391
Group S was administered physiologic serum and a mock radiation procedure; group SR, physiologic serum and radiation; group D100, 100 μg/kg dexmedetomidine and radiation; group D200, 200 μg/kg dexmedetomidine and radiation.
.001 and P = .004, respectively). In thyroid tissue, group D200 GPX levels were significantly higher than those of groups S (P = .002), SR (P = .002), and D0100 (P = .004).
significant differences were observed between groups in hepatic and lung tissues.
3.3. Alterations in CAT levels 3.2. Alterations in SOD level The SOD levels were significantly different between the 4 groups in plasma and renal and thyroid tissues (all P b .05; Table). Tissue-level changes in SOD concentration between groups are shown in Figure 1B. In plasma, SOD levels decreased slightly, but not significantly, in group SR and increased when dexmedetomidine was administered, with levels in groups D100 and D200 being significantly higher than that of group SR (P = .038 and P = .052, respectively). In renal tissue, the SOD level in group SR decreased compared with that of group S (P = .039), and there were no significant changes in the dexmedetomidine groups. In thyroid tissue, SOD levels were higher in groups D100 and group D200 than in group SR (P = .018 and P = .038, respectively). No
The CAT levels were significantly different between the 4 groups in plasma and renal, lung, and thyroid tissues (all P b .015; Table). Changes in CAT levels between groups in each tissue type are shown in Figure 1C. In plasma, radiation reduced the concentration of CAT (P = .004 for the group SR level compared with that of group S). The CAT levels in the dexmedetomidine groups were similar to group S levels and significantly higher than group SR levels. Levels in groups D100 and D200 had a P b .001 and P = .001, respectively, when compared with that of group SR. Similar to plasma, the renal tissue showed significantly decreased CAT levels following radiation treatment (P b .001 for the group SR level compared with the group S level). The CAT level in group D100 was similar to that of group S and significantly higher
Protective effects of dexmedetomidine on oxidative stress
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Fig. 1 Antioxidant enzyme levels in irradiated rats pretreated with dexmedetomidine. Plasma and various tissue levels of (A) GPX, (B) SOD, and (C) CAT were assayed 6 hours postirradiation. Group S (dotted bar) was injected with physiologic serum and underwent a sham irradiation procedure. Group SR (black bar) was injected with physiologic serum and underwent irradiation. Group D100 (striped bar) was injected with 100 μg/kg dexmedetomidine and underwent irradiation. Group D200 (gray bar) was injected with 200 μg/kg dexmedetomidine and underwent irradiation. *P b .05 compared with group S; **P b .05 compared with group SR; #both P b .05 when compared with groups S and SR; †all P b .05 when compared with groups S, SR, and D100. Plasma GPX, plasma and tissue SOD, and CAT levels were analyzed using a Tukey HSD test. Tissue GPX and tissue CAT levels were analyzed using a Tamhane T2 test.
than that of group SR (P b .001 for the D100 level compared with that of group SR). In addition, the CAT level was significantly higher in group D200 than both group SR (P = .014) and group S (P = .016). In both lung and thyroid tissue, minimal changes were observed; however, group D200 had significantly higher levels of CAT than those of group SR (P = .014 and P = .011, respectively).
3.4. Alterations in AOPP levels The AOPP levels were significantly different between the 4 groups in plasma and hepatic and lung tissues (all P ≤ .005; Table). The changes between AOPP levels in the groups by tissue type are shown in Figure 2A. In plasma, AOPP levels increased significantly following radiation treatment (P b .001 for group SR levels compared with group S levels). Dexmedetomidine administration partially inhibited the formation of
AOPP. The AOPP levels were significantly lower in groups D100 and D200 than in group SR (P = .001 and P = .006, respectively); however, they were still significantly higher than that of group S (P = .009 and P = .002, respectively). In hepatic tissue, AOPP levels were significantly lower in groups D100 and D200 than in group SR (P = .018 and P = .004, respectively). In hepatic tissue, the AOPP level was significantly higher in group SR than in group S (P = .038), and the levels in groups D100 and D200 were significantly lower than that of group SR (P = .002 and P = .003, respectively). In renal tissue, the AOPP level was significantly higher in group SR than in group S (P = .031), and the level in group D100 was significantly lower than that of group SR (P = .032). However, unlike plasma, there were no significant differences observed between group S and groups D100 and D200. There were no significant differences observed between the groups in thyroid tissues.
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Fig. 2 Levels of oxidative species in irradiated rats pretreated with dexmedetomidine. Plasma and various tissue levels of (A) AOPP, (B) MDA, and (C) N/N were assayed 6 hours postirradiation. Group S (dotted bar) was injected with physiologic serum and underwent a mock irradiation procedure. Group SR (black bar) was injected with physiologic serum and underwent irradiation. Group D100 (striped bar) was injected with 100 μg/kg dexmedetomidine and underwent irradiation. Group D200 (gray bar) was injected with 200 μg/kg dexmedetomidine and underwent irradiation. *P b .05 compared with group S; **P b .05 compared with group SR; #both P b .05 when compared with groups S and SR. Plasma and lung tissue MDA levels, and renal tissue AOPP levels were analyzed using a Tukey HSD test. Plasma and tissue AOPP, hepatic tissue MDA, and hepatic and lung tissue N/N levels were analyzed using a Tamhane T2 test. Plasma and renal tissue N/N levels were analyzed using a MannWhitney U test with Bonferroni correction. Tukey and Tamhane test significance was defined as P b .05, and U test significance was defined as P b .008.
3.5. Alterations in MDA levels The MDA levels were significantly different between the groups in plasma and hepatic and lung tissues (all P ≤ .027; Table). MDA level changes between the 4 groups based on tissue type are shown in Figure 2B. In plasma, MDA levels increased following radiation treatment (P b .001 for group SR MDA level compared with that of group S). MDA formation was reduced in dexmedetomidine-treated rats; groups D100 and D200 MDA levels were significantly lower than that of group SR (P b .001 and P = .001, respectively). In hepatic tissue, dexmedetomidine administration generally lowered MDA levels, with D100 and D200 levels being significantly lower than those of group S (P b .001 and P = .036, respectively) and group SR(P b .001 and P = .001, respectively).
