Cancer patients are frequently exposed to repeated surgical procedures under general anaesthesia. In addition to the surgical treatment, patients frequently receive chemotherapy. It is possible that the combined treatment with anaesthetic and cytotoxic drugs induces an increased toxicity risk. Knowledge of pharmacology and clinical pharmacodynamics of chemotherapeutic drugs, and their interaction with anaesthetics is generally deficient, and information on the combined action of anaesthetic and cytotoxic drugs on healthy as well as on tumour cells are lacking [1-3]. Since chemotherapeutic drugs are not selective to tumour cells; healthy cells may also be damaged, indicating the necessity for selection of anaesthetic for a surgical procedure.
With the rapid development of anticancer drugs, where each cytotoxic agent destroys the DNA of cancer cells by a variety of biological mechanisms, it is difficult to know and prevent all associated complications following from drug–drug interactions during anaesthetic management of cancer patients. Cisplatin is one of the most commonly used chemotherapeutic anticancer drugs for the treatment of many malignancies despite the fact that it is associated with many side-effects [4,5].
Sevoflurane is one of the most frequently used inhalation anaesthetics. About 5% of inhaled sevoflurane is metabolized by oxidative metabolism in liver cells, and a minor percentage is defluorinated by cytochrome P-450 in kidney. Investigations about genotoxicity and cytotoxicity of inhalational anaesthetics are often contradictory. In several studies, Sardas and colleagues [6,7] have examined the genotoxic activity of inhalation anaesthetics (i.e. sevoflurane, isoflurane, etc.) using the in vivo comet assay method. The result of these studies indicated that occupational exposure of operating room personnel to anaesthetic gases induced oxidative DNA damage of lymphocytes which are significantly decreased by using dietary antioxidants, vitamin C and vitamin A. Kvolik and colleagues  showed in vitro that halothane, isoflurane and sevoflurane used in anaesthetic doses have cytotoxic and antiproliferative effect on tumour cells. Karabiyik and colleagues  have proven on human lymphocytes of patients during anaesthesia that sevoflurane in vivo induced genotoxic effect and that full DNA repair was completed within 5 days. On the other hand, Szyfter and colleagues  concluded that sevoflurane did not exert an effect on peripheral blood lymphocytes. Hack and colleagues [11,12] suggested the necessity of use of Ehrlich ascites tumour (EAT) cells in vivo for the investigations of genotoxic and cytotoxic effect of different inhalational anaesthetics.
In this study we evaluated possible DNA damage induced in vivo by cisplatin and sevoflurane, respectively, or in combined treatment with both, using the alkaline comet assay on samples of blood, brain, liver, kidney, and also on the EAT cells of mice.
This study was approved by the Ethics Committee of The Faculty of Science and Medical School (University of Zagreb, Zagreb, Croatia) and was performed in accordance with the relevant Croatian guidelines, as well as with the Guide for the Care and Use of Laboratory Animals.
Sevoflurane (‘Sevorane') was provided by Abbott Laboratories Ltd (Queenborough, UK).
The chemotherapeutic anticancer drug cisplatin (cis-diaminedichloroplatinum(II)) was supplied by Pliva (Zagreb, Croatia).
In all, 40 male Swiss albino mice (2 months old, 20–25 g body weight) from our own conventional mouse colony were housed in groups of five in cages and were maintained on a pellet diet and water ad libitum. EAT cells were provided by the Department of Animal Physiology and were maintained in male Swiss albino mice through serial intraperitoneal inoculation in intervals (7–9 days) in an ascites form. After harvesting, tumour cell suspensions were prepared by dilution with 0.9% sodium chloride to final concentrations of 4 × 106 viable cells mL−1. Viability, confirmed by counting in a Bürker-Türk chamber using the Trypan Blue exclusion method, was always found to be at least 90%.
All mice were divided into eight experimental groups; four groups of mice were healthy and four groups of mice were EAT-bearing (injected intraperitoneally (i.p.) with 0.5 mL of EAT cells in concentration of 4 × 106 cells mL−1). The treatment of EAT-bearing mice was started 3 days after tumour inoculation. Groups of healthy, as well as groups of EAT-bearing mice, were treated for 3 consecutive days as follows. In the cisplatin group, mice were treated with cisplatin in a dose of 5 mg kg−1 via the i.p. route daily. In the sevoflurane group, mice was exposed to sevoflurane in a dose of 2.4 vol% for 2 h daily. In the combined cisplatin and sevoflurane group, mice were treated with a combination of cisplatin in a dose of 5 mg kg−1 via the i.p. route daily and afterwards exposed to sevoflurane in a dose of 2.4 vol% for 2 h daily.
