Genotoxicity of the volatile anaesthetic desflurane in human lymphocytes in vitro, established by comet assay

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1 J Appl Genet 46(3), 2005, pp Genotoxicity of the volatile anaesthetic desflurane in human lymphocytes in vitro, established by comet assay Tomasz M. Karpiñski 1, Magdalena Kostrzewska-Poczekaj 1, Ireneusz Stachecki 2, Adam Mikstacki 3, Krzysztof Szyfter 1,4 1 Laboratory of Mutagenesis, Institute of Human Genetics, Polish Academy of Sciences, Poznañ, Poland 2 Department of Anaesthesiology and the Chair of Anaesthesiology and Intensive Therapy, K. Marcinkowski University of Medical Sciences, Poznañ, Poland 3 Department of Anaesthesiology and Intensive Therapy, Regional Hospital, Poznañ, Poland 4 Department of Otolaryngology and Laryngeal Oncology, K. Marcinkowski University of Medical Sciences, Poznañ, Poland Abstract. The aim of the present study was to estimate the genotoxicity of desflurane, applied as a volatile anaesthetic. The potential genotoxicity was determined by the comet assay as the extent of DNA fragmentation in human peripheral blood lymphocytes in vitro. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA fragmentation due to cell death. Another anaesthetic, halothane, already proved to be a genotoxic agent, was used as a positive control. Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner under experimental conditions applied. The results of the study demonstrated that the genotoxicity of desflurane was comparable with that of halothane. However, considering the pharmacodynamics of both drugs, the genotoxic activity of desflurane may be connected with a less harmful effect on the exposed patients or medical staff. Key words: comet assay, desflurane, genotoxicity, halothane, inhalation anesthetics. Introduction In the current clinical practice many inhalation anaesthetics are in use, including halogenated aliphatic compounds. The oldest in this group is halothane. It was introduced as an anaesthetic in the 1960s. Search for new inhaled drugs has provided a series of halogenated aliphatic compounds, including isoflurane, sevoflurane, enflurane, desflurane and others. In previous studies, we described genotoxic effects of halothane, isoflurane and sevoflurane (Ja³oszyñski et al. 1999a, 1999b; Szyfter et al. 2004). Known detrimental effects of volatile anaesthetics are genotoxicity and cytotoxicity (Horeauf et al. 1999; Ja³oszyñski et al. 1999a; Karabiyik et al. 2001; Lamberti et al. 1989; Rozgaj et al. 2001; ªardaº et al. 1998a, 1998b; Szyfter et al. 2004). The genotoxic effect can be shown by the alkaline comet assay, which measures the induction of DNA single strand breaks in cells exposed to anaesthetics (Ja³oszyñski et al. 1999a, 1999b; Karabiyik et al. 2001; Möller et al. 2000; ªardaº et al. 1998a, 1998b; Szyfter et al. 2004). DNA strand breaks originate from the direct modification of DNA by chemical agents or their metabolites (Möller et al. 2000; Tice et al. 2000). The comet assay, known also as the single-cell gel electrophoresis, is commonly used in studies on genotoxicity. As a rapid, simple and inexpensive technique, it has been found particularly useful for preliminary estimation of the genotoxic potential of drugs by evaluation of DNA strand breaks, crosslinks and alkali-labile sites and DNA repair induced by many chemical agents (Möller et al. 2000; Tice et al. 2000; Ja³oszyñski and Szyfter 1999). In this technique the migration of DNA in an electric field, supposed to be proportional to strand breakage, is a quantitative measure Received: January 25, Revised: April 22, Accepted: May 16, Correspondence: T.M. Karpiñski, Laboratory of Mutagenesis, Institute of Human Genetics, Polish Academy of Sciences, Strzeszyñska 32, Poznañ,

