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Anesthesia of fish with benzocaine does not interfere with comet assay results
Mutation Research 534 (2003) 165–172
Anesthesia of fish with benzocaine does not interfere with comet assay results Álisson Marques de Miranda Cabral Gontijo a , Rodrigo Egydio Barreto b , Günter Speit c , Victor Alexis Valenzuela Reyes a , Gilson Luiz Volpato b , Daisy Maria Favero Salvadori a,∗ b
a Departamento de Patologia, Faculdade de Medicina, UNESP, Rubião Jr. s/n 18618-000, Botucatu, SP, Brazil Laboratory of Animal Physiology and Behavior, Departamento de Fisiologia, Instituto de Biociˆencias, Research Center on Animal Welfare—RECAW, CAUNESP, UNESP, Rubião Jr. s/n 18618-000, Botucatu, SP, Brazil c Universitätsklinikum Ulm, Abteilung Humangenetik, D-89070 Ulm, Germany
Received 7 March 2002; received in revised form 15 October 2002; accepted 23 October 2002
Abstract Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80 mg/l for ∼10 min). Furthermore, benzocaine (80–600 mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48 h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Anesthetic benzocaine (ethyl p-aminobenzoate); Single cell gel (SCG)/comet assay; DNA damage; Fish; Nile tilapia; Animal welfare
1. Introduction Aquatic organisms are extensively used in environmental monitoring [1–9]. In fish, blood erythrocytes are mainly used as sentinel markers of genotoxic ex∗ Corresponding author. Tel.: +55-14-68228255; fax: +55-14-68212346. E-mail address: [email protected] (D.M. Favero Salvadori).
posure [7]. Usually, the blood for genotoxicity assessment is collected by methods without previous anesthesia (e.g. cardiac or caudal vein puncture), which are potential discomfort and pain inducers. Although pain perception and analgesia in non-malmmalian vertebrates have not been studied adequately, anatomic, behavioral, physiologic, and biochemical studies indicate the probability of pain perception in non-mammalian vertebrates analogous to that in mammals and support
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 2 7 6 - 0
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the use of analgesia and anesthetic for invasive or painful procedures [10]. Several studies have provided evidence that na¨ıve fish may recognize by visual cues potentially harmful agents (e.g. predator threat) and trigger stress response to ‘fight or flight’ [11–14], although others claim that awareness of fear is impossible for fishes due to the absence of cerebral cortical structures crucial for the experience of fear, which are also necessary for pain perception [15]. Nevertheless, there is a general agreement that fish display robust, unconscious, neuroendocrine, and physiological stress responses to noxious stimuli, making avoidance of potentially injurious stress responses an important issue in considerations about fish welfare [15]. Thus, anesthesia of fish during aquatic biomonitoring or laboratory studies would contribute to improve fish welfare. However, the anesthetic itself might induce primary DNA damage, interfere with other genotoxic effects, and thus confound experimental results. In the present study, we evaluated the in vivo and in vitro genotoxicity of the anesthetic benzocaine (ethyl p-aminobenzoate; CAS 94-09-7) by the single cell gel (SCG)/comet assay with erythrocytes of the cichlid fish Nile tilapia, Oreochromis niloticus. We further investigated possible in vitro interactions with two known mutagens: the alkylating agent methyl methanesulfonate (MMS) and the oxidatively damaging agent hydrogen peroxide (H2 O2 ). The comet assay is a simple, quick, sensitive, and cost-effective technique to estimate global DNA damage in environmental monitoring [8,16], and has already been employed to assess exposure to various DNA-damaging chemicals in other fish [17,18]. Benzocaine was chosen due to its common use in fish management [19–23], usually efficiently applied by bath exposure [24]. If benzocaine is not genotoxic and does not interact with mutagens, blood collection for genotoxicity assessment would be possible with previous anesthesia, since ethical committees for animal experimentation recommend that animals should be freed from pain, discomfort, and distress [25]. 2. Materials and methods 2.1. Fish Adult Nile tilapia, Oreochromis niloticus (Linnaeus, 1759), were acclimatized for about 5 months in a
