Effects of Using Sevoflurane with Nitrous Oxide and Remifentanil on Dna Damage

Acta Medica Mediterranea, 2014, 30: 775

EFFECTS OF USING SEVOFLURANE WITH NITROUS OXIDE AND REMIFENTANIL ON DNA DAMAGE

FILIZ ALKAYA SOLMAZ1, ÖZLEM SELVI CAN1, ELA KADIOGLU2, SEMRA SARDAS3, OYA OZATAMER4 1Ankara University Medical Faculty, Department of Anesthesiology and Reanimation, Ankara - 2Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara - 3Department of Toxicology, Faculty of Pharmacy, Marmara University, Istanbul - 4Ankara University Medical Faculty, Department of Anesthesiology and Reanimation, Ankara, Turkey ABSTRACT

Aims: In this study, the effect of DNA damage in the course of sevoflurane anesthesia along with remifentanil and nitrous oxide, was investigated via Comet Assay technique. Materials and methods: The study included 52 patients. After approving informed consent form all participants, patients were randomly divided into three groups to provide different anesthesia maintenance. Anesthesia induction was carried out with propofol and vecuronium bromide in all groups. After intubation, Group SN (Sevoflurane-Nitrousoxide) received 50%02+50% N2O and sevoflurane 2-4%. Group SR (Sevoflurane-Remifentanil) received remifentanil by infusion at rate of 0.2mcg/kg/min, sevoflurane 2- 4%+100% O2. Group C (Control) sevoflurane 2-4% and 100% O2 for maintenance. Blood samples were obtained from all patients before induction, 120th minute of operation, and on postoperative day one and five. Results: Genotoxic damage was observed in all groups. When the groups were compared, no statistically significant difference was observed between the groups. However, while the DNA damage regressed to preoperative values on the 5th postoperative day in Group SR, it did not regress to preoperative values in Groups SN and C. Conclusion: It has been demonstrated that nitrous oxide combined with sevoflurane for anesthesia administration is not an ideal agent, and that an alternative opioid with short-acting agent remifentanil, can be used. It has also been theorized, due to remifentanil’s contribution in Group SR’s anesthesia administration, that remifentanil can lead to using lower doses of sevoflurane and positively impact the recovery of genotoxic damage.

Key words: Sevoflurane, Remifentanil, Nitrous Oxide, DNA damage, Genotoxicity, Comet assay. Received February 18, 2014; Accepted May 19, 2014

Introduction Controlled studies investigating teratogenic and carcinogenic effects of anesthetics on personnel In vivo and in vitro studies on chromosomal have followed and ASA (American Society of changes due to exposure to anesthetic gases have Anesthesiologists) data revealed that cancer risks proven that inhalation anesthetics can have muta- increase in female operating room personnel(6). genic and carcinogenic effects by effecting human Sevoflurane is a preferred anesthetic agent, espe- genetic material(1-4). The potential risks posed on cially in pediatric patients and in outpatient surg- operating room personnel who are exposed to eries, because it is comfortable to use in induction, inhalation agents at work place have raised interest eliminated rapidly, and has muscle-relaxing effects. in this topic. Russian anesthesiologist Vaisman was However, the toxic byproduct compound A the first to report through experimental and epi- that is formed through degradation of soda lime and demiologic studies in 1967, that it creates health baralime during anesthesia, limits its use. risks to work in environments polluted with anes- Compound A becomes significant especially in thetic gas waste, and that operating room personnel uses of low-flow anesthesia circuits and in long have certain health problems associated with anes- lasting interventions(7,8). thetic gases(5). 776 Filiz Alkaya Solmaz, Özlem Selvi Can et Al

