Research Article Sevoflurane Induces DNA Damage Whereas Isoflurane Leads to Higher Antioxidative Status in Anesthetized Rats Thalita L. A. Rocha,1,2 Carlos A. Dias-Junior,1,2 Jose S. Possomato-Vieira,1 Victor H. Gonçalves-Rizzi,1 Flávia R. Nogueira,2 Kátina M. de Souza,2 Leandro G. Braz,2 and Mariana G. Braz2 1Department of Pharmacology, Biosciences Institute of Botucatu, Sao Paulo State University (UNESP), Distrito de Rubião Júnior, s/n, 18618-000 Botucatu, SP, Brazil 2Department of Anesthesiology, Botucatu Medical School, Sao Paulo State University (UNESP), Distrito de Rubião Júnior, s/n, 18618-970 Botucatu, SP, Brazil Correspondence should be addressed to Carlos A. Dias-Junior; carlosjunior@ibb.unesp.br Received 29 January 2015; Revised 6 May 2015; Accepted 10 May 2015 Academic Editor: Blanca Laffon Copyright © 2015 Thalita L. A. Rocha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Taking into account that there are controversial antioxidative effects of inhalational anesthetics isoflurane and sevoflurane and absence of comparison of genotoxicity of both anesthetics in animal model, the aim of this study was to compare DNA damage and antioxidant status in Wistar rats exposed to a single time to isoflurane or sevoflurane. The alkaline single-cell gel electrophoresis assay (comet assay) was performed in order to evaluate DNA damage in whole blood cells of control animals (unexposed; n = 6) and those exposed to 2% isoflurane (n = 6) or 4% sevoflurane (n = 6) for 120min. Plasma antioxidant status was determined by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.There was no statistically significant difference between isoflurane and sevoflurane groups regarding hemodynamic and temperature variables (P > 0.05). Sevoflurane significantly increased DNA damage compared to unexposed animals (P = 0.02). In addition, Wistar rats anesthetized with isoflurane showed higher antioxidative status (MTT) than control group (P = 0.019). There were no significant differences in DNA damage or antioxidant status between isoflurane and sevoflurane groups (P > 0.05). In conclusion, our findings suggest that, in contrast to sevoflurane exposure, isoflurane increases systemic antioxidative status, protecting cells from DNA damage in rats. 1. Introduction The possibility of health hazards resulting from exposure to volatile anesthetics has been extensively discussed during the last decade. There are some epidemiological data suggesting neurotoxic, hepatotoxic, and nephrotoxic side effects from inhalational anesthetics [1, 2]. Several studies have pointed out genetic damage in operating room personnel exposed to trace concentrations of anesthetic gases [3–5]. Isoflurane (C 3 H 2 ClF 5 O) and sevoflurane (C 4 H 3 F 7 O) are inhalational anesthetics widely used in current clinical prac- tice. Both halogenated anesthetics have advantages because of low blood-gas partition coefficients, being sevoflurane with lower solubility (0.65) than isoflurane (1.4), allowing rapid induction and awakening from anesthesia [6]. The genotoxicity and mutagenicity of isoflurane have been evaluated in vitro and in clinical studies showing conflicting results [7–9]. Similar findings are described for sevoflurane [10–12]. Literature is scarce regarding the possible genotoxic effects of isoflurane or sevoflurane in experimental studies. Moreover, no report yet has compared the genotoxi- city of isoflurane and sevoflurane in animal model. So far, the alkaline single-cell gel electrophoresis assay, also known as comet assay, has been extensively used to deter- mine the extent of DNA damage, including strand breaks, alkali-labile sites, DNA cross-linking, and incomplete exci- sion repair sites in mammalian cells [13]. Fragments of DNA migrate farther in response to an electric field, so that the nucleoids resemble a “comet” with a brightly fluorescent head and tail region [14]. This is a rapid, simple, sensitive, and Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 264971, 6 pages http://dx.doi.org/10.1155/2015/264971 http://dx.doi.org/10.1155/2015/264971 2 BioMed Research International reliable biochemical technique for evaluating DNA injury after exposure to toxicants. It is still controversial whether the volatile anesthetics lead to oxidative stress. Many reports on occupational exposure to anesthetics have shown they can impair antioxidant status [5, 15, 16]. On the other hand, some clinical studies have shown volatile anesthetics do not alter redox status [12, 17]. Because of the absence of reports on genotoxicity together with controversial antioxidative effects of halogenated anes- thetics in vivo, the aim of the current study was to compare systemic DNA damage and antioxidant status in rats exposed to either isoflurane or sevoflurane, without undergoing surgery procedure. 