1200 l tank (one fish/4 l). During this period, the water temperature averaged 24 ◦ C, with continuous aeration through a biological filter, and the photoperiod was set up from 06:00 to 18:00 h. Ad libitum feeding was provided. 2.2. Anesthetic Benzocaine (ethyl p-aminobenzoate; CAS 94-09-7) was purchased form Sigma. A stock solution was prepared by the dilution of 6 g in 500 ml of ethanol. 2.3. Comet assay standardization for the Nile tilapia erythrocytes In fish, 97% of total blood cells are erythrocytes [26]. Therefore, no cell separation method was performed and whole blood cells are referred to as erythrocytes from hereafter. In this study, all whole blood samples (500 1) were collected with a heparinized syringe by cardiac puncture, immediately light protected, kept on ice, diluted 1:10 in ice-cold PBS, and processed by the comet assay within 30 min. The comet assay was conducted according to published recommendations [27]. However, due to unique characteristics of the material investigated, modifications were included, which are described below. In order to set the comet assay for Nile tilapia erythrocytes, cell suspension aliquots were treated on ice with H2 O2 (0, 2, and 10 M) for 5 min, and evaluated for a dose-response using different protocols. For each sample, 0.5 l of cell suspension were added to 70 l of 0.5% low melting point agarose, layered onto a slide pre-coated with 1.5% normal melting point agarose [28], and covered with a coverslip. After brief agarose solidification on ice, the coverslip was carefully removed and slides were immersed into lysis solution (2.5 M NaCl2 , 100 mM Na2 EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate with 1% Triton X-100, and 10% DMSO added just before use) for at least 1 h, at 4 ◦ C. Afterwards, slides were washed in ice-cold PBS for 5 min in order to remove excess of salt and detergents, left in electrophoresis buffer (0.3 mM NaOH, 1 mM EDTA, pH > 13) for different periods (5–40 min) for DNA unwinding, and electrophoresed in the same buffer for different periods (0.66 V/cm for 5–40 min). Following electrophoresis, slides were neutralized in 400 mM Tris–HCl (pH 7.5),
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fixed in absolute ethanol (both for 5 min), and stored until analysis. All of these steps were carried out under dim indirect light. The dried microscope slides were stained with ethidium bromide (20 g/ml in dH2 O; 50 l per slide), covered with a coverslip, and immediately analyzed at 400× magnification in a fluorescence microscope under green light. Analysis was performed by image analysis (Comet Assay 2.2, Perceptive Instruments, Suffolk, UK), where the mean tail moment (product of tail DNA/total DNA by the tail center of gravity) tail migration (distance from the end of the head to the end of the tail), and tail intensity (percentage DNA in tail) of 50 cells per sample were determined. Different periods of electrophoresis were compared by ANOVA, and post hoc compared by the Tukey HSD test in order to set the comet assay parameters to where a significant dose response to H2 O2 in the Nile tilapia erythrocytes was observed. Throughout this study, diluted and treated aliquots were tested for viability by trypan blue exclusion, and constantly >90% of cells excluded trypan. 2.4. In vitro treatments The comet assay was carried out as described above, however erythrocytes were treated in vitro with increasing concentrations of benzocaine (80, 160, and 600 mg/l) for 10 min, at room temperature. In further experiments, cells were treated with increasing concentrations of the alkylating agent MMS (0.001–0.1 M in PBS; for 10 min, at 37 ◦ C), or increasing concentrations of H2 O2 (1–100 M for 5 min on ice) in the presence or absence of benzocaine (80 mg/l). MMS, H2 O2 , and benzocaine experiments were performed with blood of three Nile tilapias each. 2.5. In vivo study design Twelve Nile tilapia were isolated one per glass aquarium (28 cm × 11.4 cm × 19.6 cm) and were randomly designated into four experimental groups (three fish each), two of which where exposed to MMS (7.5 mg/l) during 24 h. Of these, one was transported to an anesthesia tank containing benzocaine solution (80 mg/l). According to Munday and Wilson [23], the criteria for benzocaine efficacy was of total loss of equilibrium, with no visible response to