In lung tissue, the MDA level in group SR was higher than that of group S (P = .022), and the levels in group D200 were lower than that of group SR (P = .034). There were no significant differences observed between groups in renal and thyroid tissues.
3.6. Alterations in N/N levels The N/N levels were significantly different between groups in plasma and hepatic, renal, and lung tissues (all P ≤ .015; Table). Changes in N/N levels between the 4 groups by tissue type are shown in Figure 2C. In plasma, dexmedetomidine administration generally lowered the concentration of N/N. N/N levels were significantly lower in groups D100 and D200 than in both group S (P b .001 and P = 0,000, respectively) and
Protective effects of dexmedetomidine on oxidative stress group SR (P = .001 and P = .000, respectively). In hepatic tissue, N/N levels were lower than in group D200 than in group SR (P = .047). In renal tissue, N/N levels were significantly lower in groups D100 and group D200 than in group SR (P = .006 and P = .006, respectively). Similarly, in lung tissue, N/N levels were also lower in group D100 and D200 than in group SR (P = .032 and P = .027, respectively). There were no significant differences between groups in thyroid tissues.
3.7. Alterations in IMA levels The IMA levels showed no differences among any of the groups in plasma (Table).
4. Discussion Consistent with previous studies [6,7], we observed that ionizing radiation caused oxidative stress by increasing oxidative metabolites, such as AOPP, N/N, MDA, and IMA, and decreasing antioxidant defense mechanisms, including GPX, SOD, and CAT, in plasma and hepatic, renal, lung, and thyroid tissues. However, dexmedetomidine administration provided considerable protection from the oxidative stress caused by ionizing radiation. This is the first study investigating the protective effects of dexmedetomidine on oxidative stress induced by ionizing radiation. This protection is likely due to the antioxidant effects of dexmedetomidine, which may decrease oxidative species levels in cells. Therefore, using dexmedetomidine during procedures in which patients are exposed to ionizing radiation may ameliorate the destructive effects of radiation in addition to acting as a sedative and analgesic. The destructive effect of ionizing radiation is primarily caused by ROS generation in cells through the production of proinflammatory cytokines [17]. Radiation destroys proteins and lipids and leads to DNA and lipid membrane damage. Previous studies have indicated that ROS and oxidative stress may contribute to cytotoxicity as well as metabolic and morphological changes in animals and humans [18]. For example, Koc et al [7] administered 6.0 Gy of ionizing radiation to rats and found significantly increased MDA levels and decreased SOD and GPX activity in the rats' liver tissue. The authors hypothesized that exposing tissue to ionizing radiation induces toxic metabolite production and endogenous antioxidant enzyme degradation. These results are consistent with those of our study. In our study, we observed increased levels of MDA and AOPP in plasma and in renal and lung tissue in group SR compared with those of group S. Moreover, we observed decreased GPX, SOD, and CAT levels in plasma and renal tissue. Based on these data, we expected that decreasing ROS or increasing antioxidant status may provide protection from ionizing radiation damage. Antioxidant agents can decrease ROS production and increase levels of useful enzyme and antioxidant species that
583 contribute to the endogenous antioxidant defense system. Several intravenous anesthetic agents have been shown to act as ROS scavengers in various oxidative-stress conditions [2,19,20]. Dexmedetomidine, a selective and potent α2 receptor agonist, is currently used as a sedoanalgesia agent. It is frequently administered to patients in intensive care units and during various surgical, endoscopic, and radiologic procedures [11,21,22]. Similarly, it can be used as a sedoanalgesia agent during radiotherapy. Dexmedetomidine has been shown to have potent antioxidant properties against oxidative stress in vitro and in vivo in both humans and animals. Its protective effect against oxidative damage was demonstrated in several organs, such as lung [23], liver [24], kidney [25], skeletal muscle [26], and plasma [27]. Tufek et al [24] investigated the protective effects of dexmedetomidine against hepatic ischemia reperfusion injury in liver and remote organs by measuring oxidant and antioxidant capacity and oxidative-stress index. In that study, dexmedetomidine reduced oxidative stress in plasma, liver, and remote organs. Kocoglu et al [25] looked at the effects of dexmedetomidine on rat kidney and found that it had an oxidative damage preventive effect in their model. In another study, Geze et al [27] induced oxidative stress in ventilated rats by introducing a pneumoperitoneum in the peritoneal cavity. They administered 100 μg of dexmedetomidine before oxidative-stress induction in one of the groups and then measured IMA and performed histological analyses of lung tissue. Dexmedetomidine prophylaxis reduced IMA production and neutrophil infiltration in lung tissues. Cui et al [23] investigated dexmedetomidine's protective effects against lung alveolar epithelial cell apoptosis in vitro. They demonstrated that dexmedetomidine prevented ROS generation, cytochrome C release, and cell cycle–arrest induction by H2O2. Based on these studies, we hypothesized that dexmedetomidine may have an effect on antioxidant and oxidant status and generation of toxic metabolites following ionizing radiation treatment. In our study, ionizing radiation decreased GPX, SOD, and CAT in plasma and renal and thyroid tissue and partially decreased them in hepatic and lung tissue compared with the sham group. Dexmedetomidine administration before irradiation helped maintain the levels of these proteins and protected antioxidant status. Moreover, dexmedetomidine attenuated oxidative stress by maintaining lower levels of AOPP, MDA, and N/N in plasma and hepatic, renal, and lung tissues during irradiation, either by preventing their formation or by neutralizing the species as they formed. One of the challenges to preventing oxidative injury is determining the dose of the antioxidant agent, in this case dexmedetomidine. For example, Hoffman et al [28] evaluated dexmedetomidine protective effects in a transient cerebral ischemia rat model. Rats were administered 10 μg/kg or 100 μg/kg of dexmedetomidine before ischemia, and they found that dexmedetomidine decreased plasma catecolamine levels in a dose-dependent manner. In previous studies, the protective effect was investigated using different doses of dexmedetomidine. In practice, lower doses could fail to protect against