Increased use of Sevoflurane in the recent receive premedication. Electrocardiogram, non- years, and the positive genotoxicity results of other invasive blood pressure (systolic, diastolic and halogenated anesthetics in similar structure have mean), heart rates, end- tidal sevoflurane (ETSVF), increased research on sevoflurane’s genotoxic end- tidal carbon dioxide (ETCO2) and peripheral effects. Though sevoflurane’s possible toxic effects transcutaneous oxygen saturation (SpO ) were on various organs have been determined, there is no 2 monitored. Propofol 1.5-2.5 mg/kg and vecuronium clear evidence on the mechanisms of its interaction bromide (Norcuron-Organon, 0.1 mg/kg) were with genetic material. Still, the genotoxic effect is administered intravenously for anaesthesia induc- assumed to be caused by Compound A that is in tion. After intubation ventilation was mechanically vinyl ether structure and is one of the compounds conducted with intermittent positive pressure venti- formed via sevoflurane’s interaction with carbon lation (IPPV) ensuring the ET values would be dioxide absorbents(9). CO2 Comet assay technique is more and more 34±4 mmHg. Anaesthesia maintenance in Group accepted in genotoxic screenings. Comet technique SN (Sevoflurane-Nitrous Oxide group) was provid- is used in many fields of toxicology from aging to ed with 50% O2 + 50% N2O and sevoflurane (2-4% genetic toxicology and molecular epidemiology. concentration). While there are numerous genotoxicity studies on Group SR (Sevoflurane-Remifentanil group) inhalation anesthetics use on non-cancerous cases, received remifentanil by infusion at rate of 0.2 there were not any studies found in the literature on mcg/kg/min, sevoflurane (2-4% concentration), and genotoxicity caused by opioid added to sevoflurane. 100% O2; N2O was not administered. The goal of this study is to determine the ratio Group C (Control group) sevoflurane (2-4% of DNA damage and its postoperative course of the concentration), in 100% O2 and N2O or opioid genotoxic effects of sevoflurane’s combined use agents were not administered. The neuromuscular with nitrous oxide and remifentanil on patients blockage was reversed with neostigmin 0.07 mg/kg undergoing tympanoplasty and myringoplasty due and atropine 0.5 mg intravenously if neuromuscular to benign pathologies such as chronic otitis media. blockage persisted after surgery.

Material and methods Blood sampling Venous blood was collected using a Patients heparinized syringe in patients before anesthesia The study was performed on 52 patients (ASA (sample BA). grades 1 and 2), aged 20-50 years, who were under- Additional venous blood samples were taken going operation for elective myringoplasty and from all patients at minute 120 (sample 1) after the tympanoplasty. All patients were assigned random- start of anesthesia. Postoperative samples were ly into one of the three groups. The study was taken on the following first (sample 2) and fifth approved by the local ethics committee and a postoperative day (sample 3). signed informed consent form was obtained from All samples were coded so that the scorer was participants after the purpose and the methods of unaware of the exposure status of the subject and the study were explained at the preoperative visit. the time of withdrawal. Blood samples were A survey (Form I), particularly investigating processed on the same day since there was a risk exposure to potential risk factors that may cause that prolonged storage of the sample (more than 4 DNA damage, was developed and administered. days) releases proteolytic enzymes and causes loss Smokers, and those with malignancy and another of cell viability(10,11). systemic or genetic conditions likely to increase DNA damage were excluded from the study. All Chemicals patients were otherwise healthy and had not All chemicals were purchased from Sigma received regular medication before operation. Chemical unless otherwise stated. Superfrost 1.0- 1.2 mm thick microscope slides from Merck were Anesthetics management used. Normal and low melting point agarose were Following an 8-hour fasting period, a 5 cc obtained from Gibco. Dulbecco’s phosphate- heparinized blood sample was collected from all buffered saline (PBS), without Mg and Ca, was patients included in the study group and did not from ICN Flow. Effects of using sevoflurane with nitrous oxide and remifentanil on Dna damage 777