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handling which is equivalent to the classical stage II anesthesia [29], and the handleable stage used by Gilderhus and Marking [30]. In this study, efficient anesthesia occurred typically at 3 min of bath exposure, albeit the fish were maintained exposed until cessation of opercula movements (after ∼10 min). This was particularly carried out to satisfy all criteria of anesthesia, such as complete anesthesia, e.g. [31]. The three remaining groups served as controls for the experiment: one was only handled with no exposure to MMS or to benzocaine; another was only exposed to benzocaine, and the last was only exposed to MMS. Blood samples were collected by cardiac puncture immediately after anesthesia or handling and processed by the comet assay, as described above. In a second experiment, the in vivo time-course of a potential genotoxic effect of benzocaine alone was studied in six Nile tilapias (one fish per aquarium: 40 cm × 25 × 20 cm cm). These fish were randomly designated out of a group of 12 fish selected by size and placed in the experimental aquaria. The remaining six fish served as controls. All fish were fed ad libitum every day. Before being transported to the anesthesia tank containing benzocaine solution (80 mg/l), a blood sample was collected by cardiac puncture from each fish in order to obtain basal DNA damage levels of each group. After anesthesia, the animals were immediately returned to experimental aquaria for recovery, which occurred after 4.19 ± 0.12 min. Post-anesthesia blood samples of each fish were collected after 30 min, 2, 8, 24 and 48 h. As controls, blood samples were collected from the other six fish with no anesthesia, at the same time intervals, with exactly the same handling procedure. The comet assay was conducted as described above. In the first experiment, the standard length of the fish ranged from 16.7 to 18.3 cm, and body weight from 166.7 to 196.8 g; in the second, standard length ranged from 9.4 to 11.3 cm, body weight from 28.03 to 50.31 g. During the experimental period, aquaria were provided with continuous aeration through a biological filter. Overall water conditions were as follows: temperature averaged 25.6 ± 0.8 ◦ C; pH 6.6–6.8; nitrite and ammonia were lower than 0.5 and 0.01 mg/l, respectively; and dissolved oxygen ranged from 5.84 to 6.13 mg/l. Photoperiod was regulated by a timer
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Table 1 Comet assay standardization for the Nile tilapia erythrocytes exposed to H2 O2 in vitro
from 06:00 to 18:00 h, with artificial illumination (a fluorescent light, 590 lx).
H2 O2 (M)
2.6. Statistical analysis
0 2 10
Electrophoresis time 5 min
10 min
20 min
3.06 ± 1.48 a 1.48 ± 1.22 a 5.23 ± 2.31 b
2.24 ± 1.07 a 4.72 ± 1.56 b 12.31 ± 5.45 c
2.55 ± 1.16 a 9.35 ± 2.65 b 11.12 ± 6.43 b
Mean (±S.D.) tail moments that do not share a same letter in an electrophoresis time interval are statistically different (P < 0.05).
Table 2 Comet assay of Nile tilapia blood erythrocytes exposed to benzocaine in vitro for 10 min DNA migration (mean ± S.D.)
Benzocaine (mg/l)
0 80 160 600 Trend test a b
Tail momenta
Tail intensitya
0.74 ± 1.17 ± 1.12 ± 1.04 ± N.S.b
6.90 9.25 9.17 9.31 N.S.
0.21 0.45 0.28 0.13
± ± ± ±
Statistical analysis was performed as recommended by Lovell et al. [32]. Namely, for the in vivo experiment, the mean values were statistically analyzed by ANOVA, and when necessary were post hoc compared by the Tukey HSD test. The possibility to look for single-cell distribution is one the biggest advantages of the comet assay. To gain insight into the dispersion of comets within each fish, we calculated the coefficient of variance (CV; S.D./mean tail moment). This way, each fish had a CV value indicating if the migration pattern was homogenous or heterogeneous. Except were mentioned, mean values were considered
0.83 3.17 1.53 0.89
Data of three independent repeats. N.S.: not significant.
Table 3 Comet assay with Nile tilapia blood erythrocytes exposed to MMS or H2 O2 in vitro in the presence or absence of the anesthetic benzocaine Agent
DNA migration (tail moment)a Without benzocaine
MMS (M) 0 0.001 0.01 0.1 H2 O2 (M) 0 1 10
0.96 1.16 2.57 11.23
± ± ± ±
0.12 0.24 0.35c 1.68c
1.42 ± 1.31 4.45 ± 1.01c 9.38 ± 1.23c
P-value
With benzocaine (80 mg/l) 1.07 1.24 4.65 8.82
± ± ± ±
0.48 0.07 3.68d 0.27c
2.05 ± 1.43 3.27 ± 1.81d 6.61 ± 1.35c
N.S.b N.S. N.S. N.S. N.S. N.S. N.S.
Data is represented as mean ± S.D. of three independent experiments. b Comparisons between aliquots exposed or not to (80 mg/l) of benzocaine (N.S.: not significant, P > 0.05). c P < 0.01. d P < 0.05. a
Fig. 1. In vivo effects on DNA migration (depicted by the tail moment) of fish erythrocytes caused by anesthesia with benzocaine (80 mg/l) of fish (n = 3 per treatment) exposed or not to MMS (7.5 mg/l) (A). Mean coefficient of variance (CV = S.D./mean of 50 comets per animal) of the data depicted in (A). Note that reduced CVs have been associated to genotoxicity in fish erythrocytes (B). Mean values (±S.D.) that do not share the same letter are statistically different (P < 0.03; ANOVA).