584 oxidative damage, whereas higher doses could produce hemodynamic adverse effects; therefore, more extensive studies of dose effects are needed. We used 2 different doses, 100 and 200 μg/kg of dexmedetomidine, to evaluate dosing effects in our study. Both doses of dexmedetomidine protected against irradiation injury by maintaining higher antioxidant enzyme levels and lower oxidative species levels. The 200-μg/kg dexmedetomidine dose had similar effects to the 100-μg/kg dexmedetomidine dose in all but a few parameters. The only difference observed was that the 200-μg/kg dose had significantly higher GPX levels in thyroid tissue. There were no statistically significant differences between the 2 groups with respect to oxidant species and antioxidant enzyme levels. Further studies with higher statistical power are needed to determine whether there were no differences or the differences were not observable in our study design. Another challenge to preventing oxidative injury is the drug administration time. In previous studies, dexmedetomidine was administered either before oxidative stress [29] or during the procedure [25]. Ideally, all tissues should be saturated with dexmedetomidine before irradiation to maximize its protective effect against oxidative stress. A previous study also noted that antioxidant administration before radiation administration could potentiate its beneficial effect [6]. Therefore, we administered dexmedetomidine to rats before irradiation in our study. Notably, although the deleterious effects of radiation initiate rapidly, within hours to months [8], its clinical manifestation can be observed many months or years afterwards. We focused our study on the early effects of ionizing radiation on biochemical markers and demonstrated that dexmedetomidine had a protective effect on radiation-induced oxidative injury based on the assumption that preventing these early effects may circumvent later clinical manifestations. From a clinical standpoint, many patients have comorbid conditions when the radiation for therapeutic or diagnostic purposes is applied. Thus, dexmedetomidine administration for sedation or analgesia during these procedures could prevent downstream complications produced by radiation. Our findings support the clinical use of dexmedetomidine to help prevent oxidative injury arising from ionizing radiation; however, there are limitations in this study. First, we only investigated biomarkers in plasma and tissues using molecular assays. Histopathological examination of tissue samples could present a clearer picture of the events occurring at the wholecell level. Second, we only studied the biomarkers at the 6hour postirradiation time point, and some of the deleterious effects of ionizing radiation could present at a later time. Future studies should include later time points to determine the extent and duration of the protective effects conferred by dexmedetomidine against radiation-induced oxidative injury. In conclusion, ionizing radiation used for therapeutic or diagnostic purpose can induce oxidative stress, which decreases antioxidant enzyme levels and generates harmful metabolites. The anesthetic dexmedetomidine may be a promising antioxidant agent that can be coupled with these procedures to
D. Kutanis et al. prevent oxidative injury. Further comprehensive investigations, including randomized controlled trials, are needed to confirm these observations in a clinical setting.
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Protective effects of dexmedetomidine on oxidative stress [21] Mahmoud M, Gunter J, Donnelly LF, Wang Y, Nick TG, Sadhasivam S. A comparison of dexmedetomidine with propofol for magnetic resonance imaging sleep studies in children. Anesth Analg 2009;109:745-53. [22] Mahmoud M, Mason KP. Dexmedetomidine: review, update, and future considerations of paediatric perioperative and periprocedural applications and limitations. Br J Anaesth 2015;115:171-82. [23] Cui J, Zhao H, Wang C, Sun JJ, Lu K, Ma D. Dexmedetomidine attenuates oxidative stress induced lung alveolar epithelial cell apoptosis in vitro. Oxid Med Cell Longev 2015;2015:358396. [24] Tufek A, Tokgoz O, Aliosmanoglu I, Alabalik U, Evliyaoglu O, Ciftci T, et al. The protective effects of dexmedetomidine on the liver and remote organs against hepatic ischemia reperfusion injury in rats. Int J Surg 2013;11:96-100. [25] Kocoglu H, Ozturk H, Ozturk H, Yilmaz F, Gulcu N. Effect of dexmedetomidine on ischemia-reperfusion injury in rat kidney: a histopathologic study. Ren Fail 2009;31:70-4.
585 [26] Dong X, Xing Q, Li Y, Han X, Sun L. Dexmedetomidine protects against ischemia-reperfusion injury in rat skeletal muscle. J Surg Res 2014;186: 240-5. [27] Geze S, Cekic B, Imamoglu M, Yoruk MF, Yulug E, Alver A, et al. Use of dexmedetomidine to prevent pulmonary injury after pneumoperitoneum in ventilated rats. Surg Laparosc Endosc Percutan Tech 2012;22: 447-53. [28] Hoffman WE, Kochs E, Werner C, Thomas C, Albrecht RF. Dexmedetomidine improves neurologic outcome from incomplete ischemia in the rat. Reversal by the alpha 2-adrenergic antagonist atipamezole. Anesthesiology 1991;75:328-32. [29] Wang ZX, Huang CY, Hua YP, Huang WQ, Deng LH, Liu KX. Dexmedetomidine reduces intestinal and hepatic injury after hepatectomy with inflow occlusion under general anaesthesia: a randomized controlled trial. Br J Anaesth 2014;112:1055-64.
