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DNA damage evaluated by the alkaline comet assay in lymphocytes of humans anaesthetized with isoflurane
Mutation Research 418 Ž1998. 1–6
DNA damage evaluated by the alkaline comet assay in lymphocytes of humans anaesthetized with isoflurane S. S¸ ardas¸ a
a,)
, L. Karabıyık b, N. Aygun ¨ a, A.E. Karakaya
a
Department of Toxicology, Gazi UniÕersity, Faculty of Pharmacy, 06330 Ankara, Turkey b Department of Anaesthesia, Turkish State Railways Hospital, 06330 Ankara, Turkey Received 3 February 1998; revised 22 April 1998; accepted 15 July 1998
Abstract In the present paper, we report data on the possible DNA damage, induced in vivo by isoflurane using the alkaline single cell gel electrophoresis technique ŽSCGE-comet assay. in patients beforerafter anaesthesia and in control group. Twelve patients, aged 22–66 years old, were anaesthetized for elective abdominal surgery with isoflurane in oxygen for 120–162 min Žmean: 133.2 min.. Venous blood samples were obtained from the patients before the induction of anaesthesia, at 60 and 120 min of anaesthesia and on the first, third and fifth following days of anaesthesia. SCGE was examined in 100 cells from each specimen graded as undamaged, intermediate and tailed nuclei. The number of undamaged nucleus was almost same in control and in patients before anaesthesia. However, significant differences were observed in proportion of undamaged, intermediate and tailed nucleus of patients at 60 and 120 min of anaesthesia and on the first day. DNA damage started to return to normal rates after the third day of anaesthesia and were almost identical with the rates of control group five days later. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Comet assay; Single cell gel electrophoresis; DNA damage; DNA single strand break; Cytogenetic effect; Isoflurane
1. Introduction The possibility of a health hazard from anaesthetic agents has been discussed extensively for the last decade w1–4x. Several hints from experimental and epidemiological studies give rise to the suspicion that genotoxic side effects can arise from inhalational anaesthetics w5,6x. The introduction of isoflurane into clicinal practice was delayed initially by the suggestion of Corbett in 1976 that it might produce liver tumors in rats w7x, but these findings ) Corresponding author. Tel.: q90-312-212-9909; Fax: q90312-2222-326
could not be confirmed in a larger study by Eger at al. in 1978 w8x. Isoflurane was approved subsequently for clinical use in North America, and was considered to be superior in many ways, to other inhalation anaesthetics w9x. The sister chromatid exchange ŽSCE. assay has been widely applied in investigations of individuals exposed to anaesthetic agents by various investigators w3,10–14x. In this work we evaluated the genotoxic effects of isoflurane in human lymphocytes by using the single cell gel electrophoresis ŽSCGE-comet. assay. This assay has been shown to be a sensitive method for the evaluation of DNA damage Žsingle-strand breaks and alkali labile damage. in individual cells w15,16x.
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 9 9 - 0
2
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
The technique of comet assay involves embedding cells in agarose gel on microscope slides and lysing with detergent and high salt. Slides are then soaked in an alkaline solution to allow cleavage of DNA at alkali labile sites. During electrophoresis under alkaline conditions, cells with damaged DNA display increased migration of DNA from the nucleus towards the anode. Broken DNA migrates further in the electric field, and the cell then resembles a ‘comet’ with a brightly fluorescent head and a tail region which increases as damage increases. Therefore the technique is also called ‘comet assay’. Images are visualized by fluorescence microscopy after ethidium bromide staining. Prior to the present study, neither patients nor operating room staff exposed to anaesthetic gases had been subjected to comet assay.