2 320 T.M. Karpiñski et al. of genotoxicity (Duez et al. 2003). The genotoxic activity of anaesthetic gases has already been studied by the comet assay (Ja³oszyñski et al. 1999a; Karabiyik et al. 2001; ªardaº et al. 1998a, 1998b; Szyfter et al. 2004). Another line of evidence of genotoxic activity of halogenated compounds applied in anaesthesiology comes from numerous cytogenetic observations. Some published reports document an increased number of chromosome aberrations (Karelova et al. 1992; Lamberti et al. 1989; Rozgaj et al. 2001, 1999), increase in micronuclei formation (Robbiano et al. 1998) and a high rate of sister chromatid exchanges (Karelova et al. 1992). However, a lack of genotoxicity of some halogenated anaesthetics has been reported as well (Husum et al. 1983, 1986). Cytogenetic alterations were observed in mammalian cells after in vivo and in vitro exposure to various anaesthetics (Robbiano et al. 1998). The effects of occupational exposure to inhalation anaesthetics are not sufficiently documented but some reports on nephrotoxicity (Lucchini et al. 1996), hepatotoxicity and carcinogenicity have been published (Robbiano et al. 1998). Some metabolites of anaesthetic gases are associated with toxic effects in certain tissues, e.g. in the liver, kidney or brain (Lucchini et al. 1996; Walker 1996). Anaesthetics can increase the frequency of spontaneous abortion among female operating room personnel or affect human reproduction in other ways (Boivin 1997, Rosenfeld and Loose-Mitchell 1998). The present work is an extension of the former studies on genotoxicity of halogenated anaesthetics. The goal of this study was to estimate the genotoxicity of desflurane (1,2,2,2-tetrafluoroethyl-difluoromethylether) and to confront it with the already proven genotoxic activity of halothane (2-bromo-2-chloro-1, 1,1-trifluoroethane) (Ja³oszyñski et al. 1999a; Ja³oszyñski et al. 1999b; Szyfter et al. 2004). The potential genotoxicity was studied as the extent of DNA degradation measured by the comet assay. Material and methods Chemicals All chemicals were of analytical grade, purchased from: Serva: normal melting point agarose, Triton X-100 and 4,6-diamidino-2-phenylindol 2 HCl (DAPI); Sigma: dimethyl sulfoxide (DMSO) and tris; Bio-Rad: low melting point agarose; IITD Poland: RPMI 1640 medium without L-glutamine; Aqua-Medica Poland: Gradisol L; Leciva Czech Republic: halothane (Narcotan); and Baxter Belgium: desflurane (Suprane). Peripheral blood lymphocytes: isolation and treatment Human peripheral blood lymphocytes (PBL) were obtained from two healthy, year old, non-smoking volunteers. The cells were separated by the standard method, including centrifugation over Gradisol L at 1200 rpm for 15 min. The cells were suspended in the RPMI 1640 medium without L-glutamine and exposed to halothane and desflurane at a concentration of 0.1 mm, 1 or 10 mm in 1% DMSO on ice. Because of a high volatility of both anaesthetics and to prevent repair of induced DNA lesions, the treatment was carried out at 4 o C in sealed plastic tubes. After exposure for 5, 10, 30 or 60 min, the cells were settled down by a 10-min spinning (700 rpm) and washed twice with fresh medium. The negative controls were samples with water or with 1% DMSO. The positive controls were samples with halothane. This agent was also used as a positive control in our previous study (Szyfter et al. 2004). Slides were prepared in duplicate. Alkaline comet assay The alkaline comet assay was conducted as described by Ja³oszyñski et al. (1999a) and Szyfter et al. (2004). Briefly, the PBL suspension (30 L) was mixed with 70 L of 1% low melting point agarose in the RPMI 1640 medium at 37 o C. The mixture was pipetted onto microscope slides previously pre-coated with a layer of 1% normal agarose. The slides were immersed in lysis solution (2.5 M NaCl, 0.1 M Na 2 EDTA, 10 mm tris, 1% of freshly added Triton X-100, ph 10) for 1 h to remove proteins. The slides were then placed in a horizontal electrophoretic tank in cold buffer (4 o C, 3 M NaOH, 1 mm Na 2 EDTA, ph 13) for 40 min to allow DNA unwinding. The electrophoresis was carried out in the same solution for 30 min (at 300 ma, 0.56 V/cm). After electrophoresis, slides were removed from the tank and immersed in neutralization buffer (0.4 M tris, ph 7.5) and stained with DAPI (2 µg ml 1 in distilled water). Image analysis Slides were examined with an Axiophot fluorescence microscope (Opton, Germany) with