Cell preparation comets in category HM) and expressed as total 30 μl blood from each donor was added to comet score (TCS), which summarizes the damage phosphate buffer saline (PBS) in an Eppendorf and frequencies as referred by Collins(13). was underlayed with Histopaque 1077 for lympho- The overall score for each slide was therefore cyte isolation. Cells were immediately centrifuged between 0 (undamaged) and 200 (maximally dam- at 200xg for 3 minutes at 4oC. Lymphocytes were aged) in arbitrary units. One slide reader performed retrieved from just above the boundary between analysis blindly, without knowledge of the groups PBS and Histopaque and were added to PBS and in order to minimize variability. centrifuged at 200xg for 3 minutes at 4oC. Cells were checked for viability by trypan blue exclusion Statistical analysis test. Each analysis was done in duplicate, and car- Statistical analysis was performed using a sta- ried out immediately after sample collection with- tistical package programme (SPSS 11.5, Chicago, out freezing or storing. IL, USA). Chi-square test and one-way ANOVA for demographic and anamnestic evaluations; and t-test Comet assay for within group and one-way ANOVA for between The basic alkaline technique of Singh et al. group comet essay comparisons between the groups. was followed with minor modifications(12). Briefly, All data displayed is in mean ± standard deviation the cells were mixed with 75 µl of 0.5% low-melt- (SD) form. p< 0.05 was considered as statistically ing agarose in phosphate-buffered saline and imme- significant. diately pipetted onto agarose-coated slides, spread out with a cover slip, and maintained at 4oC for 10 Results minutes to solidify. After removal of the cover slip, the slides were immersed in cold lysing solution There was no significant difference between (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% groups in terms of patients’ demographic character- sodium sarcosinate, pH 10.0) with 1% Triton X-100 istics (Table 1) and hemodynamic variables.in all and 10% DMSO added just before use for a mini- patients during this study. mum of 1 hour at 4oC. Group SN Group SR Group C (n=18) (n=18) (n=16) The slides were removed from the lysing solu- Gender (female/male) 12/6 12/6 15/1 tion, drained, and placed in a horizontal gel elec- Age (years) 33.7± 12.7 37.1± 10.3 33.7± 9.1 trophoresis tank (Pharmacia GNA 100) with fresh Weight (kg) 67.1± 12.5 68.4± 11.9 69± 15.9 Duration of anaesthesia 163.5± 34.7 178± 39.7 161.8± 39.5 electrophoresis buffer (1 mM Na2EDTA and 300 (min) Duration of operation mM NaOH, pH 13) for 40 minutes to allow the 151.5±34.7 171.6± 39.2 155.3± 40.6 unwinding of the DNA and the expression of alkali- (min) Data are displayed as mean ± S.D.; P< 0.05 (between groups, labile damage. Electrophoresis was conducted at by one-way ANOVA and Chi-square test). 1.6 Vcm-1 for 20 minutes (300 mA). After elec- Table 1: Demographic characteristics of patients and trophoresis, the slides were washed three times with control group. neutralizing buffer (0.4 M Tris, pH 7.5) and stained All patients were administered 1.5-2.5 mg/kg for 10 minutes with 20µg/ml ethidium bromide. propofol at induction. The total propofol administered to groups SN, SR and C were 177.50 ± 33.22 Microscopic analysis and slide scoring mg, 184.17 ± 30.54 mg, and 183.44 ± 40.48 respec- A total of 100 cells were analyzed using two tively. The patients were administered 0.1 mg/kg slides per subject for comet assay at x400 magnifi- vecuronium bromide and the total amount adminis- cation under a fluorescent microscope (Zeiss, tered were 10.72 ± 1.97mg, 10.11 ± 1.23 mg, and Oberkochen, West Germany, and RG) equipped 10.06 ± 1.57 mg for groups SN, SR, and C respec- with an excitation filter of 546 nm and a barrier fil- tively. The dosage was not different between ter of 590 nm. One hundred cells were classified by groups. eye into three categories on the basis of the extent The occupational distribution was not different of migration as; no migration (NM), low migration between the groups. There was no difference in (LM) and high migration (HM). terms of amount of time worked at the related busi- The number of comets in each sample was cal- ness. Between the groups, self-reports on exposure to culated (0x number of comets in category NM +1x chemicals at home or at work place did not differ. number of comets in category LM+2x number of 778 Filiz Alkaya Solmaz, Özlem Selvi Can et Al