potent opioid analgesic and vecuronium bromide ŽNorcuron-Organon, 0.1 mgrkg. which is a non-depolarising neuromuscular bloker were administered intravenously for the induction of general anaesthesia. Also tracheal intubation was performed for each patient. Volatile anaesthetic isoflurane ŽForane-Abbot, 1–1.5%. in O 2 was administered by inhalation. The duration of isoflurane administration ranged from 120 to 162 min Žmean: 133.2 min.. A second venous blood sample was taken from all patients at 60 min after start of anaesthesia, a third sample at 120 min of anaesthesia and other samples were taken on the following first, third and fifth day. All samples were coded so that investigators were unaware of the time of withdrawal. Also, all patientsrcontrols were chosen among non-smokers in order to limit confounding factors. 2.2. Chemicals
2. Materials and methods 2.1. Donors The study was performed on 12 patients, aged 22–66 years old Žmean: 43.5 years old. who underwent operation for elective abdominal surgery. All patients were otherwise healthy and had not received regular medication before operation. Informed consent was obtained from all patients at the preoperative visit. There were 10 women and 2 men in patient group and 12 matching healthy people Ž9 women and 2 men. participated as control group for this study. None of the patients were anaesthetized prior to this study. All patients met the Class 1 and Class 2 criteria of patient classification of the American Society of Anesthesiologists w17x ŽClass 1: a normal healthy patient, no disease other then surgical pathology, no systemic disturbances; Class 2: a patient with mild systemic disease, systemic disturbance due to surgical condition. and were not premedicated. During the study, electrocardiogram, blood pressure Žsystolic, diastolic and mean., heart rates and peripheric oxygen saturation ŽSpO 2 . were monitored by Datascope passport, USA. Venous blood was sampled in a heparinized syringe before anaesthesia. Thiopentone Sodium ŽPentothal Sodium-Abbott, 6 mgrkg. as hypnotic agent and fentanyl citrate ŽFentanyl Citrate-Abbott, 1 mg. as
All chemicals were purchased from the Sigma Chemical unless otherwise stated. Lymphocyte Seperation Medium was from ICN Flow and TC-199 from Gibco. Superfrost 1.0–1.2 mm thick microscope slides from Merck were used. Normal and low melting point agarose were obtained from Gibco. Dulbecco’s phosphate-buffered salts ŽPBS., without Mg and Ca, was from ICN Flow. 2.3. Peripheral blood lymphocyte preparation Five milliliter of blood was carefully layered over 8 ml Lymphocyte Separation Medium and centrifuged at 2000 = g for 15 min. After the plasma layer was removed and saved, the buffy coat was carefully removed and the cells were washed with TC-199 medium and then collected by 10 min centrifugation at 1000 = g. Lymphocytes were resuspended at approximately 10 7 per milliliter in TC-199 medium with 20% vrv plasma and 10% vrv DMSO. Lymphocytes were transferred to microfuge tubes and stored at y808C. 2.4. The alkaline comet assay (single cell gel electrophoresis) Slides were prepared in duplicate. A 120 ml normal 0.5% agarose in PBS was layered on to a
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
precleaned microscope slide, immediately covered with a coverslip and allowed to solidify. Approximately 1500 lymphocytes were mixed with 75 ml of 0.5% low melting point agarose in PBS at 378C, the coverslip removed and the mixture added to slide. The coverslip was replaced and the slide placed on ice for 5 min. After solidification of the agarose the coverslip was gently removed, a top layer of 75 ml low melting point agarose added, the coverslip replaced and the slide returned to ice. Once the top layer had solidified, the coverslip was removed and the slide gently immersed in cold lysing solution Ž2.5 M NaCl, 100 mM EDTA, 1% N lauryl sarcosine, 10 mM Tris–HCl pH 10 to which 1% Triton X-100 and 10% DMSO had been added fresh.. The slides were left at 48C for at least 1 h. 2.4.1. Electrophoresis and staining The slides were removed from the lysing solution and placed close together in a horizontal gel electrophoresis tank ŽPharmacia GNA 100. near the anode. It was filled with fresh electrophoresis buffer Ž300 mM NaOH, 1 mM EDTA, pH 13. to a level of 0.25 cm above the slides which were then to soak for 40 min in the alkali. Electrophoresis was carried out 40 min at 19 V, 300 mA using a consort E 425 electrophoresis power supply. All the above steps were carried out under dimmed yellow light to avoid any additional DNA damage occurring. After electrophoresis, the slides were gently removed from the tank and washed three times with neutralizing buffer Ž0.4 M Tris–HCl, pH 7.5. allowing the slides to sit for 5 min each time. Excess liquid was blotted from each slide and the DNA was stained with 50 ml ethidiumbromide Ž40 mgrml.. A clean coverslip was then placed over the slide. Slides were stored in a closed container at 48C and analyzed within 24 h, gel dehydration over longer storage times led to deterioration in slide quality. 2.4.2. DNA migration (comet) assessment At least 100 cells in two slides per subject were analyzed at 200 = magnification under a fluorescent microscope ŽZeiss. equipped with an exitation filter of 546 nm and a barrier filter of 590 nm. We determined the degree of damage by grading the nuclei as; undamaged, intermediate Žat low damage levels, stretching of attached strands of DNA, rather
3
than migration of individual pieces is likely to occur. and tailed Žwith increasing numbers of breaks, DNA pieces migrate freely from the nucleus forming comet images.. Statistical comparisons between the grade of DNA damages in controlrpatient group were analysed by using Student’s t-test, Mann Whitney U test and Wilcoxon test. 3. Results and discussion All parameters monitored were within acceptable normal clinical limits in all patients during this study. The number of undamaged, intermediate and tailed nuclei among 100 nuclei examined for each individual and the mean values are presented in Table 1. SCGE examined in twelve patients before the induction of anaesthesia were not statistically different from the healthy control group Ž p ) 0.05 for undamaged, intermediate and tailed nuclei.. At 60 min of anaesthesia the mean tailed nuclei were 6.8 " 3.8; 10.2 " 4.6 after 120 min of anaesthesia; 6.1 " 3.0 on the first day after anaesthesia, 4.7 " 3.8; on the third day and 3.4 " 3.3 on the fifth day after anaesthesia. The SCGE for comet imaged cells at the 60 min of anaesthesia Ž6.8 " 3.8. significantly increased and reached to highest value Ž10.2 " 4.6. at 120 min. The mean value of tailed nuclei in both sampling times were significantly different Ž p 0.05. from the mean value of tailed nuclei in patients observed before anaesthesia. However, the increased mean value of tailed nuclei started to decrease on the postoperative first Ž6.1 " 3.0., third Ž4.7 " 3.8. day and the mean value for tailed nuclei observed on the fifth day Ž3.4 " 3.3. was not statistically different from the mean tailed nuclei evaluated before anaesthesia Ž4.0 " 3.5. ŽTable 1.. These differences can be clearly observed in Fig. 1 by the frequency histogram of grade of damage in control and patient group for different sampling times. The photograph in Fig. 2 shows the grade of damage and belongs to a patient during anaesthesia with isoflurane Ž120 min. and Fig. 3 belongs to the same patient on the fifth day after the operation. Nonsmoker subjects were selected since smoking plays a causative role in the higher incidence of DNA damages Žintermediater tailed nuclei. when evaluated by comet technique w18x. This has been also shown by Husum et al. w14x
T 2 4 3 1 2 0 1 4 2 2 0 4 2.1 "1.4
82 81 74 96 89 90 82 90 89 92 99 97 88.4 "7.4
14 9 17 4 9 8 9 6 7 4 1 2 7.5 "4.6
I 4 10 9 0 2 2 9 4 4 4 0 1 4.0 "3.5
T
U
7 4 7 5 9 1 1 3 10 6 3 5 5.0 "2.8
I
U 91 92 90 94 89 99 98 93 88 91 97 91 92.7 "3.5
Sample BA Ž n s 12 . Žbefore anaesth..