2. Materials and Methods 2.1. Animals. This study was approved by the Ethical Com- mittee for Animal Research (protocol number 684) from the Biosciences Institute of Botucatu from Sao Paulo State University (UNESP). All animals were treated in accordance with the recommendations of the Ethical Principles approved by the Brazilian Society of Science in Laboratory Animals. A total of 18 male Wistar rats, weighing 300–350 g, were provided by the Biosciences Institute of Botucatu, UNESP. The animals were maintained at the Department of Pharma- cology (UNESP) with restricted-access rooms at a controlled temperature (23 ± 2∘C) and on a 12 h light-dark cycle. The animals were given free access to a standard chow diet and drinking water ad libitum, and their age was 10 weeks on the day of exposure. 2.2. ExperimentalDesign. Theratswere assigned randomly to one of three groups, each of which consisted of 6 animals that were unexposed (control, C group) or exposed to different volatile anesthetics: isoflurane (Isoforine, Cristalia, Sao Paulo, Brazil; ISO group) or sevoflurane (Sevorane, Abbott, Buenos Aires, Argentina; SEVO group). Isoflurane and sevoflurane concentrations were recorded by means of an infrared analyzer (Vamos Plus; Dräger, Lübeck, Germany). Induction of anesthesia with 3% isoflu- rane or 5% sevoflurane at a continuous oxygen flow (2 L/min) was performed in a glass chamber connected to an anesthesia machine (AI; Insight, Ribeirao Preto, Brazil). Having con- firmed immobility and loss of righting reflex, the animals were placed in ventral recumbency onheat pad for preventing hypothermia (Heat Pad; Insight, Ribeirao Preto, Brazil). The anesthetic plan was maintained by face mask with 2% isoflurane or 4% sevoflurane. Subsequently, a polyethylene catheter (PE50) was inserted into the left carotid for evaluation of the mean, systolic, and diastolic arterial pressure. Data were recorded using a data acquisition system (MP150CE; Biopac Systems Inc., Goleta, CA) connected to a computer (Acknowledge 3.2, for Windows). Heart rate values were derived from the blood pressure recordings and processed online.The absence of somatic motor reflexes in response to tail pitching or blinking in response to a low-pressure corneal stimulation indicated deep anesthesia and analgesia. Body temperature was measured using a probe inserted in the rectum of each Anesthesia Carotid artery catheterization Hemodynamic parameters Hemodynamic stabilization Euthanasia and blood sample collection Time (min) (5󳰀) (10–15󳰀) T30 T60 T90 T120 Figure 1: Experimental design for rats exposed to 2% isoflurane or 4% sevoflurane for 120min. rat, which was connected to a monitor (DX 2023 monitor; Dixtal Biomedica, Sao Paulo, Brazil). The data acquisitions were initiated 30min after anesthesia onset [18]. The experimental design for anesthetized rats is presented in Figure 1. 2.3. Blood Collection. Blood was collected in EDTA tubes from each decapitated rat from all groups, and comet assay was carried out immediately. Part of the blood was cen- trifuged to obtain the plasma, which was aliquoted and stored until evaluation of antioxidant status (MTT assay). All the procedures were performed under dim light to prevent additional DNA damage. 