SN GROUP *p< 0.05 Patient No Sample BA (before anaesth) Sample 1 (120 min) Sample 2 (1st day) Sample 3 (5th day) aNM: no migration; LM: low NM LM HM TM NM LM HM TM NM LM HM TM NM LM HM TM migration; HM: high migra- 1 95 4 1 6 92 6 2 10 96 4 0 4 83 9 8 25 tion l; TM: total migration 2 93 7 0 7 90 9 1 11 95 4 1 6 95 5 0 5 sample BA: before anaesthe- 3 91 8 1 10 90 7 2 11 90 9 1 11 91 9 0 9 sia; sample 1: 120th min 4 91 9 0 9 88 10 2 14 89 8 3 14 93 7 0 7 during anaesthesia; sample 2: 5 90 10 0 10 85 14 1 16 82 15 3 21 89 9 2 13 6 86 12 2 16 83 13 4 21 84 14 2 18 85 13 2 17 first day after anaesthesia; 7 89 9 2 13 90 9 1 11 86 12 2 16 87 12 1 14 sample 3: fifth day after anae- 8 85 12 3 18 81 15 4 23 74 19 6 31 80 14 6 26 sthesia. 9 83 15 2 19 79 18 3 24 82 16 2 20 84 13 3 19 bThe mean comet response 10 86 10 4 18 83 14 3 20 82 15 3 21 84 13 3 19 11 96 4 0 4 93 6 1 8 92 6 2 10 92 6 2 10 scored as TM = NM x 0 + 12 97 3 0 3 94 5 1 7 95 5 0 5 96 4 0 4 LMx1 + HM x 2; sample BA: 13 93 6 1 8 96 3 1 5 94 6 0 6 86 13 1 15 before anaesthesia; sample 1: 14 88 12 0 12 87 12 1 14 88 10 2 14 88 11 1 13 120th min during anaesthesia; 15 92 8 0 8 89 10 1 12 88 10 2 14 90 9 1 11 sample 2: first day after anae- 16 90 9 1 11 88 10 2 14 85 12 3 18 88 10 2 14 17 94 6 0 6 91 8 1 10 91 7 2 11 93 6 1 8 sthesia; sample 3: fifth day 18 90 9 1 11 88 10 2 14 85 12 3 18 88 10 2 14 after anaesthesia. mean 90.5 8.5 1 10.6 88.1* 9.9* 1.8* 13.6* 87.6* 10.2* 2.06* 14.3* 88.4* 9.6 1.9* 13.5* ±SD 3.88 3.16 1.18 4.89 4.55 3.87 1.04 5.4 5.7 4.45 1.43 6.9 4.36 3.07 2.1 6.1 Table 2: Individual grade of DNA damage evaluated by Comet (100 cells) in Group SN and the mean ± S.D. comet response in Group SNb.