Control Ž n s 12 .
49 62 59 67 64 61 68 80 77 79 81 81 69.0) H "10.5
U 44 23 33 31 29 35 21 13 14 14 17 16 24.1) H "10.0
I
Sample 1 Ž60 min . T 7 15 8 2 7 4 11 7 9 7 2 3 6.8) H "3.8
57 50 47 49 56 53 59 66 65 69 75 75 60.0) q "9.8
U 28 29 41 46 34 40 28 30 26 22 16 17 29.7) q "9.2
I 15 21 12 5 10 7 13 4 9 9 9 8 10.1) q "4.6
T
Sample 2 Ž120 min .
51 64 51 74 54 74 64 78 69 89 84 84 70.3) H "12.3
U 41 27 43 19 37 20 25 22 25 9 10 12 24.1) H "11.4
I
Sample 3 Ž1st day . T 8 9 6 7 9 6 11 0 6 2 6 4 6.3) "3.1
69 84 71 81 92 89 84 87 89 – 97 86 84.4) "8.3
U
17 9 24 12 8 8 10 11 7 – 2 11 10.8) q "5.7
I
Sample 4 Ž3rd day .
14 7 5 7 0 3 6 2 4 – 1 3 4.7) "3.8
T
70 89 70 95 90 – 84 – 87 93 98 95 87.1 "9.9
U
19 7 24 4 9 – 11 – 10 5 1 5 9.5 "7.0
I
Sample 5 Ž5th day .
11 4 6 1 0 – 5 – 3 2 1 0 3.4 "3.3
T
) p - 0.05 Žwith control: Student’s t-test and Mann Whitney U test.. q: p - 0.05 Žwith sample BA: Paired t-test and Wilcoxon test.. U: Undamaged, I: Intermediate, T: Tailed. Sample BA: before anaesthesia, Sample 1: 60 min during anaesthesia, Sample 2: 120 min during anaesthesia, Sample 3: first day after anaesthesia, Sample 4: third day after anaesthesia, Sample 5: fifth day after anaesthesia.
1 2 3 4 5 6 7 8 9 10 11 12 Mean "SD
Subject number
Table 1 Individual and Mean " SD grade of DNA damage evaluated by SCGE Ž100 cells. in control and patient group
4 S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
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Fig. 1. The frequency histogram of grade of damage in control and patient group Žmean" SD.. ) p - 0.05 Žwith control: Student’s t-test and Mann Whitney U test., q: p - 0.05 Žwith sample BA: Paired t-test and Wilcoxon test.. Sample BA: before anaesthesia, Sample 1: 60 min during anaesthesia, Sample 2: 120 min during anaesthesia, Sample 3: first day after anaesthesia, Sample 4: third after anaesthesia, Sample 5: fifth day after anaesthesia.
by sister chromatid exchange ŽSCE. in lymphocytes of smokers anaesthetized with isoflurane, and smokers are reported to have increased sister chromatid exchanges in lymphocytes in peripheral blood when exposed to potential SCE inducing agents w19–21x. Most of the published cytogenetic data in humans for anaesthetic gases are usually on chromosome alterations, sister chromatid exchanges w3,12,13,22– 24x. However, examination of SCE in peripheral blood lymphocytes of patients before and after anaesthesia with isoflurane in nitrous oxide revealed no indication of a mutagenic action of short-term
exposure to these agents in anaesthetic concentrations w11,14x. DNA damage was detected by nucleoid sedimentation in human lymphocytes stimulated with pokeweed mitogen after enflurane exposure in vitro by Reitz et al. w25x and reported that the method of nucleoid sedimentation helps to show DNA damage in proliferating cells after exposure to volatile anaesthetics or therapeutic gases. Also DNA single strand breaks in peripheral lymphocytes of neurosurgical patients were determined before and after 180 min of general anaesthesia with isoflurane and the fre-
Fig. 2. Representative tailed and intermediate nuclei from a patient Žsubject no. 9. during operation anaesthetized with isoflurane. Sample time: 120 min of anaesthesia.