After treatments performed during 3 consecutive days, the animals were sacrificed by cervical dislocation for evaluation of DNA damage. Each experiment included a parallel control group of healthy and EAT-bearing mice. Samples of blood and all tissue samples from the mice were collected for analysis. The tumour cells for analysis were obtained by abdominal cavity lavage. Anaesthesia was maintained with sevoflurane (2.4 vol%) in oxygen (3 L min−1) in a specially designed incubator connected to an anaesthetic machine (Sulla 800; Dräger, Germany) using a sevoflurane vaporizer. The sevoflurane concentration was monitored using a respiration monitor (PM 8050; Dräger). The fresh gas was flowing in one direction, without rebreathing, and exhaled gases were released through the exhaust pipe into the atmosphere. Nitrous oxide was not used in order to avoid additional DNA lesions. The depth of anaesthesia was considered satisfactory when mice were sleeping calmly, breathing spontaneously and not moving their tails.
All the chemicals used were of analytical grade. Normal melting point (NMP) agarose and low melting point (LMP) agarose were supplied by Sigma. Fully frosted microscope slides from Surgipath (Richmond, IL, USA) were used. Triton X-100, dimethyl sulphoxide (DMSO), sodium sarcosinate, sodium EDTA (ethylenediaminetetraacetic acid) and Tris-HCl were supplied by Sigma. Sodium chloride (p.a.), sodium hydroxide (p.a.) and ethidium bromide (p.a.) were supplied by Kemika (Zagreb, Croatia).
The Comet assay (single-cell gel electrophoresis) under alkaline conditions (pH > 13), as a simple visual method for the measuring of DNA damage, was used for analysis of samples. Blood samples were collected using a micropipette, after the tail vein was cut. The brain, liver and kidney tissues were collected from mice and pressed through the screen in the homogenization buffered solution at pH 7.5 (0.075 M NaCl and 0.024 M EDTA) and then cooled to 4°C (in the ratio 1 g tissue : 1 mL buffer).
All collected samples (peripheral blood, brain, liver, kidney and EAT cells) were analysed using a modification of the method of Singh and colleagues , as described in the following procedure. A freshly prepared cell suspension, obtained as a mixture of cells with 100 μL of 0.5% LMP agarose, were placed onto precleaned microscope slides, previously precoated with 300 μL of 0.6% NMP agarose. After cooling on ice for 10 min, slides were covered with 0.5% LMP agarose. After the agarose gel had solidified, slides were immersed for 1 h in ice-cold lysis solution, consisting of 100 mM EDTA, 2.5 M NaCl, 10 mM Tris-HCl and 1% sodium sarcosinate, adjusted to pH 10 with 1% Triton X-100 and 10% DMSO, added just prior to use. Prior to electrophoresis, slides were removed from the lysing solution and placed for 20 min in a horizontal electrophoresis unit (near the anode) filled with an alkaline buffer, in order to allow the unwinding of DNA and to express alkali-labile damage. The electrophoresis alkaline solution consisted of 1 mM EDTA and 300 mM NaOH, pH 13. After the unwinding of DNA electrophoresis was carried out in the same solution for 20 min at 25 V (300 mA). Alkali unwinding and electrophoresis were performed at 4°C under dimmed light. After electrophoresis, the alkali in the gels was neutralized by the rinsing of slides with Tris buffer (0.4 M Tris-HCl, pH 7.5). After neutralization, slides were stained with the fluorescent dye, ethidium bromide (20 μg mL−1), covered and stored in sealed boxes at 4°C for analysing.
A total of 500 cells of the same kind from each group was analysed under a fluorescent microscope (Opton, Germany) at 160× magnification. Electrophoresis at high pH results in structures resembling comets, as observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks [14,15]. Despite modern evaluation software for precise estimates of DNA damage, the human eye also provides the successful determination of degrees of damage, according to comet appearance. DNA migration can be determined visually by the categorization of comets into different ‘classes' of migration, or by using an eyepiece micrometer to estimate image or tail length [14,16]. We determined the DNA migration visually by the categorization of comets into different classes of migration in a value from 0 to 4 (arbitrary units), according to the degree of DNA damage. The comet tail lengths were evaluated to determine the DNA damage [9,15].
Results from the Comet assay were analysed using descriptive statistical methods and presented in Table 1. Non-parametric Kruskal–Wallis ANOVA & Median test were used for statistical analysis of differences between particular groups and posthoc analysis was performed with the Mann–Whitney U-test. The software package Statistica 6.0 (StatSoft, Inc., Tulsa, OK, USA; for Windows) was used. Statistical significance was set at P < 0.05.
The median and range of comet tail lengths, expressed in arbitrary units (a.u.) in the range 0–4 are presented in Table 1.
In the peripheral blood leucocytes (PBL) of healthy animals, the median comet tail lengths observed in cisplatin- or sevoflurane-treated group were of similar value and were significantly increased in comparison with the control group (P < 0.001). On the other hand, in PBL of the EAT-bearing mice, an increase of the comet tail length was observed in the cisplatin group as compared to the sevoflurane group (P < 0.001). The combined treatment exerted the strongest genotoxic effect in both healthy (P < 0.001) and EAT-bearing mice (P < 0.001) groups as compared with cisplatin or sevoflurane treatment alone. A statistically significant difference (P < 0.001) in PBL DNA lesions between control healthy and tumour-bearing mice was observed.