SR GROUP *p<0.05 Patient No Sample BA (before anaesth) Sample 1 (120 min) Sample 2 (1st day) Sample 3 (5th day) aNM: no migration; LM: low NM LM HM TM NM LM HM TM NM LM HM TM NM LM HM TM migration; HM: high migra- 1 92 6 2 10 79 9 12 33 80 10 10 30 82 12 6 24 tion l; TM: total migration 2 91 7 2 11 86 12 2 16 91 8 1 10 92 6 2 10 sample BA: before anaesthe- 3 91 7 2 11 86 10 4 18 87 10 3 16 90 9 1 11 th 4 90 10 0 10 89 9 2 13 89 10 1 12 91 7 2 11 sia; sample 1: 120 min 5 91 8 1 10 88 10 2 14 85 12 3 18 91 7 2 11 during anaesthesia; sample 6 89 9 2 13 86 10 4 18 90 9 1 11 91 8 1 10 2: first day after anaesthesia; 7 93 7 0 7 89 9 2 13 86 13 1 15 89 10 1 12 sample 3: fifth day after 8 89 10 1 12 86 13 1 15 84 14 2 18 88 10 2 14 anaesthesia. 9 81 13 6 25 83 14 3 20 86 10 4 18 84 12 4 20 b 10 93 7 0 7 89 10 1 12 85 13 2 17 93 6 1 8 The mean comet response 11 92 8 0 8 89 10 1 12 86 12 2 16 90 10 0 10 scored as TM = NM x 0 + LM 12 89 9 2 13 88 10 2 14 86 10 4 18 89 10 1 12 x 1 + HM x 2; sample BA: 13 92 8 0 8 89 10 1 12 86 12 2 16 90 10 0 10 before Anaesthesia; sample 1: 14 91 8 1 10 84 13 3 19 84 13 3 19 86 12 2 16 120th min during anaesthesia; 15 88 12 0 12 86 12 2 16 84 13 3 19 92 8 0 8 16 91 6 3 12 89 9 2 13 87 10 3 16 90 7 3 13 sample 2: first day after anae- 17 90 9 1 11 89 9 2 13 88 8 4 16 89 8 3 14 sthesia; sample 3: fifth day 18 90 9 1 11 88 10 2 14 85 13 2 17 86 13 1 15 after anaesthesia. mean 90.1 8.5 1.3 11.1 86.83* 10.5* 2.67* 15.8* 85.94* 11.1* 2.7* 16.7* 88.8 9.2 1.7 12.7 ±SD 2.6 1.8 1.4 3.91 2.7 1.58 2.49 4.96 2.64 1.87 2.04 4.19 3.1 2.2 1.5 4.08 Table 3: Individual grade of DNA damage evaluated by Comet (100 cells) in Group SRa and the mean ± S.D. comet response in Group SRb.

None of the participants consumed cigarettes Also no significant difference was observed in or alcohol. Between the groups, the number of terms of days for last radiologic assessment among smokers at home was not different. groups. History of cancer and hereditary disease in The groups did not differ in terms of medicine patients and their primary and secondary relatives; no use and types of medication used within the past difference was observed between the groups. Other two months. None of the cases reported regular vit- than the reasons for the surgery, the groups did not dif- amin use history for longer than 6 months. The fer significantly with respect to the diseases they had dietary choices and cooking method preferences did within the past two years and the time since recovery. not show a difference between the groups. The number of radiologic assessments was 2.11 ± Postoperative analgesic and antibiotic use and 1.64, 1.50 ± 0.61 and 1.81 ± 1.32 for groups SN, SR, duration of use were not different between the and C respectively and there was no statistically sig- groups. Throughout the surgery, ETC02 values were nificant difference between the groups (p> 0.05). Effects of using sevoflurane with nitrous oxide and remifentanil on Dna damage 779

C GROUP *p<0.05 Sample 3 aNM: no migration; LM: low Patient No Sample BA (before anaesth) Sample 1 (120 min) Sample 2 (1st day) (5th day) migration; HM: high migra- NM LM HM TM NM LM HM TM NM LM HM TM NM LM tion l; TM: total migration 1 97 3 0 3 82 16 2 20 81 9 10 29 91 6 sample BA: before anaesthe- 2 91 9 0 9 86 13 1 15 88 10 2 14 91 8 3 87 12 1 14 88 10 2 14 87 10 3 16 89 10 sia; sample 1: 120 min during 4 88 10 2 14 85 12 3 18 91 7 2 11 92 6 anaesthesia; sample 2: first 5 86 13 1 15 83 15 2 19 87 12 1 14 88 10 day after anaesthesia; sample 6 88 9 3 15 84 13 3 19 85 12 3 18 86 12 3: fifth day after anaesthesia. 7 89 8 3 14 87 9 4 17 84 10 6 22 88 8 bThe mean comet response sco- 8 92 6 2 10 87 9 4 17 84 12 4 20 85 12 9 95 4 1 6 89 9 2 13 87 10 3 16 92 6 red as TM = NM x 0 + LM x 1 10 89 9 2 13 88 10 2 14 86 12 2 16 89 10 + HM x 2; sample BA: before 11 90 9 1 11 84 14 2 18 84 13 3 19 88 10 anaesthesia; sample 1: 120th 12 90 9 1 11 89 10 1 12 86 13 1 15 89 10 min during anaesthesia; sam- 13 91 7 2 11 89 8 3 14 85 12 3 18 86 13 ple 2: first day after anaesthe- 14 85 14 1 16 82 16 2 20 83 13 4 21 85 12 15 90 10 0 10 86 13 1 15 86 13 1 15 89 10 sia; sample 3: fifth day after 16 91 6 0 6 88 10 2 14 86 13 1 15 89 10 anaesthesia. mean 89.9 8.6 1.2 11.1 86.06* 11.6* 2.2* 16.1* 85.7* 11.3* 3.0* 17.4* 88.5 9.5 ±SD 3.06 2.98 1 3.72 2.46 2.65 0.93 2.61 2.23 1.77 2.29 4.21 2.25 2.22 Table 4: Individual grade of DNA damage evaluated by Comet (100 cells) in Group Ca and the mean ± S.D. comet response in Group Cb.