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statistically significant at an α level of 0.05. To determine the concentration-related effect significance in vitro, multiple pair-wise comparisons were conducted between the control data and each dose using the Student’s t test as the trend test with a corrected α-value [α/number of comparisons] [33]. Data of benzocaine simultaneously treated aliquots were compared with controls by ANOVA.
3. Results 3.1. Standardization of the comet assay for fish erythrocytes After 20 min of resting in alkaline buffer, erythrocyte nuclei were fully unwound, with no nuclear core detectable. Electrophoresis for 10 min led to a clear and concentration-related effect on DNA migration after treatment with H2 O2 in contrast to the shorter or longer electrophoresis times (Table 1). After 40 min of electrophoresis, samples were unscorable
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with extensive tails that overlaid each other. Therefore, all comet assays were conducted with 20 min alkaline DNA unwinding and 10 min for electrophoresis. 3.2. In vitro effects Treatment of fish erythrocytes with benzocaine alone (80–600 mg/l) did not induce genotoxic effects in the comet assay. Although the values found after benzocaine treatment were somewhat higher than the controls, these differences were not statistically significant (P > 0.20), and did not indicate any concentration–effect relationship (Table 2). Fish blood erythrocytes were further assayed together with MMS or H2 O2 to look for possible interactions with the anesthetic benzocaine. A clear concentration-related effect was observed for both DNA-damaging agents (P < 0.05), and there was no significant difference in the presence of the anesthetic (P > 0.10) (Table 3). DNA damage was unscorable at the highest concentration of H2 O2 (100 M) tested because of comets
Fig. 2. DNA migration (tail moment) from erythrocytes of Nile tilapia anesthetized by bath exposure (∼10 min) to benzocaine (80 mg/l). Control (䉬) and anesthetized groups (䉫) (n = 6, each); P = 0.72; ANOVA (A). Percentage of comets with high levels of DNA damage in blood erythrocytes of Nile tilapia (mean ± S.E.; n = 6) anesthetized with benzocaine. Gray columns (control group); black columns (group exposed to benzocaine (80 mg/l); P = 0.33; ANOVA) (B).
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with virtually all DNA in the tail, which overlaid each other. This was regardless of benzocaine co-treatment. Therefore, this concentration was not included into the statistical analysis. 3.3. In vivo effects The 24 h bath exposure to MMS induced significant DNA migration (P < 0.03) in fish erythrocytes. This effect was not altered by the subsequent exposure to benzocaine (P > 0.40; Fig. 1A). Moreover, in fish not exposed to MMS, anesthetization with benzocaine did not induce DNA migration in comparison with fish not anesthetized (P > 0.30; Fig. 1A). Significantly reduced CVs were observed only for the groups exposed to MMS (Fig. 1B). In the time-course experiment, no significant difference was observed for DNA migration (P = 0.72) in anesthetized Nile tilapia erythrocytes when compared with controls throughout the sampling period of 48 h (Fig. 2A). Also, the percentage of comets with high levels of damage did not differ in fish exposed to benzocaine (P > 0.30) (Fig. 2B). The distribution of the migration of comets was very heterogeneous in vivo. As a reduction in comet migration heterogeneity may be indicative of an effect, we therefore compared the mean of individual CVs per sampling time for both controls and anesthetized fish and found high, albeit not significantly different CVs (P = 0.45) during the sampling period (Fig. 3).
Fig. 3. Tail moment distribution, depicted by the mean±S.D. of the coefficient of variance (CV = S.D./mean of 50 comets per animal) of erythrocytes of six Nile tilapia per group. Gray columns (control group); black columns (group exposed to benzocaine (80 mg/l); P = 0.45).