Evaluation of the level of DNA damage (according to their comet tail length) induced by cisplatin and sevoflurane, indicates that the brain cells are the most sensitive type of cells (Table 1) as there was a highly significant increase (P < 0.001) of the tail length in brain cells in both treatment groups (cisplatin or sevoflurane) vs. control group. However, a significant decrease (P < 0.001) of the tail length in brain cells in the combined treatment group of cisplatin–sevoflurane was observed in comparison to the cisplatin group. A similar tendency was also observed in brain cells of EAT-bearing mice. Comet photographs in Figure 1 show representative DNA damage of brain cells, in the range 0–4.
The results show that cisplatin and sevoflurane exerted statistically significant genotoxic effects on the liver cells of healthy and EAT-bearing mice cells as compared to a control group. Cisplatin induced a statistically significant genotoxic effect on liver cells as compared to sevoflurane (P < 0.001). The combined treatment produced the strongest genotoxic effect in both animal groups (P < 0.001).
Genotoxicity of cisplatin and sevoflurane on kidney cells of normal and EAT-bearing mice was significant (P < 0.001) as compared with the control group. Unexpectedly, the kidney cells were more sensitive to treatment with sevoflurane than to cisplatin (P < 0.001). Combined treatment with cisplatin and sevoflurane induced stronger genotoxic effect as compared to cisplatin treatment alone in the group of EAT-bearing mice (P < 0.001).
Cisplatin and sevoflurane exhibited significant genotoxicity on EAT cells compared to control group (P < 0.001). It is particularly important to mention that the combined treatment expressed statistically lower genotoxic effect on tumour cells than cisplatin alone (P < 0.001).
Both chemotherapeutic and anaesthetic drugs used in the present study (cisplatin and sevoflurane) showed the potential to induce different level of DNA damage, depending on type of cells. The detection of DNA damage is an indication of genotoxicity. In this study we used a simple and sensitive method, the alkaline comet assay of Singh and colleagues  to determine the genotoxicity of cisplatin and sevoflurane given alone or in combination in two experimental groups of animals, healthy and EAT-bearing mice. The in vivo alkaline comet assay has potential advantages over other in vivo genotoxicity test methods due to relative ease of application to any tissue that provides a single cell suspension and its sensitivity for detecting low levels of DNA damage . The alkaline comet assay can be used to detect DNA damage, such as strand breaks, as well as alkali-labile sites, DNA–DNA and DNA–protein crosslinks.
The results of our investigation (Table 1) have shown that sevoflurane and cisplatin induced strong in vivo genotoxic effect on PBL, kidney, brain, liver and tumour cells of mice. The combined treatments of mice with sevoflurane and cisplatin had a synergistic effect on PBL, liver cells and kidney cells. On the other hand, the combination had the opposite effect in brain and EAT cells. This study confirmed that PBL, brain and kidney cells of untreated EAT-bearing mice were more sensitive in this assay than cells from healthy control mice. This is in agreement with the conclusion reached by Kopjar and colleagues , who showed that peripheral blood lymphocytes from tumour patients demonstrated a higher basal level of the DNA lesions compared to the healthy volunteers.
The results on genotoxicity of kidney cells demonstrated that treatment with sevoflurane induced higher levels of DNA damage than treatment with cisplatin alone, although in previous studies cisplatin exhibited potentiation of nephrotoxicity . These results are not in agreement with results by Lee and colleagues [19,20] who showed that functional capacity of kidney after 1 h of treatment with volatile anaesthetic expressed anti-apoptotic and anti-inflammatory effects. In our study, a genotoxic effect of sevoflurane was demonstrated after repeated treatment with anaesthetic given for 2 h in consecutive 3-day treatments when the genotoxicity test was performed 30 min after the last treatment. The difference in genotoxicity between cisplatin and sevoflurane could be explained by the lack in functional capacity of the kidney cells to repair after sevoflurane since cisplatin was able to cause more damage to kidney cells as expressed by double strand breaks throughout the treatment that caused either apoptotic or necrotic cell damage. The damaged cells caused by cisplatin during the first and the second treatment disappeared and were not available for comet testing in this study.
As one possible explanation of the mechanism in which fluorinated anaesthetics induce direct DNA damage, Jaloszynski and colleagues  suggested a direct DNA interaction, in which the most probable alkaline-labile modification may be an alkylation at N7 purine bases. Other explanations for that genotoxicity of fluorinated anaesthetics is that they undergo metabolic oxidation processes that might produce highly reactive products . On the other hand, the anticancer activity of cisplatin is believed to result from its interaction with DNA by the mechanism of binding with N7 of purine bases, forming cisplatin–DNA adducts that inhibit fundamental cellular processes .
In conclusion, these results indicated that sevoflurane may influence the genotoxic action of cisplatin, in healthy and tumour cells. Further studies should be directed to clarify the possible mechanism underlying the interaction, which would be of particular interest for daily clinical practice in the treatment of cancer patients.
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