Sample BA Sample 1 Sample 2 Sample 3 aThe mean comet response scored as TM = NM x 0 + LM x 1 + (before anaesthia) (120 min) (1st day) (5th day) HM x 2; sample BA: before anaesthesia; sample 1: 120 min Group C 11.1±3.7 16.1±2,6* 17,4±4,2*+ 12.6±3.1* during anaesthesia; sample 2: first day after anaesthesia; sample 3: fifth day after anaesthesia Group SR 11.1±3.9 15.8±4.9* 16.7±4.1*+ 12.7±4.0 *P< 0.05 in comparison to preoperative values th Group SN 10.6±4.8 13.6±5.4* 14.3±6.9*+ 13.5±6.1* +P< 0.05 in comparison to preoperative, 120 minute, and postoperative day 5 values. Table 5: The mean ± S.D. comet response in control and patients groupsa. not different between the groups; however, the Discussion ETSVF values were lower in Group SR compared to the other two groups. While the mean blood pres- Many in vivo and in vitro experimental and sure measures were lower in Groups SN and SR clinical cytogenetic studies have been conducted, compared to Group C, heart pace was lower in demonstrating that anesthetic gas exposure causes Group SR compared to Groups SN and C. Oxygen chromosomal changes(1-3). Current data from a saturation did not reveal a significant difference in majority of these studies suggest that anesthetic within and between group comparisons. None of gases have genotoxicity potential. the patients remained hypoxic in the perioperative Alkaline Comet Assay is applicable for study- period. The 100 lymphocyte cells counted in each ing DNA damage and reparation, biomonitoriza- blood sample was examined in three categories: tion, and genotoxicity(14-16). This technique can be NM (no migration, no damage), LM (low migra- used on various somatic cells. tion, low damage, stretch) and HM (high migration, In our study, we preferred to examine lympho- high damage, and comet). Total migration (TM) cytes obtained from peripheral blood as it is easier was calculated using the following formula: to collect and there is more knowledge/experience TM=NMX0+LMX1+HMX2 (Tables 2-4). about/on them. While no genotoxic feature of In between-groups TM comparisons, the pre- inhalation agents were observed in studies using operative TM mean did not yield a significant dif- prior techniques, DNA damage has been detected ference. Except for the 5th postoperative day score more clearly and distinctly using this technique. of the remifentanil group, the mean total comet Sensitivity of this technique allows for observing score of all groups were higher than that of preoper- damage on a single strand of DNA(17,18). ative values, and the maximum values were Parallel to advancements in genotoxicity observed on the 1st postoperative day. screenings, while initial studies reported that The 5th postoperative day mean total comet halothane had a slight mutagenic effect and that score was not different than preoperative values in enflurane, isoflurane and nitrous oxide did not have Group SR (Table 5). mutagenic effects(19,20). 780 Filiz Alkaya Solmaz, Özlem Selvi Can et Al