3 321 IMAC-CCD S30 camera and ISIS 3 v 2.00 image analysis system (MetaSystems Hard- and Software, Altlussheim, Germany). The anaesthetic-induced and spontaneous strand breaks were measured as total comet length (increase in DNA migration). Median values were calculated for 100 comets per slide. Statistics The data were transformed to estimate the statistical significance by the Mann-Whitney U-test with the use of Graph Pad Prism 4 software. Results were considered significant when p < Results and discussion The comets resulting from DAPI staining, which visualize the extent of DNA damage, are presented in Figure 1 and 2. They show that desflurane and halothane are capable of inducing DNA fragmentation in human peripheral blood lymphocytes in vitro. Considerable differences between both negative controls (with water and with DMSO) and samples exposed to all concentrations of both anesthetics were observed. DMSO alone was used as an additional negative control because both halothane and desflurane were dissolved in 1% Figure 1. Nuclei of peripheral blood lymphocytes from the control probe DMSO due to their low solubility in water. The exposure of PBL to halothane and desflurane (Table 1) markedly increased DNA migration in a dose-dependent manner. The increase in mean comet length induced by each of the two drugs was significantly different (p < 0.01) from the control (for halothane U = 1.000, p = ; for desflurane U = 2.000, p = ). The difference between samples treated with halothane and Figure 2. Comet tails of peripheral blood lymphocytes after 30-min exposure to 1 mm halothane desflurane was not statistically significant (U = 57, p = ). The analysis of anaesthetic-induced genotoxicity showed that the most harmful was a short (5 minutes) treatment (Figures 3 5). A prolongation of treatment was followed by a tendency to shorten the comet tails but still remaining longer than in water or DMSO experiments. This can be related to DNA repair proceeding during exposure even at low temperature. Another explanation could be the shrinking effect of DMSO towards the cells, which has already been reported. DMSO acts a protective agent and is able to suppress the side effects of anaesthetics during long co-exposure times (Ja³oszyñski et al. 1999a; Szyfter et al. 2004). New inhaled anaesthetic agents permit a rapid induction and faster emergence from anaesthesia than halothane. Because these agents are eliminated more rapidly than older drugs, special attention must be paid to postoperative analgesia, as it requires earlier analgesic administration (Walker 1996). Halothane may result in an unpredictable hepatotoxicity, possibly due to a reactive free radical metabolite. However, incidence of such hepatotoxicity is extremely rare (1 in 10,000 30,000 patients). This drug is also associated with cardiovascular system depression, sensitization of the heart to catecholamines, production of extrasystoles and transient arrhythmias, and relaxation of skeletal muscles. Desflurane is often used in ambulatory surgery. At high doses it decreases peripheral vascular resistance and respiration in a similar way as halothane (Rosenfeld and Loose-Mitchell 1998). In medical practice the detrimental action of both drugs is different. At least three features,

4 322 T.M. Karpiñski et al. Table 1. Effect of halothane and desflurane on the extent of DNA degradation in peripheral blood lymphocytes, measured by the comet assay (mean comet length ± SD, in ìm) Treatment Exposure time (minutes) Water ± ± ± ± % DMSO not determined ± ± ± mm halothane ± ± ± ± mm halothane ± ± ± ± mm halothane ± ± ± ± mm desflurane ± ± ± ± mm desflurane ± ± ± ± mm desflurane ± ± ± ± namely: blood/gas partition coefficient, lipid/gas partition coefficient and percentage of elimination activity of volatile anaesthetics. For inhaled anaesthetic gases, high blood/gas partition coefficients result in a slower elimination. The blood/gas partition coefficient is 2.3 for halothane and 0.42 for desflurane (Hatch 1999; Walker 1996). Lipid/gas partition coefficients describe the solubility in cell lipid membrane and an ability to penetrate into cells. Lipid/gas partition coefficients for desflurane and halothane are 19 and 224, respectively. Therefore, halothane can better penetrate Figure 3. Genotoxic effect of halothane shown in PBL in vitro. A measure of genotoxicity was an induction of DNA migration (µm, median) in comet assay. Figure 4. Genotoxic effect of desflurane shown in PBL in vitro. A measure of genotoxicity was an induction of DNA migration (µm, median) in comet assay.