Dexmedetomidine acts as an oxidative damage prophylactic in rats exposed to ionizing radiation☆ Dilek Kutanis (Assistant Professor)a,⁎, Engin Erturk a , Ahmet Besir a , Yucel Demirci b , Selcuk Kayir c , Ali Akdogan a , Birgul Vanizor Kural d , Zumrut Bahat e , Emine Canyilmaz e , Hanife Kara d a
Karadeniz Technical University, Medical Faculty, Department of Anaesthesiology and Intensive Care, Trabzon, Turkey Giresun University, Medical Faculty, Department of Anaesthesiology and Intensive Care, Giresun, Turkey c Hitit University, Corum Education and Research Hospital, Corum, Turkey d Karadeniz Technical University, Medical Faculty, Department of Biochemistry, Trabzon, Turkey e Karadeniz Technical University, Medical Faculty, Department of Radiation Oncology, Trabzon, Turkey b
Received 4 March 2016; revised 16 May 2016; accepted 7 June 2016
Keywords: Ionizing radiation; Oxidative damage; Dexmedetomidine
Abstract Study objective: To investigate the effects of dexmedetomidine on oxidative injury caused by ionizing radiation. Design: Randomized controlled experimental study. Setting: Department of radiation oncology and research laboratory of an academic hospital. Interventions: Twenty-eight rats were randomized to 4 groups (n = 7 per group). Group S rats were administered physiologic serum; group SR rats were administered physiologic serum and 10 Gy external ionizing radiation. Groups D100 and D200 were administered 100 and 200 μg/kg dexmedetomidine intraperitoneally, respectively, 45 minutes before ionizing radiation. Measurements: Liver, kidney, lung, and thyroid tissue and serum levels of antioxidant enzymes (glutathione peroxidase [GPX], superoxide dismutase, and catalase) and oxidative metabolites (advanced oxidation protein products, malondialdehyde, and nitrate/nitrite, and serum ischemia-modified albumin) were measured 6 hours postprocedure. Main results: In group SR, IR decreased antioxidant enzyme levels and increased oxidative metabolite levels (P b .05). In plasma, antioxidant enzyme levels were higher and oxidative metabolite levels were lower in groups D100 and D200 than in group SR (P b .01). In tissues, hepatic and lung GPX levels were higher in groups D100 and D200 than in group SR (P b .001). Renal and thyroid GPX levels were higher in D200 than in group SR (P b .01). Thyroid superoxide dismutase levels were higher in groups D100 and D200 than in group SR (P b .01). Renal, lung, and thyroid catalase levels were higher in group D200 than in group SR (P b .01). Hepatic, renal, and lung advanced oxidation protein products and malondialdehyde levels were lower in groups D100 and D200 than in group SR (P b .01). Hepatic, renal, and lung nitrate/nitrite levels were lower in group D200 than in group SR (P b .05).
☆
We declare that there is no conflict of interest. ⁎ Corresponding author at: Karadeniz Technical University, Faculty of Medicine, Department of Anaesthesiology and Intensive Care, 61080 Trabzon, Turkey. Tel.: +90 462 3775898. E-mail address: [email protected] (D. Kutanis). http://dx.doi.org/10.1016/j.jclinane.2016.06.031 0952-8180/© 2016 Elsevier Inc. All rights reserved.
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D. Kutanis et al. Conclusions: Dexmedetomidine preserves the antioxidant enzyme levels and reduces toxic oxidant metabolites. Therefore, it can provide protection from oxidative injury caused by ionizing radiation. © 2016 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
People are often exposed to radiation during medical or paramedical procedures. Ionizing radiation is used for surgery, angiography, and several imaging protocols. It is also frequently used as a cancer treatment. However, these procedures can produce unwanted adverse effects. The basic mechanism of action for ionizing radiation is DNA damage that induces cell death [1]. Ionizing radiation creates reactive oxygen species (ROSs) through water ionization and subsequent reactions with chemicals in the immediate cellular environment. ROSs react with membrane lipids, proteins, and DNA, which is lethal to cells [2,3]. In addition, ROSs negatively impact antioxidant defense mechanisms in cells by reducing the activity of antioxidant enzymes, especially glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) [3,4]. Moreover, some destructive oxidative species, such as advanced oxidation protein products (AOPP), malondialdehyde (MDA), nitrite/nitrate (N/N), and ischemia-modified albumin (IMA), are produced during ionizing radiation reactions. Several recent studies have investigated methods to minimize oxidative damage [2] induced by ionizing radiation [2,5-8]. Reducing the amount of ionizing radiation the patient is exposed to is one method of minimizing damage; however, this technique is not a viable damage-reduction method for cancer radiotherapy due to negative effects on tumor response. Antioxidant drugs are another damage-reduction method because they inhibit ROS formation or inactivate ROSs after ionization has occurred. Anesthetics [9] and anesthesia methods are frequently used during procedures involving radiation, such as radiotherapy [10], invasive imaging applications, or surgery under anesthesia [11]. Several urologic and orthopedic surgical procedures and invasive interventional procedures are performed with radiation imaging. Sedoanalgesia, which combines sedation and analgesia, is commonly used for these types of procedures [11]. For example, sedoanalgesia is frequently used when implanting catheters for high-dose radiation brachytherapy [12]. Dexmedetomidine, an α2 adrenergic receptor agonist, has sedative, anxiolytic, and analgesic effects. Thus, it is ideal for sedoanalgesia procedures [10]. Dexmedetomidine has been shown to have antioxidant properties in experimental studies [13], and 1 study showed that dexmedetomidine enhanced the SOD activity of human blood [14]. In this randomized prospective study, we investigated the effects of dexmedetomidine on ionizing radiation damage. To this end, we measured the plasma and tissue levels of GPX, SOD, CAT, AOPP, MDA, N/N, and IMA in rats treated with dexmedetomidine before exposure to ionizing radiation.
2.1. Animals and groups All methods were approved by the Ethical Committee of Laboratory Animal Research of Karadeniz Technical University (protocol no. 4-2013). Ten- to 12-week-old SpragueDawley male rats weighing 250-300 g were used in this study. The rats were divided into 4 groups. Group S was administered physiologic serum and were not irradiated (n = 7). Group SR was administered physiologic serum and radiation (n = 7). Group D100 was administered 100 μg/kg dexmedetomidine and radiation (n = 7). Group D200 was administered 200 μg/kg dexmedetomidine and radiation (n = 7). Rats were housed in transparent polycarbonate cages, maintained on a 12-hour light-dark cycle, and kept at 20°C-22°C ± 2°C with 45%-65% humidity. Rats were fed ad libitum with standard food and fresh tap water.