Fig. 3. Undamaged nuclei from the same patient Žsubject no. 9. after operation. Sample time: fifth day after the anaesthesia with isoflurane.
6
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
quency of DNA single strand breaks appeared to be significantly enhanced immediately after anaesthesia w26x. Since the introduction of comet assay by Singh et al. w15x, it has become accepted as a rapid, simple, and sensitive visual technique for measuring DNA breakage which has found application in DNA damage and repair studies, biomonitoring, and determination of genotoxicity. The detection of DNA singlestrand breaks is an instantaneous indication of genotoxicity. However, there are no published data on anaesthetic gases in humans yet using this technique. We conclude that comet assay in peripheral blood lymphocytes of patients during anaesthesia with isoflurane indicates genotoxic effect before DNA repair begins. However, DNA repair is not always perfect such as patients with genetic defects in DNA repair, i.e., xeroderma pigmentosum patients with isoflurane may induce irreversible DNA damage. Therefore determination of DNA damage in patients especially of repeated anaesthesia is particularly important and finally further studies by the comet assay are needed to clarify the grade of damage that might appear with different anaesthetics such as halothane, enflurane, sevoflurane.
w11x
w12x
w13x
w14x
w15x
w16x
w17x
w18x
w19x
References w1x L. Waskell, A study of the mutagenicity of anaesthetics and their metabolites, Mutation Res. 57 Ž1978. 141–153. w2x C.P. Larson, Mortality among anaesthesiologists, Anesthesiology 51 Ž1979. 193–194. w3x S. S¸ ardas¸, H. C¸ uhruk, A.E. Karakaya, Y. Atakurt, Sister chromatid exchanges in operating room personnel, Mutation Res. 279 Ž1992. 117–120. w4x I.J. Kant, M.V. Rijssen-Moll, P.J.A. Borm, Simulation of nitrous oxide concentrations in operating and recovery rooms, Ann. Occup. Hyg. 34 Ž1990. 575–583. w5x J.M. Baden, V.F. Simmon, Mutagenic effects of inhalational anaesthetics, Mutation Res. 75 Ž1980. 169–189. w6x J.R. Marier, Halogenated hydrocarbon environmental pollution: the special case of halogenated anaesthetics, Environ. Res. 28 Ž1982. 212–239. w7x T.H. Corbett, Cancer and congenital anomalies associated with anesthetics, Ann. N.Y. Acad. Sci. 271 Ž1976. 58–66. w8x E.I. Eger, A.E. White, C.L. Brown, C.G. Biava, T.H. Corbett, W.C. Stevens, A test of the carcinogenicity of enflurane, isoflurane, halothane, methoxyflurane and nitrous oxide in mice, Anesth. Analg. 57 Ž1978. 678–694. w9x E.I. Eger, Isoflurane: a review, Anesthesiology 55 Ž1981. 559–576. w10x B. Husum, H.C. Wulf, E. Niebuhr, Sister chromatid ex-
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changes in lymphocytes after anaesthesia with halothane or enflurane, Acta Anaesthesiol. Scand. 25 Ž1981. 97–98. B. Husum, N. Valentin, H.C. Wulf, A. Halaburt, E. Niebuhr, Sister chromatid exchanges in cigarette smokers: effects of halothane, isoflurane or subarachnoid blockade, Br. J. Anaesth. 57 Ž1985. 1100–1103. B. Husum, H.C. Wulf, E. Niebuhr, Sister chromatid exchanges in human lymphocytes after anaesthesia with fluroxene, Br. J. Anaesth. 54 Ž1982. 987–990. A.E. White, S Takehisa, E.I. Eger, S. Wolff, W.C. Stevens, Sister chromatid exchanges induced by inhaled anaesthetics, Anesthesiology 50 Ž1979. 426–430. B. Husum, H.C. Wulf, E. Niebuhr, A. Kyst, N. Valentin, Sister chromatid exchanges in lymphocytes of humans anaesthetized with isoflurane, Br. J. Anaesth. 56 Ž1984. 559–564. N.P. Singh, M.T. Mc Coy, R.R. Tice, E.L. Scheneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 Ž1988. 184–191. P.L. Olive, J.P. Banath, R.E. Durand, Heterogenecity in radiation-induced DNA damage and repair. In tumor and normal cells measured using the comet assay, Radiat. Res. 122 Ž1990. 86–94. V.J. Collins, Records, mortality, operative risk and medicolegal considerations, in: Principles of anesthesiology, 3, LeaFebiger, Philadelphia, 1993, pp. 29–66. S. S¸ ardas¸, D. Walker, D. Akyol, A.E. Karakaya, Assessment of smoking-induced DNA damage in lymphocytes of smoking mothers of newborn infants using the alkaline single cell gel electrophoresis technique, Mutation Res. 335 Ž1995. 