2.4. Genotoxicity Assay. Before performing comet assay, cell viability was determined by trypan blue dye exclusion [19]. Theprotocol used for the alkaline single-cell gel electrophore- sis assay (comet assay) followed the guidelines previously proposed [13]. Briefly, 10 𝜇L of fresh peripheral blood cells was added to 100 𝜇L of 0.5% low-melting point agarose at 37∘C, layered onto a precoated slide with 1.5% regular agarose in duplicate, and covered with a coverslip. After brief agarose solidification in a refrigerator, the coverslip was removed and slides were immersed in lysis solution (2.5M NaCl, 100mM EDTA, and 10mM Tris, pH 10, with 1% Triton X-100 and 10% dimethyl sulfoxide) overnight. Slides were then washed in phosphate-buffered saline (PBS) for 5min and immersed in a freshly prepared alkaline buffer (pH 1mM EDTA and 300mM NaOH, pH > 13) for 20min and the electrophoresis was carried out using the same solution conducted for 20min at 25V and 300mA. Following this step, the slides were neutralized in 0.4M Tris-HCl (pH 7.5) for 15min, fixed in absolute ethanol for 5min, and stored at room temperature until analysis.The slides were stained with Sybr Gold and a total of 100 randomly captured nucleoids per animal (50 from each slide) were examined blindly by one expert observer at 400xmagnification using a fluorescent microscope connected to an image analysis system (Comet Assay IV, Perceptive Instruments, UK) that was calibrated previously according to the manufacturer’s instructions. The parameter tail moment was considered to measure DNA damage (arbitrary units). 2.5. Evaluation of Antioxidant Status. Direct reductions in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium BioMed Research International 3 Table 1: Hemodynamic variables in anesthetized rats with 2% isoflurane or 4% sevoflurane (ISO and SEVO, resp.). Data were recorded at 30 (𝑇 30 ), 60 (𝑇 60 ), 90 (𝑇 90 ), and 120min (𝑇 120 ) of anesthesia. Data are presented as mean (S.D.). HR: heart rate; SAP: systolic arterial pressure; MAP: mean arterial pressure; DAP: diastolic arterial pressure. 𝑃 > 0.05 among time points in the same group and between groups regarding a specific time point. Variables Groups Time points 𝑇 30 𝑇 60 𝑇 90 𝑇 120 Heart rate (beats/min) ISO 312 (30) 326 (30) 327 (44) 324 (46) SEVO 323 (45) 338 (32) 357 (26) 365 (29) SAP (mmHg) ISO 87 (13) 90 (9) 87 (14) 86 (16) SEVO 103 (19) 98 (15) 98 (12) 105 (18) MAP (mmHg) ISO 78 (9) 84 (6) 80 (9) 79 (12) SEVO 89 (21) 84 (17) 84 (12) 87 (17) DAP (mmHg) ISO 67 (10) 73 (7) 73 (5) 65 (11) SEVO 76 (20) 70 (15) 71 (11) 74 (15) bromide) were measured as previously described [20] with slight modifications. Briefly, 100 𝜇L of plasma was mixed with 12.5𝜇L of dye solution (5mg/mL in PBS); the final volume was adjusted to 200𝜇L with PBS, and the mixture was incubated for 60min at 37∘C. The reaction was termi- nated by the addition of 750𝜇L of 0.04M hydrochloric acid in isopropanol. The tubes were centrifuged for 10min at 1000×g, the supernatant was collected, and the absorbance was measured at 570 nm. 2.6. Statistical Analysis. Hemodynamic and temperature data were compared between isoflurane and sevoflurane groups and within each group using ANOVA followed by a Tukey test or the 𝑡-test. For body weight, comet assay, and MTT data, since they showed a normal distribution, ANOVA was applied to compare the three groups, followed by Tukey test, when necessary. A probability value 𝑃 < 0.05 was considered statistically significant. 3. Results Table 1 shows no statistically significant difference between isoflurane and sevoflurane groups regarding hemodynamic variables (𝑃 > 0.05). In addition, body weight did not statistically differ among groups, and rectal temperature data did not differ between isoflurane and sevoflurane groups (data not shown; 𝑃 > 0.05). None of the animals died during anesthesia. Cell viability was higher than 98% for all groups (99.7% for control group, 98.2% for isoflurane group, and 98.9% for sevoflurane group). The results of the comet assay in the peripheral blood of rats are shown in Figure 2. Sevoflurane significantly increased DNA damage compared to the control (𝑃 = 0.02). DNA damage was slightly higher in isoflurane group compared to the control, but with no significant difference (𝑃 > 0.05). No significant differences regarding DNAdamage were found between isoflurane and sevoflurane groups (𝑃 > 0.05). C ISO SEVO D N A d am ag e ( a.u .) 