2.2. Anesthesia and radiation Rats were initially anesthetized by injecting 10 mg/kg xylazine hydrochloride (Rompun 23, 32 mg/mL, Bayer) and 50 mg/kg ketamine (Ketalar 50 mg/mL, Pfizer) intraperitoneally (IP) before irradiation and blood and tissue extraction. For groups S and SR, physiologic serum was injected IP. For groups D100 and D200, 100 and 200 μg/kg dexmedetomidine (Precedex 100 μg/mL, Abbott Park, IL), respectively, was injected IP. At 45 minutes postinjection, all rats were transported in metal-free polycarbonate cages to the Radiotherapy Department at Karadeniz Technical University. Groups SR, D100, and D200 were removed from their cages and exposed to 10 Gy of external ionizing radiation using a cobalt-60 teletherapy machine (80-cm fixed source to surface distance, 2.5cm depth). Group S underwent a sham irradiation where the rats went through the same handling steps as the other groups but were not irradiated. After the procedure, rats were returned to their cages.
2.3. Plasma and tissue extraction At 6-hours postprocedure, all rats were sacrificed using cardiac puncture blood collection. Blood was collected and stored in tubes containing no anticoagulant. Subsequently, plasma was collected after centrifugation at 1800g for 10 minutes. Aliquots of plasma were prepared and stored in Eppendorf tubes at −80°C for downstream tests. Liver, right kidney, right lung, and thyroid tissue samples were extracted and stored in Eppendorf tubes at −80°C for downstream tests.
Protective effects of dexmedetomidine on oxidative stress
2.4. Biochemical assessments 2.4.1. GPX activity The GPX activity was determined using the Cayman Chemical Glutathione Peroxidase Assay Kit (catalog no. 703102; Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, this kit is a glutathione reductase––coupled assay that uses H2O2 as the substrate. The activity was measured by tracking the NADPH oxidation rate spectrophotometrically at 340 nm. 2.4.2. SOD activity The SOD activity was determined using the Cayman Chemical Superoxide Dismutase Assay kit (catalog no. 706002) according to the manufacturer's instructions. Briefly, the kit uses a tetrazolium salt to detect superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the amount of enzyme needed to produce 50% dismutation of the superoxide radicals. 2.4.3. CAT activity The CAT activity was measured using the Cayman Chemical Catalase Assay Kit (catalog no. 707002) according to the manufacturer's instructions. Briefly, CAT can use alcohols as electron donors to decompose H2O2, forming water and an aldehyde. CAT activity can be measured spectrophotometrically at 540 nm using a chromagen to determine the aldehyde concentration in solution. 2.4.4. Serum and tissue AOPP The AOPP levels were measured using the Rat AOPP ELISA (enzyme-linked immune sorbent assay) kit (catalog no. CSB-EQ027429RA; Cusabio Biotech, Wuhan, Hubei Province, China) according to the manufacturer's instructions. 2.4.5. N/N levels The N/N levels were measured using the Cayman Chemical Nitrate/Nitrite Colorimetric Assay Kit (catalog no. 780001) according to the manufacturer's instructions. Briefly, total nitrite reacts with Griess reagent (sulfanilamide and naphthalene–ethylenediamined hydrochloride), and the reaction is measured spectrophotometrically at 540 nm. 2.4.6. Plasma IMA levels The IMA levels in plasma were assessed using the Rat IMA ELISA kit (catalog no: CSB-E13620 r; Cusabio Biotech). The results are reported in units per milliliter. 2.4.7. Plasma MDA activity assay The MDA concentration in rat plasma samples was determined based on lipid peroxidation using the method described by Yagi [15]. Tetramethoxypropane was used as a standard, and MDA levels were presented as nanomoles per milliliter. 2.4.8. Tissue MDA activity assay Tissues were weighed and homogenized in ice-cold 1.15% w/v KCl. The homogenate was centrifuged at 2000g for 10
579 minutes. The MDA levels in tissue samples were determined using the method described by Mihara and Uchiyama [16]. Tetramethoxypropane was used as a standard, and tissue MDA levels were presented as nanomoles per gram wet tissue.
2.5. Statistical analysis Statistical analysis was performed using SPSS version 13.0 (International Business Machines Corp, Armonk, NY). Tests on individual samples were performed in triplicate, and the mean value of individual samples was used to calculate group values. Compatibility of the variables to normal distribution was evaluated using analytical methods (1-sample KolmogorovSmirnov tests). All group values are presented as the mean ± SD. Kruskal-Wallis test was used as a nonparametric analysis of variance (ANOVA) which was used to compare 4 different samples. One-way ANOVA was used as a parametric ANOVA which was used to compare 4 different samples. For intergroup comparisons with ANOVA, we clarified this discrepancy by using the Tukey honestly significant difference (HSD) and Tamhane T2 tests. Tukey HSD test was performed on tissue SOD levels; renal AOPP levels; lung MDA levels; and plasma GPX, SOD, CAT, and MDA levels. Post hoc Tamhane T2 tests were performed on tissue GPX, CAT, and AOPP levels; hepatic MDA levels; hepatic and lung N/N levels; and plasma AOPP levels. Statistical significance was defined as a P b .05 for both tests. For intergroup comparisons with Kruskal-Wallis, we clarified this discrepancy by using the Mann-Whitney U test with Bonferroni correction. This test was performed on plasma and renal N/N levels. In this case, a P b .008 was considered as statistically significant.