213–217. B. Lambert, M. Nordenskjold, ¨ B. Werelius, Increased frequency of sister chromatid exchanges in cigarette smokers, Hereditas 88 Ž1978. 147–149. S. S¸ ardas¸, S. Gok, ¨ A.E. Karakaya, Increased frequency of sister chromatid exchanges in the peripheral lymphocytes of cigarette smokers, Toxicol. In Vitro 5 Ž1991. 263–265. S. S¸ ardas¸, S. Gok, ¨ A.E. Karakaya, Sister chromatid exchanges in the lymphocytes of nurses handling antineoplastic drugs, Toxicol. Lett. 55 Ž1991. 311–315. B. Husum, E. Niebuhr, H.C. Wulf, I. Norgaard, Sister chromatid exchanges and structural chromosome aberrations in lymphocytes in operating room personnel, Acta Anaesthesiol. Scand. 27 Ž1983. 262–265. M.O. Bradley, I.C. Hsu, C.C. Harris, Relationship between sister chromatid exchange and mutagenicity, toxicity and DNA damage, Nature 282 Ž1979. 318–320. K. Holmberg, B. Lambert, J. Lindsten, S. Soderhall, DNA ¨ and chromosome alterations in lymphocytes of operating room personnel and in patients before and after inhalation anaesthesia, Acta Anaesthesiol. Scand. 26 Ž1982. 531–539. M. Reitz, K.D. Gupta, L. Brandt, Detection of DNA damage in stimulated human lymphocytes after enflurane exposure in vivo, Environ. Res. 59 Ž1992. 476–484. M. Reitz, E.A. Rumpf, E. Lanz, DNA single strand breaks in peripheral human lymphocytes after anesthesia with isoflurane-nitrous oxide-oxygen, Arzneim-Forsch.rDrug. Res. 43 Ž12. Ž1993. 1258–1261.
DNA damage evaluated by the alkaline comet assay in lymphocytes of humans anaesthetized with isoflurane S. S¸ ardas¸ a
a,)
, L. Karabıyık b, N. Aygun ¨ a, A.E. Karakaya
a
Department of Toxicology, Gazi UniÕersity, Faculty of Pharmacy, 06330 Ankara, Turkey b Department of Anaesthesia, Turkish State Railways Hospital, 06330 Ankara, Turkey Received 3 February 1998; revised 22 April 1998; accepted 15 July 1998
Abstract In the present paper, we report data on the possible DNA damage, induced in vivo by isoflurane using the alkaline single cell gel electrophoresis technique ŽSCGE-comet assay. in patients beforerafter anaesthesia and in control group. Twelve patients, aged 22–66 years old, were anaesthetized for elective abdominal surgery with isoflurane in oxygen for 120–162 min Žmean: 133.2 min.. Venous blood samples were obtained from the patients before the induction of anaesthesia, at 60 and 120 min of anaesthesia and on the first, third and fifth following days of anaesthesia. SCGE was examined in 100 cells from each specimen graded as undamaged, intermediate and tailed nuclei. The number of undamaged nucleus was almost same in control and in patients before anaesthesia. However, significant differences were observed in proportion of undamaged, intermediate and tailed nucleus of patients at 60 and 120 min of anaesthesia and on the first day. DNA damage started to return to normal rates after the third day of anaesthesia and were almost identical with the rates of control group five days later. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Comet assay; Single cell gel electrophoresis; DNA damage; DNA single strand break; Cytogenetic effect; Isoflurane
1. Introduction The possibility of a health hazard from anaesthetic agents has been discussed extensively for the last decade w1–4x. Several hints from experimental and epidemiological studies give rise to the suspicion that genotoxic side effects can arise from inhalational anaesthetics w5,6x. The introduction of isoflurane into clicinal practice was delayed initially by the suggestion of Corbett in 1976 that it might produce liver tumors in rats w7x, but these findings ) Corresponding author. Tel.: q90-312-212-9909; Fax: q90312-2222-326
could not be confirmed in a larger study by Eger at al. in 1978 w8x. Isoflurane was approved subsequently for clinical use in North America, and was considered to be superior in many ways, to other inhalation anaesthetics w9x. The sister chromatid exchange ŽSCE. assay has been widely applied in investigations of individuals exposed to anaesthetic agents by various investigators w3,10–14x. In this work we evaluated the genotoxic effects of isoflurane in human lymphocytes by using the single cell gel electrophoresis ŽSCGE-comet. assay. This assay has been shown to be a sensitive method for the evaluation of DNA damage Žsingle-strand breaks and alkali labile damage. in individual cells w15,16x.