0.10 0.08 0.06 0.04 0.02 0.00 ∗ Figure 2: DNA damage (mean ± S.D.) detected by comet assay in whole blood cells of animals unexposed to anesthetics (C = control) or exposed to 2% isoflurane (ISO) or 4% sevoflurane (SEVO). ∗𝑃 = 0.02 versus control. C ISO SEVO 0.3 0.2 0.1 0.0 M TT (O .D .5 7 0 nm ) ∗ Figure 3: Plasma antioxidant status (mean ± S.D.) of animals unexposed to anesthetics (C = control) or exposed to isoflurane (ISO) or sevoflurane (SEVO). ∗𝑃 = 0.019 versus control. Rats anesthetized with isoflurane showed higher antiox- idative status (MTT) than control group (𝑃 = 0.019). There was no significant difference regarding antioxidant capacity between isoflurane and sevoflurane groups (𝑃 > 0.05; Figure 3). 4. Discussion The main findings of the current study are that sevoflurane induced DNA damage whereas isoflurane led to a higher antioxidative status in Wistar rats exposed for 120min. The maintenance of hemodynamic stability and rectal temperature were similar in both anesthetics and were rele- vant since alterations in these parameters may influence the results. The novelty of this study consists of comparing two differ- ent halogenated anesthetics widely used, in rats exposed to a single time for 120min.Thus, different from clinical practice, we can isolate the role of anesthetic agents from surgery to try to understand the systemic effects of these drugs. Information about genotoxicity of modern halogenated anesthetics is still 4 BioMed Research International insufficient. Thus, the current study indicates for the first time that rats exposed once to sevoflurane have increased systemic genetic damage within a few hours, when compared to unexposed animals. The concentrations of 2% and 4% of isoflurane and sevoflurane, respectively, have already been used inWistar rats, allowing an adequate anesthesia plan [21– 23]. Male mice repeatedly exposed to 2.4% sevoflurane (2 h daily, for 3 days) presented more DNA damage in leukocytes detected by comet assay and blood micronucleus compared to the control [24]. Repeated sevoflurane anesthesia (3% in oxygen for 3 h/day for 3 consecutive days) was investigated in male rabbits with or without antioxidant supplemen- tation [25]. The authors found that previously vitamin E (50 I.U./day) or selenium (15 𝜇g/day) supplementation pre- vented increase of DNA damage in mononuclear cells when compared to nonsupplemented animals exposed to sevoflu- rane. Some advantages of sevoflurane in clinical practice include the very low blood and tissue solubility and a pleasant odor. However, about 5% of inhaled sevoflurane is metabolized in the liver by cytochrome P450 2E1 isoenzyme, giving rise to reactive products, which could directly trigger the generation of peroxynitrite and increase peroxides and nitric oxide [26]. It is known that free radicals or reactive oxygen species (ROS) are major oxidants that react with DNA, damaging it by various lesions, such as oxidized bases, abasic sites, and/or strand breaks [27]. Some authors have also suggested that fluorinated anesthetics, including sevoflurane, could directly lead toDNAdamage, and themost probable modification would be an alkylation of purines [8]. Additionally, sevoflurane can induce cellular apoptosis [28]. The observed increase of DNA damage in sevoflurane group may be due to genotoxicity, and not to cell toxicity, since the exposure did not decrease cell viability.Thus, possiblemecha- nisms of sevoflurane genotoxicity include direct genotoxicity and/or oxidative route by metabolism. InducedDNAdamage occurred in blood cells earlier than tissues such as liver, kidney, and brain [24]. According to the authors, blood is the first compartment to absorb sevoflurane and the hematopoietic system may be highly sensitive to genotoxic agents, in part because hematopoietic cells undergo rapid division. However, a few negative results concerning sevoflurane genotoxicity have already been reported. This anesthetic was not able to induce genetic lesions in vitro, when lymphocytes were exposed to 1mM or 10mM at 4∘C or 37∘C for 10 and 30min [29]. No changes in oxidative DNA damage were observed in adults without comorbidities who underwent minimally invasive surgeries maintained with 1.9% sevoflu- rane anesthesia [12]. Different from isoflurane, sevoflurane did not enhance plasma antioxidative status in exposed rats. Similar results were described in literature. A study showed that sevoflurane had no effects on the antioxidant system (glutathione per- oxidase