3. Results 3.1. Alterations in GPX levels The GPX levels in plasma and hepatic, renal, lung, and thyroid tissues were significantly different between the 4 groups (all P b .005; Table). Changes in plasma and tissue GPX levels among various groups are shown in Figure 1A. In plasma, GPX levels dropped when rats were exposed to radiation, and these levels recovered in the presence of dexmedetomidine. The GPX level was significantly lower in group SR than in groups S (P = .038), D100 (P = .003), and D200 (P = .004). In hepatic tissue, GPX levels were significantly higher in groups D100 and D200 than in group SR (P = .020 and P b .001, respectively). The GPX level was also higher in group D200 than in group S (P = .002). Similar to plasma, in renal tissue, the GPX level was lower in group SR than in group S (P = .023), and the level was restored in group D200 (P = .041 compared with group SR). In lung tissue, dexmedetomidine administration (groups D100 and D200) produced significantly higher GPX levels than those of group S (P = .002 and P = .009, respectively) and group SR (P =
580 Table SD)
D. Kutanis et al. Plasma and tissue levels of various antioxidant enzymes and destructive oxidative species 6 hours post–radiation treatment (mean ± Group S
Plasma
Hepatic
Renal
Lung
Thyroid
GPX SOD CAT AOPP MDA N/N IMA GPX SOD CAT AOPP MDA N/N GPX SOD CAT AOPP MDA N/N GPX SOD CAT AOPP MDA N/N GPX SOD CAT AOPP MDA N/N
502.83 0.784 1737.16 10.52 0.007 1.87 0.52 2274.48 0.243 5557.77 433.69 1.0312 14.95 1080.25 0.361 5946.76 664.48 0.7232 14.80 633.44 0.381 5897.49 118.79 0.4151 7.96 510.302 0.637 6152.72 56.16 0.8777 13.53
Group SR ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
177.97 0.086 597.20 3.78 0.0019 0.11 0.20 526.90 0.073 978.03 141.23 0.1412 1.70 284.00 0.045 1228.47 88.48 0.1566 4.11 172.74 0.179 908.75 38.50 0.1094 1.19 216.65 0.235 1440.14 27.12 0.1709 8.24
233.51 0.637 813.44 31.74 0.015 1.72 1.53 1887.75 0.220 4950.64 525.33 1.2093 18.73 614.39 0.289 1798.56 819.77 0.7905 22.41 470.27 0.392 4602.41 177.99 0.6224 11.18 466.25 0.538 5157.75 91.99 0.9749 10.37
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
47.36 0.109 242.77 9.25 0.0038 0.14 2.34 503.81 0.083 456.97 66.87 0.1516 4.3 156.50 0.029 806.69 132.01 0.0890 11.14 249.42 0.085 865.93 25.16 0.1654 2.74 171.03 0.230 1529.89 50.82 0.1795 3.00
GroupD100
GroupD200
P values
483.99 0.822 1967.72 18.01 0.007 1.28 0.51 2946.08 0.205 4964.59 399.43 0.6411 13.15 708.42 0.320 7957.09 664.69 0.6792 11.26 2170.45 0.420 6738.91 116.66 0.4515 6.92 701.63 0.915 7804.87 60.48 0.8050 8.16
629.48 0.811 1881.49 19.88 0.0081 1.20 0.48 3629.56 0.208 5336.00 328.82 0.7760 12.43 1039.59 0.346 8722.54 683.93 0.6702 12.39 2141.47 0.336 7597.96 101.57 0.4286 6.79 2156.94 0.876 8240.34 58.21 0.8467 10.76
P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
109.01 0.100 477.37 2.78 0.0027 0.16 0.08 574.19 0.059 557.59 59.55 0.0896 1.59 174.60 0.055 1550.47 103.36 0.1247 3.16 610.25 0.116 1832.53 20.69 0.1043 1.06 268.86 0.159 2052.22 27.37 0.1299 2.61
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
175.26 0.098 425.41 1.16 0.0033 0.11 0.09 493.09 0.053 985.39 89.91 0.1462 1.66 285.776 0.053 1481.43 47.91 0.2437 2.05 749.76 0.091 1687.15 33.30 0.1046 1.49 655.41 0.208 1375.04 45.25 0.1242 6.81
b .001 = .006 b .001 b .001 b .001 = .004 = .283 b .001 = .693 = .440 = .007 b .001 = .001 = .001 = .042 b .001 = .017 = .514 = .015 b .001 = .648 = .004 b .001 = .015 b .001 b .001 = .009 = .005 = .283 = .225 = .391
Group S was administered physiologic serum and a mock radiation procedure; group SR, physiologic serum and radiation; group D100, 100 μg/kg dexmedetomidine and radiation; group D200, 200 μg/kg dexmedetomidine and radiation.
.001 and P = .004, respectively). In thyroid tissue, group D200 GPX levels were significantly higher than those of groups S (P = .002), SR (P = .002), and D0100 (P = .004).
significant differences were observed between groups in hepatic and lung tissues.
3.3. Alterations in CAT levels 3.2. Alterations in SOD level The SOD levels were significantly different between the 4 groups in plasma and renal and thyroid tissues (all P b .05; Table). Tissue-level changes in SOD concentration between groups are shown in Figure 1B. In plasma, SOD levels decreased slightly, but not significantly, in group SR and increased when dexmedetomidine was administered, with levels in groups D100 and D200 being significantly higher than that of group SR (P = .038 and P = .052, respectively). In renal tissue, the SOD level in group SR decreased compared with that of group S (P = .039), and there were no significant changes in the dexmedetomidine groups. In thyroid tissue, SOD levels were higher in groups D100 and group D200 than in group SR (P = .018 and P = .038, respectively). No
The CAT levels were significantly different between the 4 groups in plasma and renal, lung, and thyroid tissues (all P b .015; Table). Changes in CAT levels between groups in each tissue type are shown in Figure 1C. In plasma, radiation reduced the concentration of CAT (P = .004 for the group SR level compared with that of group S). The CAT levels in the dexmedetomidine groups were similar to group S levels and significantly higher than group SR levels. Levels in groups D100 and D200 had a P b .001 and P = .001, respectively, when compared with that of group SR. Similar to plasma, the renal tissue showed significantly decreased CAT levels following radiation treatment (P b .001 for the group SR level compared with the group S level). The CAT level in group D100 was similar to that of group S and significantly higher
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Fig. 1 Antioxidant enzyme levels in irradiated rats pretreated with dexmedetomidine. Plasma and various tissue levels of (A) GPX, (B) SOD, and (C) CAT were assayed 6 hours postirradiation. Group S (dotted bar) was injected with physiologic serum and underwent a sham irradiation procedure. Group SR (black bar) was injected with physiologic serum and underwent irradiation. Group D100 (striped bar) was injected with 100 μg/kg dexmedetomidine and underwent irradiation. Group D200 (gray bar) was injected with 200 μg/kg dexmedetomidine and underwent irradiation. *P b .05 compared with group S; **P b .05 compared with group SR; #both P b .05 when compared with groups S and SR; †all P b .05 when compared with groups S, SR, and D100. Plasma GPX, plasma and tissue SOD, and CAT levels were analyzed using a Tukey HSD test. Tissue GPX and tissue CAT levels were analyzed using a Tamhane T2 test.
than that of group SR (P b .001 for the D100 level compared with that of group SR). In addition, the CAT level was significantly higher in group D200 than both group SR (P = .014) and group S (P = .016). In both lung and thyroid tissue, minimal changes were observed; however, group D200 had significantly higher levels of CAT than those of group SR (P = .014 and P = .011, respectively).