5 323 Figure 5. Median comet length [µm] after exposure to 0, 0.1, 1 and 10 mm halothane and desflurane for various times into cells, and the difference in solubility may result in a stronger effect on DNA, as compared to desflurane. Metabolism of anaesthetic gases accounts for a small part of their elimination: 20% for halothane, metabolism and 0.02% for desflurane. Desflurane, in contrast to halothane, is metabolized to a minimal degree and the main way of its excretion is through the lungs (Rosenfeld and Loose-Mitchell 1998; Walker 1996). In our study the effect of both anaesthetics on DNA was similar, although considering their pharmacodynamics halothane should act faster. Probably the published studies disclose a synergistic effect of DMSO with other substances, which was mentioned in some reports (Pommier et al. 1988; Gebel et al. 1999). It is likely that in the present study DMSO could increase the toxic activity of desflurane. However, this is only a supposition. Hence, the genotoxicity of both studied anaesthetics in vitro is approximately the same. However, considering the published data about the drug s pharmacodynamics (Rosenfeld and Loose-Mitchell 1998; Walker 1996; Hatch 1999), the toxicity of desflurane for patients seems to be less harmful than that of halothane. REFERENCES Boivin J-F, Risk of spontaneous abortion in women occupationally exposed to anaesthetic gases: a meta-analysis. Occup Environ Med 54: Duez P, Dehon G, Kumps A, Dubois J, Statistics of the comet assay: a key to discriminate between genotoxic effects. Mutagenesis 18: Gebel T, Koenig A, Impact of dimethyl sulfoxide and examples of combined genotoxicity in the SOS chromotest. Mutat Res 18: Hatch DJ, New inhalation agents in paediatric anaesthesia. Br J Anaesth 83: Horeauf K, Lierz M, Wiesner G, Schroegendorfer K, Lierz P, Spacek A, et al Genetic damage in operating room personnel exposed to isoflurane and nitrous oxide. Occup Environ Med 56: Husum B, Niebuhr E, Wulf H.C, Norgaard I, Sister chromatid exchanges and structural chromosome aberrations in lymphocytes in operating room personnel. Acta Anaesthesiol Scand 27: Husum B, Wulf HC, Mathiassen F, Niebuhr E, Sister chromatid exchanges in lymphocytes of dentists and chairside assistants: no indication of a mutagenic effect of exposure to waste nitrous oxide. Community Dent Oral Epidemiol 14: Ja³oszyñski P, Kujawski M, W¹sowicz M, Szulc R, Szyfter K, 1999a. Genotoxicity of inhalation anaesthetics halothane and isoflurane in human lympho-

6 324 T.M. Karpiñski et al. cytes studied in vitro using the comet assay. Mutat Res 439: Ja³oszyñski P, T¹drowska M, Kujawski M, W¹sowicz M, Szulc R, Szyfter K, 1999b. Genotoxicity of inhalation anesthetics (halothane, isoflurane and sevoflurane) in human lymphocytes studied in vivo and in vitro. Neoplasma 46, Supplement: Ja³oszyñski P, Szyfter K, Comet assay nowoczesna technika w badaniach genotoksykologicznych [Comet assay a new technique in genotoxicologic studies]. In: Koroniak H, Barciszewski J, eds. Na pograniczu chemii i biologii [At the border between chemistry and biology]. Vol. 3. Wyd. UAM, Poznañ. Karabiyik L, ªardaº S, Polat U, Kocabas NA, Karakaya AE, Comparison of genotoxicity of sevoflurane and isoflurane in human lymphocytes studiedinvivousingthecometassay.mutatres 492: Karelova J, Jablonicka A, Gavora J, Hano L, Chromosome and sister-chromatid exchange analysis in peripheral lymphocytes, and mutagenicity of urine in anesthesiology personnel. Int Arch Occup Environ Health 64: Lamberti L, Bigatti P, Ardito G, Armellino F, Chromosome analysis in operating room personnel. Mutagenesis 4: Lucchini R, Placidi D, Toffoletto F, Alessio L, Neurotoxicity in operating room personnel working with gaseous and nongaseous anesthesia. Int Arch Occup Environ Health 68: Möller P, Knudsen LE, Loft S, Wallin H, The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidem Biomarkers & Prevention 9: Pommier RF, Woltering EA, Milo G, Fletcher WS, Synergistic cytotoxicity between dimethyl sulfoxide and antineoplastic agents against ovarian cancer in vitro. Am J Obstet Gynecol 159: Robbiano L, Mereto E, Migliazzi Morando A, Pastore P, Brambilla G, Increased frequency of micronucleated kidney cells in rats exposed to halogenated anaesthetics. Mutat Res 413: 1 6. Rosenfeld G, Loose-Mitchell D, Pharmacology. Williams & Wilkins. Rozgaj R, Kasuba V, Peric M, Chromosome aberrations in operating room personnel. Am J Ind Med 35: Rozgaj R, Kasuba V, Jazbec A, Preliminary study of cytogenetic damage in personnel exposed to anesthetic gases. Mutagenesis 16: ªardaº S, Aygün N, Gamli M, Ünal Y, Ünal N, Berk N, Karakaya AE, 1998a. Use of alkaline comet assay (single cell gel electrophoresis technique) to detect DNA damages in lymphocytes of operating room personnel occupationally exposed to anaesthetic gases. Mutat Res 418: ªardaº S, Karabiyik L, Aygün N, Karakaya AE, 1998b. DNA damage evaluated by the alkaline comet assay in lymphocytes of humans anaesthetized with isoflurane. Mutat Res 418: 1 6. Szyfter K, Szulc R, Mikstacki A, Stachecki I, Rydzanicz M, Ja³oszyñski P, Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo. J Appl Genet 45: Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ and Molec Mutagenesis 35: Walker J, What is new with inhaled anesthetics: part I. J Perianesthesia Nursing 11:

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