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 9 9 - 0
2
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
The technique of comet assay involves embedding cells in agarose gel on microscope slides and lysing with detergent and high salt. Slides are then soaked in an alkaline solution to allow cleavage of DNA at alkali labile sites. During electrophoresis under alkaline conditions, cells with damaged DNA display increased migration of DNA from the nucleus towards the anode. Broken DNA migrates further in the electric field, and the cell then resembles a ‘comet’ with a brightly fluorescent head and a tail region which increases as damage increases. Therefore the technique is also called ‘comet assay’. Images are visualized by fluorescence microscopy after ethidium bromide staining. Prior to the present study, neither patients nor operating room staff exposed to anaesthetic gases had been subjected to comet assay.
potent opioid analgesic and vecuronium bromide ŽNorcuron-Organon, 0.1 mgrkg. which is a non-depolarising neuromuscular bloker were administered intravenously for the induction of general anaesthesia. Also tracheal intubation was performed for each patient. Volatile anaesthetic isoflurane ŽForane-Abbot, 1–1.5%. in O 2 was administered by inhalation. The duration of isoflurane administration ranged from 120 to 162 min Žmean: 133.2 min.. A second venous blood sample was taken from all patients at 60 min after start of anaesthesia, a third sample at 120 min of anaesthesia and other samples were taken on the following first, third and fifth day. All samples were coded so that investigators were unaware of the time of withdrawal. Also, all patientsrcontrols were chosen among non-smokers in order to limit confounding factors. 2.2. Chemicals
2. Materials and methods 2.1. Donors The study was performed on 12 patients, aged 22–66 years old Žmean: 43.5 years old. who underwent operation for elective abdominal surgery. All patients were otherwise healthy and had not received regular medication before operation. Informed consent was obtained from all patients at the preoperative visit. There were 10 women and 2 men in patient group and 12 matching healthy people Ž9 women and 2 men. participated as control group for this study. None of the patients were anaesthetized prior to this study. All patients met the Class 1 and Class 2 criteria of patient classification of the American Society of Anesthesiologists w17x ŽClass 1: a normal healthy patient, no disease other then surgical pathology, no systemic disturbances; Class 2: a patient with mild systemic disease, systemic disturbance due to surgical condition. and were not premedicated. During the study, electrocardiogram, blood pressure Žsystolic, diastolic and mean., heart rates and peripheric oxygen saturation ŽSpO 2 . were monitored by Datascope passport, USA. Venous blood was sampled in a heparinized syringe before anaesthesia. Thiopentone Sodium ŽPentothal Sodium-Abbott, 6 mgrkg. as hypnotic agent and fentanyl citrate ŽFentanyl Citrate-Abbott, 1 mg. as
All chemicals were purchased from the Sigma Chemical unless otherwise stated. Lymphocyte Seperation Medium was from ICN Flow and TC-199 from Gibco. Superfrost 1.0–1.2 mm thick microscope slides from Merck were used. Normal and low melting point agarose were obtained from Gibco. Dulbecco’s phosphate-buffered salts ŽPBS., without Mg and Ca, was from ICN Flow. 2.3. Peripheral blood lymphocyte preparation Five milliliter of blood was carefully layered over 8 ml Lymphocyte Separation Medium and centrifuged at 2000 = g for 15 min. After the plasma layer was removed and saved, the buffy coat was carefully removed and the cells were washed with TC-199 medium and then collected by 10 min centrifugation at 1000 = g. Lymphocytes were resuspended at approximately 10 7 per milliliter in TC-199 medium with 20% vrv plasma and 10% vrv DMSO. Lymphocytes were transferred to microfuge tubes and stored at y808C. 2.4. The alkaline comet assay (single cell gel electrophoresis) Slides were prepared in duplicate. A 120 ml normal 0.5% agarose in PBS was layered on to a
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
precleaned microscope slide, immediately covered with a coverslip and allowed to solidify. Approximately 1500 lymphocytes were mixed with 75 ml of 0.5% low melting point agarose in PBS at 378C, the coverslip removed and the mixture added to slide. The coverslip was replaced and the slide placed on ice for 5 min. After solidification of the agarose the coverslip was gently removed, a top layer of 75 ml low melting point agarose added, the coverslip replaced and the slide returned to ice. Once the top layer had solidified, the coverslip was removed and the slide gently immersed in cold lysing solution Ž2.5 M NaCl, 100 mM EDTA, 1% N lauryl sarcosine, 10 mM Tris–HCl pH 10 to which 1% Triton X-100 and 10% DMSO had been added fresh.. The slides were left at 48C for at least 1 h. 2.4.1. Electrophoresis and staining The slides were removed from the lysing solution and placed close together in a