Sardas et al(21) used Alkaline Comet technique combined with general anesthesia and spinal anes- and studied DNA damage in non-smoking patients thesia were administered to smokers only; pre-anes- administered isoflurane anesthesia. As a result, thesia and next day blood samples from the three using the comet method, they concluded that geno- groups did not have a difference with respect to toxic effect starts in peripheral blood lymphocytes SCE values. As a result of these studies, they con- of patients exposed to isoflurane before DNA repa- cluded that smoking alone emerged a factor ration begins. increasing DNA damage. We also excluded smok- Additionally considering that damage repara- ers from our study as we had theorized that it would tion in not always successful in patients with DNA contribute to DNA damage. reparation genetic defects with conditions such as Studies on sevoflurane use in humans are lim- diabetes, malignancy, advanced age, genetic varia- ited; there was study on sevoflurane using the tions, chemical exposure, it has been reported that Comet technique found in the literature review. isoflurane anesthesia may lead to irreversible DNA Karabiyik et al(7) compared damage in patients who damage and serious health problems and that it is received sevoflurane and isoflurane using the comet important to investigate DNA damage in patients technique and have not detected any difference who receive repeated isoflurane anesthesia(21). between the two agents. In another study on a group of benign and A study published in 1997, conducted on malignant patients administered sevoflurane anes- Chinese hamster ovary cells, mention the DNA thesia, it was reported that the nucleic structure of damage caused by Compound A, a toxic metabolite their lymphocytes were effected; the damage was at of sevoflurane(8). This is the only cytogenetic study maximized especially at 120th minute of operation of compound A conducted on mammals. In both and while this damage regressed to preoperative studies, the genotoxic effect of sevoflurane is values on postoperative day 5 in benign patients, explained by compound A’ s ability to cause struc- malignant patient groups’ lymphocyte cell DNA tural changes by binding to DNA molecules. damage was not fully repaired, so the conclusion Similar effects in our study can possibly be associ- was that the genotoxic effects of sevoflurane, like ated with toxic features of compound A. those of many other inhalation anesthetics, were While there are numerous studies on inhala- reversible in benign patients with intact DNA repa- tion anesthetics’ genotoxicity on humans conducted ration mechanisms(22). Since the reparation and on benign patients, there are no studies investigat- regression time would be longer due to defects in ing how of adding opioids to inhalation agents reparation mechanisms, patients who could have impacts genotoxicity. Prior SCE and comets tests accumulating damage effects due to genetic dis- on inhalation anesthetics revealed that genotoxic eases or malignancy that could trigger the muta- effects are maximized at 120th minute. Start to genicity and carcinogenicity of agents with known decrease by postoperative day 1, and return to pre- genotoxicity were excluded from the study. operative values on day 5. Therefore, these time Hoerauf et al.(23) studied on the SCE frequency periods were taken as reference points for the sec- of non-smoking operating room personnel exposed ond, third and fourth blood samples(3,7,25). to isoflurane and nitrous oxide for a minimum of 3 Genotoxic effects were maximized on day 1 months, determined that SCE frequency significant- and started to decrease afterwards in all groups of ly increases among these individuals compared to our study as well. the control group. Additionally, they also empha- While Allen et al.(26), aimed to investigate the sized that the genetic damage of being exposed to genotoxicity of opioids only, did not observe geno- anesthetic gas waste exposure may be equivalent to toxicity in an Ames test. It was reported that the that of a smoker who smokes 11-20 cigarettes a genotoxic effect of remifentanil (in the form of mini day. In our study, the total migration ratio of non- mutant colony formation) in in vitro L5178 tk+/- smoking patients in Groups SN and K was maxi- mouse lymphoma test, was present only in mouse mized on postoperative day 1 and this ratio had not KCS9 metabolic activation and over the dose of regressed back to preoperative values on day 5. 308mcg/ml. Such activity pattern observed in In the remifentanil group, total migration was genetic toxicology tests has been reported to be not maximized on postoperative day 1 as well but had specific to remifentanil and is observed with other regressed back to preoperative values by postopera- opioids as well. tive day 5. Husum B. et al.(24), halothane, isoflurane Effects of using sevoflurane with nitrous oxide and remifentanil on Dna damage 781