and superoxide dismutase enzymes) of anesthetized pigs [30]. Other study revealed that sevoflurane anesthesia did not alter the activities of antioxidant enzymes in the liver, brain, and lung of exposed rats [31]. Additionally, no changes in glutathione peroxidase and catalase activities in rat erythrocytes were detected after 4% sevoflurane exposure [22]. The International Agency for Research onCancer (IARC) stated that there is inadequate evidence for the carcino- genicity of isoflurane in animals. Volatile anesthetics are not classifiable as to their carcinogenicity to humans [32]. In the current study, we did not observe a significant difference between comet assay data in isoflurane and sevoflurane groups. Interestingly, any difference was also observed in systemicDNAdamage when adult patients were anesthetized with isoflurane or sevoflurane [33]. Supporting our findings, isoflurane was not found mutagenic when evaluated in the bacterial Ames test, using metabolic activation or not, or in Drosophila melanogaster [34, 35]. Contrarily, repeated exposure to isoflurane (1.7% in oxy- gen for 2 h daily for 3 consecutive days) induced genotoxicity in leukocytes and some organs of 8-week-old male Swiss albino mice [21]. Sprague-Dawley rats exposed to isoflurane (1% in air for 30min or 60min) have increased time- dependent DNA damage detected in lymphocytes [36]. In contrast, our study showed isoflurane did not increase DNA damage. Differences in isoflurane anesthesia have already been reported in Wistar and Sprague-Dawley rats [37]. Thus, besides animal strain, time (30 or 60min versus 120min) and concentration of exposure (1% versus 2%), animal age (6–8 weeks versus 10 weeks), and target cells analyzed (isolated lymphocytes versus whole blood) are some factors that could explain opposite findings concerning genotoxicity between the studies. Regarding comet assay, we evaluated DNA damage in whole blood cells. Among the advantages of using peripheral blood are the speed, the low cost, and the simplicity in performing the assay and the lower variability of the results [38–40]. It also must be highlighted that, different from sevoflu- rane, hepatic biotransformation of isoflurane is low (≤0.2%) [41]. Clinical studies performed by our research group indi- cated absence of systemic DNA breaks or oxidative DNA damage in patients under isoflurane anesthesia [9, 17]. Despite the increase of oxidative stress parameters such as lipid and protein oxidation in rats exposed to isoflurane for 60min, Kim et al. [36] detected any alteration during the first 30min. The authors could not show evidence of an association between DNA damage and oxidative stress parameters. Differently, in the current study, we have shown rats anesthetized with isoflurane presented higher plasma antioxidative status. Interestingly, patients undergoing mini- mally invasive surgery lasting 120min showed slight increase of plasma antioxidant capacity during isoflurane anesthesia [17]. Much is still unknown about the possible mechanisms by which isoflurane can have antioxidative properties. It has already been reported that anesthetics can modulate heme oxygenase- (HO-) 1, which exerts anti-inflammatory and antioxidative effects [42]. Isoflurane can induce HO-1 via nuclear factor kappa B (NF𝜅B) [43]. It is already known that anesthetic preconditioning and protection from tissue ischemic injury involve ROS, but the mechanisms are unknown [44]. Thus, isoflurane may provide a benefit against ischemia-reperfusion (IR) injury. BioMed Research International 5 A study provided evidence that induction of the cytopro- tective enzyme HO-1 by nontoxic and clinically approved isoflurane concentration protected rat livers from IR injury [45]. This anesthetic can attenuate oxidative stress and has neuroprotective effects in vitro, but it may work through indirect mechanisms to reduce oxidative stress-induced cell injury [46]. The pretreatment with isoflurane protected car- diomyocytes from damage by oxidative stress; sarcolemmal and mitochondrial Adenosine Triphosphate- (ATP-) sen- sitive potassium channels play essential and distinct roles in protection afforded by this anesthetic [47]. 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