horizontal gel electrophoresis tank ŽPharmacia GNA 100. near the anode. It was filled with fresh electrophoresis buffer Ž300 mM NaOH, 1 mM EDTA, pH 13. to a level of 0.25 cm above the slides which were then to soak for 40 min in the alkali. Electrophoresis was carried out 40 min at 19 V, 300 mA using a consort E 425 electrophoresis power supply. All the above steps were carried out under dimmed yellow light to avoid any additional DNA damage occurring. After electrophoresis, the slides were gently removed from the tank and washed three times with neutralizing buffer Ž0.4 M Tris–HCl, pH 7.5. allowing the slides to sit for 5 min each time. Excess liquid was blotted from each slide and the DNA was stained with 50 ml ethidiumbromide Ž40 mgrml.. A clean coverslip was then placed over the slide. Slides were stored in a closed container at 48C and analyzed within 24 h, gel dehydration over longer storage times led to deterioration in slide quality. 2.4.2. DNA migration (comet) assessment At least 100 cells in two slides per subject were analyzed at 200 = magnification under a fluorescent microscope ŽZeiss. equipped with an exitation filter of 546 nm and a barrier filter of 590 nm. We determined the degree of damage by grading the nuclei as; undamaged, intermediate Žat low damage levels, stretching of attached strands of DNA, rather
3
than migration of individual pieces is likely to occur. and tailed Žwith increasing numbers of breaks, DNA pieces migrate freely from the nucleus forming comet images.. Statistical comparisons between the grade of DNA damages in controlrpatient group were analysed by using Student’s t-test, Mann Whitney U test and Wilcoxon test. 3. Results and discussion All parameters monitored were within acceptable normal clinical limits in all patients during this study. The number of undamaged, intermediate and tailed nuclei among 100 nuclei examined for each individual and the mean values are presented in Table 1. SCGE examined in twelve patients before the induction of anaesthesia were not statistically different from the healthy control group Ž p ) 0.05 for undamaged, intermediate and tailed nuclei.. At 60 min of anaesthesia the mean tailed nuclei were 6.8 " 3.8; 10.2 " 4.6 after 120 min of anaesthesia; 6.1 " 3.0 on the first day after anaesthesia, 4.7 " 3.8; on the third day and 3.4 " 3.3 on the fifth day after anaesthesia. The SCGE for comet imaged cells at the 60 min of anaesthesia Ž6.8 " 3.8. significantly increased and reached to highest value Ž10.2 " 4.6. at 120 min. The mean value of tailed nuclei in both sampling times were significantly different Ž p 0.05. from the mean value of tailed nuclei in patients observed before anaesthesia. However, the increased mean value of tailed nuclei started to decrease on the postoperative first Ž6.1 " 3.0., third Ž4.7 " 3.8. day and the mean value for tailed nuclei observed on the fifth day Ž3.4 " 3.3. was not statistically different from the mean tailed nuclei evaluated before anaesthesia Ž4.0 " 3.5. ŽTable 1.. These differences can be clearly observed in Fig. 1 by the frequency histogram of grade of damage in control and patient group for different sampling times. The photograph in Fig. 2 shows the grade of damage and belongs to a patient during anaesthesia with isoflurane Ž120 min. and Fig. 3 belongs to the same patient on the fifth day after the operation. Nonsmoker subjects were selected since smoking plays a causative role in the higher incidence of DNA damages Žintermediater tailed nuclei. when evaluated by comet technique w18x. This has been also shown by Husum et al. w14x
T 2 4 3 1 2 0 1 4 2 2 0 4 2.1 "1.4
82 81 74 96 89 90 82 90 89 92 99 97 88.4 "7.4
14 9 17 4 9 8 9 6 7 4 1 2 7.5 "4.6
I 4 10 9 0 2 2 9 4 4 4 0 1 4.0 "3.5
T
U
7 4 7 5 9 1 1 3 10 6 3 5 5.0 "2.8
I
U 91 92 90 94 89 99 98 93 88 91 97 91 92.7 "3.5
Sample BA Ž n s 12 . Žbefore anaesth..
Control Ž n s 12 .
49 62 59 67 64 61 68 80 77 79 81 81 69.0) H "10.5
U 44 23 33 31 29 35 21 13 14 14 17 16 24.1) H "10.0
I
Sample 1 Ž60 min . T 7 15 8 2 7 4 11 7 9 7 2 3 6.8) H "3.8
57 50 47 49 56 53 59 66 65 69 75 75 60.0) q "9.8
U 28 29 41 46 34 40 28 30 26 22 16 17 29.7) q "9.2
I 15 21 12 5 10 7 13 4 9 9 9 8 10.1) q "4.6
T
Sample 2 Ž120 min .
51 64 51 74 54 74 64 78 69 89 84 84 70.3) H "12.3
U 41 27 43 19 37 20 25 22 25 9 10 12 24.1) H "11.4
I
Sample 3 Ž1st day . T 8 9 6 7 9 6 11 0 6 2 6 4 6.3) "3.1
69 84 71 81 92 89 84 87 89 – 97 86 84.4) "8.3
U
17 9 24 12 8 8 10 11 7 – 2 11 10.8) q "5.7
I
Sample 4 Ž3rd day .
14 7 5 7 0 3 6 2 4 – 1 3 4.7) "3.8
T
70 89 70 95 90 – 84 – 87 93 98 95 87.1 "9.9
U
19 7 24 4 9 – 11 – 10 5 1 5 9.5 "7.0
I
Sample 5 Ž5th day .
11 4 6 1 0 – 5 – 3 2 1 0 3.4 "3.3
T
) p - 0.05 Žwith control: Student’s t-test and Mann Whitney U test.. q: p - 0.05 Žwith sample BA: Paired t-test and Wilcoxon test.. U: Undamaged, I: Intermediate, T: Tailed. Sample BA: before anaesthesia, Sample 1: 60 min during anaesthesia, Sample 2: 120 min during anaesthesia, Sample 3: first day after anaesthesia, Sample 4: third day after anaesthesia, Sample 5: fifth day after anaesthesia.