3.4. Alterations in AOPP levels The AOPP levels were significantly different between the 4 groups in plasma and hepatic and lung tissues (all P ≤ .005; Table). The changes between AOPP levels in the groups by tissue type are shown in Figure 2A. In plasma, AOPP levels increased significantly following radiation treatment (P b .001 for group SR levels compared with group S levels). Dexmedetomidine administration partially inhibited the formation of
AOPP. The AOPP levels were significantly lower in groups D100 and D200 than in group SR (P = .001 and P = .006, respectively); however, they were still significantly higher than that of group S (P = .009 and P = .002, respectively). In hepatic tissue, AOPP levels were significantly lower in groups D100 and D200 than in group SR (P = .018 and P = .004, respectively). In hepatic tissue, the AOPP level was significantly higher in group SR than in group S (P = .038), and the levels in groups D100 and D200 were significantly lower than that of group SR (P = .002 and P = .003, respectively). In renal tissue, the AOPP level was significantly higher in group SR than in group S (P = .031), and the level in group D100 was significantly lower than that of group SR (P = .032). However, unlike plasma, there were no significant differences observed between group S and groups D100 and D200. There were no significant differences observed between the groups in thyroid tissues.
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Fig. 2 Levels of oxidative species in irradiated rats pretreated with dexmedetomidine. Plasma and various tissue levels of (A) AOPP, (B) MDA, and (C) N/N were assayed 6 hours postirradiation. Group S (dotted bar) was injected with physiologic serum and underwent a mock irradiation procedure. Group SR (black bar) was injected with physiologic serum and underwent irradiation. Group D100 (striped bar) was injected with 100 μg/kg dexmedetomidine and underwent irradiation. Group D200 (gray bar) was injected with 200 μg/kg dexmedetomidine and underwent irradiation. *P b .05 compared with group S; **P b .05 compared with group SR; #both P b .05 when compared with groups S and SR. Plasma and lung tissue MDA levels, and renal tissue AOPP levels were analyzed using a Tukey HSD test. Plasma and tissue AOPP, hepatic tissue MDA, and hepatic and lung tissue N/N levels were analyzed using a Tamhane T2 test. Plasma and renal tissue N/N levels were analyzed using a MannWhitney U test with Bonferroni correction. Tukey and Tamhane test significance was defined as P b .05, and U test significance was defined as P b .008.
3.5. Alterations in MDA levels The MDA levels were significantly different between the groups in plasma and hepatic and lung tissues (all P ≤ .027; Table). MDA level changes between the 4 groups based on tissue type are shown in Figure 2B. In plasma, MDA levels increased following radiation treatment (P b .001 for group SR MDA level compared with that of group S). MDA formation was reduced in dexmedetomidine-treated rats; groups D100 and D200 MDA levels were significantly lower than that of group SR (P b .001 and P = .001, respectively). In hepatic tissue, dexmedetomidine administration generally lowered MDA levels, with D100 and D200 levels being significantly lower than those of group S (P b .001 and P = .036, respectively) and group SR(P b .001 and P = .001, respectively).
In lung tissue, the MDA level in group SR was higher than that of group S (P = .022), and the levels in group D200 were lower than that of group SR (P = .034). There were no significant differences observed between groups in renal and thyroid tissues.
3.6. Alterations in N/N levels The N/N levels were significantly different between groups in plasma and hepatic, renal, and lung tissues (all P ≤ .015; Table). Changes in N/N levels between the 4 groups by tissue type are shown in Figure 2C. In plasma, dexmedetomidine administration generally lowered the concentration of N/N. N/N levels were significantly lower in groups D100 and D200 than in both group S (P b .001 and P = 0,000, respectively) and
Protective effects of dexmedetomidine on oxidative stress group SR (P = .001 and P = .000, respectively). In hepatic tissue, N/N levels were lower than in group D200 than in group SR (P = .047). In renal tissue, N/N levels were significantly lower in groups D100 and group D200 than in group SR (P = .006 and P = .006, respectively). Similarly, in lung tissue, N/N levels were also lower in group D100 and D200 than in group SR (P = .032 and P = .027, respectively). There were no significant differences between groups in thyroid tissues.