In our study, while comet technique was used Form I to examine sevoflurane’s genotoxic damage, we aimed at investigating the damage caused by added 1. Time worked in this business: remifentanil and how much of this damage is 2. Within the past ten years, have you been exposed to any che- reversible postoperatively. As a result, we demon- micals through inhalation or direct skin contact at home or at strated that the group receiving remifentanil has work place? Yes/No th damage increase in 120 minute of the operation, Name of the chemical(s): that this damage is maximized on postoperative day Duration of exposure: 1, and that it regresses to preoperative values on Frequency of exposure: postoperative day 5. In nitrous oxide and control 3. Is smoking allowed in your current environment (home or groups, the damage was detected at 120th minute of work place)? the operation, was maximized on postoperative day Yes/No Number of smokers: 1, and had regressed on postoperative day 5 but had not returned to preoperative values yet. 4. Do you drink alcohol? It is observed that using nitrous oxide to lower Yes/No Frequency: the inhalation agent dose and to provide a more sta- Amount: glass(es)/day ble anesthesia is not harmless and it adds to the sevoflurane damage. Propofol was used as intra- 5. Do you or your family members have any genetic diseases (diabetes, cancer, etc.)? venous agent during induction in our study. Since Yes/No this medication has antioxidant features and the Name of disease(s): dose was equal across the groups, it was reasoned Time since diagnosis: that it would not impact the results. The type and 6. Disease(s) within the past two years: duration of analgesics and antibiotics used postop- Name of disease(s) eratively was not different between the groups and Time since diagnosis: no genotoxic characteristic of these medications 7. Vaccinations within the past year: was referenced in literature(23,27). We reasoned that radiologic assessments would not impact conclu- 8. Did you have any radiologic assessments within the past year? sions, as the groups did not differ in terms of num- Yes /No ber of assessments and the number of days since Number of assessments: last assessment. Assessment date(s): We were scrupulous to avoid preoperative 9. Name(s) and duration of medication(s) used within the past hypoxia in patients during the study. By doing so, 2 months: we aimed at eliminating the negative impact hypoxic cell damage or oxidative damage could have on 10. Did you have long term vitamin use within the past 6 months? the test. To minimize exposure to chemical agents Yes/No in our study, we surveyed out participants to evalu- Vitamin type(s): ate environmental factors, and did not find a differ- 11. Your dietary habits: ence between the groups. Since DNA damage is Mostly vegetarian / Mostly meat reportedly higher in smokers than non-smokers, smokers were excluded from the study(28,29). 12. Preferred cooking method: Boiling / Frying/Grilling As a result of this study, while calling atten- tion to sevoflurane’s possible genotoxic characteris- 13.Surgical history: tics, we also demonstrated that: Comet test is Diagnoses and dates: General anesthesia history: applicable in determining and following genotoxic Operation information: behavior in patients with genetic diseases, malig- Operation duration: nancy potential and additional risk factors; remifen- Anesthesia duration: Name(s) and dose(s) of anesthetic(s) used: tanil and nitrous oxide added to sevoflurane anes- History hypoxia: thesia of minimum of 2 hours impacts the lymphocyte nucleus structure in benign patients, but this 14. Postoperative medication use: Analgesic(s) and antibiotic(s) – dose(s): impact does not vary across groups; and is highest Chemotherapy agents: in all three groups on postoperative day 1 and Other medication(s) and dose(s): regresses to preoperative values in Group SR on 782 Filiz Alkaya Solmaz, Özlem Selvi Can et Al postoperative day 5, while it did not regress to pre- 8) Eger EI, Laster MJ, Winegar R, Han C, Gong D. operative values in the other two groups. Compund A induces sister chromatid exchanges in Chinese hamsters ovarial cells. Anesthesiology 1997; Total migration values between the groups 86: 918-22. were not different between the groups across each 9) Jones R. M. Desflurane and sevoflurane: Inhalation blood sample (p> 0.05). anaesthetics for this decade? Br. J. Anaesth 1990; 65: We determined that the genotoxic effects of 527-536. sevoflurane are reversible in individuals with intact 10) Martin, V.J. Gren, M.H.L., Schmezer, P. The single cell gel electrophoresis assay. 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