1 2 3 4 5 6 7 8 9 10 11 12 Mean "SD
Subject number
Table 1 Individual and Mean " SD grade of DNA damage evaluated by SCGE Ž100 cells. in control and patient group
4 S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
5
Fig. 1. The frequency histogram of grade of damage in control and patient group Žmean" SD.. ) p - 0.05 Žwith control: Student’s t-test and Mann Whitney U test., q: p - 0.05 Žwith sample BA: Paired t-test and Wilcoxon test.. Sample BA: before anaesthesia, Sample 1: 60 min during anaesthesia, Sample 2: 120 min during anaesthesia, Sample 3: first day after anaesthesia, Sample 4: third after anaesthesia, Sample 5: fifth day after anaesthesia.
by sister chromatid exchange ŽSCE. in lymphocytes of smokers anaesthetized with isoflurane, and smokers are reported to have increased sister chromatid exchanges in lymphocytes in peripheral blood when exposed to potential SCE inducing agents w19–21x. Most of the published cytogenetic data in humans for anaesthetic gases are usually on chromosome alterations, sister chromatid exchanges w3,12,13,22– 24x. However, examination of SCE in peripheral blood lymphocytes of patients before and after anaesthesia with isoflurane in nitrous oxide revealed no indication of a mutagenic action of short-term
exposure to these agents in anaesthetic concentrations w11,14x. DNA damage was detected by nucleoid sedimentation in human lymphocytes stimulated with pokeweed mitogen after enflurane exposure in vitro by Reitz et al. w25x and reported that the method of nucleoid sedimentation helps to show DNA damage in proliferating cells after exposure to volatile anaesthetics or therapeutic gases. Also DNA single strand breaks in peripheral lymphocytes of neurosurgical patients were determined before and after 180 min of general anaesthesia with isoflurane and the fre-
Fig. 2. Representative tailed and intermediate nuclei from a patient Žsubject no. 9. during operation anaesthetized with isoflurane. Sample time: 120 min of anaesthesia.
Fig. 3. Undamaged nuclei from the same patient Žsubject no. 9. after operation. Sample time: fifth day after the anaesthesia with isoflurane.
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S. S¸ardas¸ et al.r Mutation Research 418 (1998) 1–6
quency of DNA single strand breaks appeared to be significantly enhanced immediately after anaesthesia w26x. Since the introduction of comet assay by Singh et al. w15x, it has become accepted as a rapid, simple, and sensitive visual technique for measuring DNA breakage which has found application in DNA damage and repair studies, biomonitoring, and determination of genotoxicity. The detection of DNA singlestrand breaks is an instantaneous indication of genotoxicity. However, there are no published data on anaesthetic gases in humans yet using this technique. We conclude that comet assay in peripheral blood lymphocytes of patients during anaesthesia with isoflurane indicates genotoxic effect before DNA repair begins. However, DNA repair is not always perfect such as patients with genetic defects in DNA repair, i.e., xeroderma pigmentosum patients with isoflurane may induce irreversible DNA damage. Therefore determination of DNA damage in patients especially of repeated anaesthesia is particularly important and finally further studies by the comet assay are needed to clarify the grade of damage that might appear with different anaesthetics such as halothane, enflurane, sevoflurane.
w11x
w12x
w13x
w14x
w15x
w16x
w17x
w18x
w19x
References w1x L. Waskell, A study of the mutagenicity of anaesthetics and their metabolites, Mutation Res. 57 Ž1978. 141–153. w2x C.P. Larson, Mortality among anaesthesiologists, Anesthesiology 51 Ž1979. 193–194. w3x S. S¸ ardas¸, H. C¸ uhruk, A.E. Karakaya, Y. Atakurt, Sister chromatid exchanges in operating room personnel, Mutation Res. 279 Ž1992. 117–120. w4x I.J. Kant, M.V. Rijssen-Moll, P.J.A. Borm, Simulation of nitrous oxide concentrations in operating and recovery rooms, Ann. Occup. Hyg. 34 Ž1990. 575–583. w5x J.M. Baden, V.F. Simmon, Mutagenic effects of inhalational anaesthetics, Mutation Res. 75 Ž1980. 169–189. w6x J.R. Marier, Halogenated hydrocarbon environmental pollution: the special case of halogenated anaesthetics, Environ. Res. 28 Ž1982. 212–239. w7x T.H. Corbett, Cancer and congenital anomalies associated with anesthetics, Ann. N.Y. Acad. Sci. 271 Ž1976. 58–66. w8x E.I. Eger, A.E. White, C.L. Brown, C.G. Biava, T.H. Corbett, W.C. Stevens, A test of the carcinogenicity of enflurane, isoflurane, halothane, methoxyflurane and nitrous oxide in mice, Anesth. Analg. 57 Ž1978. 678–694. w9x E.I. Eger, Isoflurane: a review, Anesthesiology 55 Ž1981. 559–576. w10x B. Husum, H.C. Wulf, E. Niebuhr, Sister chromatid ex-
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