3.7. Alterations in IMA levels The IMA levels showed no differences among any of the groups in plasma (Table).
4. Discussion Consistent with previous studies [6,7], we observed that ionizing radiation caused oxidative stress by increasing oxidative metabolites, such as AOPP, N/N, MDA, and IMA, and decreasing antioxidant defense mechanisms, including GPX, SOD, and CAT, in plasma and hepatic, renal, lung, and thyroid tissues. However, dexmedetomidine administration provided considerable protection from the oxidative stress caused by ionizing radiation. This is the first study investigating the protective effects of dexmedetomidine on oxidative stress induced by ionizing radiation. This protection is likely due to the antioxidant effects of dexmedetomidine, which may decrease oxidative species levels in cells. Therefore, using dexmedetomidine during procedures in which patients are exposed to ionizing radiation may ameliorate the destructive effects of radiation in addition to acting as a sedative and analgesic. The destructive effect of ionizing radiation is primarily caused by ROS generation in cells through the production of proinflammatory cytokines [17]. Radiation destroys proteins and lipids and leads to DNA and lipid membrane damage. Previous studies have indicated that ROS and oxidative stress may contribute to cytotoxicity as well as metabolic and morphological changes in animals and humans [18]. For example, Koc et al [7] administered 6.0 Gy of ionizing radiation to rats and found significantly increased MDA levels and decreased SOD and GPX activity in the rats' liver tissue. The authors hypothesized that exposing tissue to ionizing radiation induces toxic metabolite production and endogenous antioxidant enzyme degradation. These results are consistent with those of our study. In our study, we observed increased levels of MDA and AOPP in plasma and in renal and lung tissue in group SR compared with those of group S. Moreover, we observed decreased GPX, SOD, and CAT levels in plasma and renal tissue. Based on these data, we expected that decreasing ROS or increasing antioxidant status may provide protection from ionizing radiation damage. Antioxidant agents can decrease ROS production and increase levels of useful enzyme and antioxidant species that
583 contribute to the endogenous antioxidant defense system. Several intravenous anesthetic agents have been shown to act as ROS scavengers in various oxidative-stress conditions [2,19,20]. Dexmedetomidine, a selective and potent α2 receptor agonist, is currently used as a sedoanalgesia agent. It is frequently administered to patients in intensive care units and during various surgical, endoscopic, and radiologic procedures [11,21,22]. Similarly, it can be used as a sedoanalgesia agent during radiotherapy. Dexmedetomidine has been shown to have potent antioxidant properties against oxidative stress in vitro and in vivo in both humans and animals. Its protective effect against oxidative damage was demonstrated in several organs, such as lung [23], liver [24], kidney [25], skeletal muscle [26], and plasma [27]. Tufek et al [24] investigated the protective effects of dexmedetomidine against hepatic ischemia reperfusion injury in liver and remote organs by measuring oxidant and antioxidant capacity and oxidative-stress index. In that study, dexmedetomidine reduced oxidative stress in plasma, liver, and remote organs. Kocoglu et al [25] looked at the effects of dexmedetomidine on rat kidney and found that it had an oxidative damage preventive effect in their model. In another study, Geze et al [27] induced oxidative stress in ventilated rats by introducing a pneumoperitoneum in the peritoneal cavity. They administered 100 μg of dexmedetomidine before oxidative-stress induction in one of the groups and then measured IMA and performed histological analyses of lung tissue. Dexmedetomidine prophylaxis reduced IMA production and neutrophil infiltration in lung tissues. Cui et al [23] investigated dexmedetomidine's protective effects against lung alveolar epithelial cell apoptosis in vitro. They demonstrated that dexmedetomidine prevented ROS generation, cytochrome C release, and cell cycle–arrest induction by H2O2. Based on these studies, we hypothesized that dexmedetomidine may have an effect on antioxidant and oxidant status and generation of toxic metabolites following ionizing radiation treatment. In our study, ionizing radiation decreased GPX, SOD, and CAT in plasma and renal and thyroid tissue and partially decreased them in hepatic and lung tissue compared with the sham group. Dexmedetomidine administration before irradiation helped maintain the levels of these proteins and protected antioxidant status. Moreover, dexmedetomidine attenuated oxidative stress by maintaining lower levels of AOPP, MDA, and N/N in plasma and hepatic, renal, and lung tissues during irradiation, either by preventing their formation or by neutralizing the species as they formed. One of the challenges to preventing oxidative injury is determining the dose of the antioxidant agent, in this case dexmedetomidine. For example, Hoffman et al [28] evaluated dexmedetomidine protective effects in a transient cerebral ischemia rat model. Rats were administered 10 μg/kg or 100 μg/kg of dexmedetomidine before ischemia, and they found that dexmedetomidine decreased plasma catecolamine levels in a dose-dependent manner. In previous studies, the protective effect was investigated using different doses of dexmedetomidine. In practice, lower doses could fail to protect against
584 oxidative damage, whereas higher doses could produce hemodynamic adverse effects; therefore, more extensive studies of dose effects are needed. We used 2 different doses, 100 and 200 μg/kg of dexmedetomidine, to evaluate dosing effects in our study. Both doses of dexmedetomidine protected against irradiation injury by maintaining higher antioxidant enzyme levels and lower oxidative species levels. The 200-μg/kg dexmedetomidine dose had similar effects to the 100-μg/kg dexmedetomidine dose in all but a few parameters. The only difference observed was that the 200-μg/kg dose had significantly higher GPX levels in thyroid tissue. There were no statistically significant differences between the 2 groups with respect to oxidant species and antioxidant enzyme levels. Further studies with higher statistical power are needed to determine whether there were no differences or the differences were not observable in our study design. Another challenge to preventing oxidative injury is the drug administration time. In previous studies, dexmedetomidine was administered either before oxidative stress [29] or during the procedure [25]. Ideally, all tissues should be saturated with dexmedetomidine before irradiation to maximize its protective effect against oxidative stress. A previous study also noted that antioxidant administration before radiation administration could potentiate its beneficial effect [6]. Therefore, we administered dexmedetomidine to rats before irradiation in our study. Notably, although the deleterious effects of radiation initiate rapidly, within hours to months [8], its clinical manifestation can be observed many months or years afterwards. We focused our study on the early effects of ionizing radiation on biochemical markers and demonstrated that dexmedetomidine had a protective effect on radiation-induced oxidative injury based on the assumption that preventing these early effects may circumvent later clinical manifestations. From a clinical standpoint, many patients have comorbid conditions when the radiation for therapeutic or diagnostic purposes is applied. Thus, dexmedetomidine administration for sedation or analgesia during these procedures could prevent downstream complications produced by radiation. Our findings support the clinical use of dexmedetomidine to help prevent oxidative injury arising from ionizing radiation; however, there are limitations in this study. First, we only investigated biomarkers in plasma and tissues using molecular assays. Histopathological examination of tissue samples could present a clearer picture of the events occurring at the wholecell level. Second, we only studied the biomarkers at the 6hour postirradiation time point, and some of the deleterious effects of ionizing radiation could present at a later time. Future studies should include later time points to determine the extent and duration of the protective effects conferred by dexmedetomidine against radiation-induced oxidative injury. In conclusion, ionizing radiation used for therapeutic or diagnostic purpose can induce oxidative stress, which decreases antioxidant enzyme levels and generates harmful metabolites. The anesthetic dexmedetomidine may be a promising antioxidant agent that can be coupled with these procedures to
D. Kutanis et al. prevent oxidative injury. Further comprehensive investigations, including randomized controlled trials, are needed to confirm these observations in a clinical setting.
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