4. Discussion We have standardized the comet assay for whole blood erythrocytes of the cichlid fish Nile tilapia, Oreochromis niloticus, which is the most cultivated cichlid in the world [34]. We have also provided evidence of no DNA-damaging effect of the anesthetic benzocaine (an analog of procaine) on these cells. In vitro treatments of erythrocytes with increasing concentrations of benzocaine did not produce a significant induction of DNA damage. It is important to notice that the alkaline version of the comet assay used here is sensitive for a wide variety of DNA lesions. Among them are DNA strand breaks, alkali-labile lesions including abasic sites, and incomplete-repair sites. According to the proposed in vitro comet assay testing guideline [27], cells should be exposed for 3–6 h and the use of different exposure duration should be justified whenever negative data are obtained. Fish are usually considered anesthetized at 3 min of bath exposure. Moreover, it is known that plasma clearance of benzocaine is extremely rapid, being eliminated in its majority within the first 20 min after an intra-arterial bolus administration [35]. Thus, longer in vitro exposure periods would not be biologically relevant. To our knowledge, there is no published study that addressed the genotoxicity of benzocaine. Nevertheless, other analogs of procaine, all typically used as local anesthetics in humans, have been tested for genotoxicity and mutagenicity in different organisms such as bacteria, Drosophila melanogaster, and mammals, with no clear genotoxic effect [36,37]. Cocaine, also similar in structure and in function to benzocaine, was itself at best a weak clastogen in vitro [38], and was found not to be mutagenic or carcinogenic in rodents [39]. Thus, these reports on procaine analogs do not suggest relevant genotoxic effects of this group of compounds. Clear concentration-related effects were obtained for DNA damage in erythocytes exposed in vitro to two known genotoxins. Alkylating agents are expected to be the most potent and abundant chemical DNA-damaging agents found in our environment [40]. In this study, MMS served as a model for alkylation damage. DNA damage induced by MMS in erythrocytes is not altered by the simultaneous treatment with the anesthetic benzocaine in vitro. Furthermore, as a model for oxidative damage, which can happen endogenously or exogenously, H2 O2 was applied and
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there was also no evidence for an interaction with benzocaine. Correspondingly, in the in vivo tests, blood erythrocytes did not reveal increased DNA damage following benzocaine bath exposure. Likewise, DNA damage induced by MMS was not altered by anesthesia prior to sampling. Moreover, benzocaine anesthesia does not seem to have a late effect on DNA migration of blood erythrocytes, as evidenced by a 48 h follow-up. In this experiment, we observed substantial dispersion in the distribution of comet migration within each fish, as depicted by the individual CV. This has been reported for other fish and seems to be a feature not related to the technique [41]. In several studies, lower withinand between-animal variability (i.e. intra- and interindividual variability, respectively) were observed in fish exposed to chemical DNA-damaging agents, in comparison with controls [8,9,16]. Thus, apart from inducing DNA migration, MMS also homogenizes comet distribution, whereas benzocaine neither affects comet migration nor distribution. Exposure to MMS also decreased between-animal variability from ∼70 to <20% (Fig. 1A; S.D./mean), while previous anesthesia did not effect this variability. Besides, along the time-course experiment, the comet distribution was not significantly affected, corroborating the absence of an effect by benzocaine exposure. According to published studies, the within- and between-animal variability would be different in exposed groups, if there were an effect. In the literature, concentrations of benzocaine used for anesthesia by bath exposure commonly range from 20 to 100 mg/l and vary according to fish species e.g. [19–22]. The concentration tested herein was efficient at 3 min of bath exposure for the Nile tilapia. Fish were exposed for up to 10 min to 80 mg/l of benzocaine, a rather high concentration for other fish. No increase in basal DNA damage was observed after this prolonged and high-dose exposure. The exposure of Nile tilapia to higher concentrations, as 160 or 320 mg/l, might reduce the time for anesthesia and opercular cessation to approximately 3 and 1.5 min, respectively. However, under these concentrations, the fish are in high risk for cardiac arrest (data not shown). Even though higher concentrations were not tested in vivo, we consider that further testing would not be informative, and suggest the concentration tested herein to be safe for anesthesia of fish when using blood erythrocytes for
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genotoxicity monitoring. This view is also supported by our in vitro findings that did not indicate any significant increase in DNA damage in blood erythrocytes exposed to extremely high doses of benzocaine (up to 600 mg/l) for 10 min. Finally, it is valuable to notice that no cytotoxicity was evinced by benzocaine exposure in vivo or in vitro. In conclusion, based on the in vivo and in vitro experiments, which demonstrate that the anesthetic benzocaine does not have a genotoxic or co-genotoxic effect, we conclude that the use of benzocaine is safe and can be recommended for comet assay studies with Nile tilapia blood erythrocytes. The use of anesthetized fish contributes to the amelioration of animal welfare, because it avoids unnecessary suffering.
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[25] H.W. Gonyou, Why the study of animal behavior is associated with the animal welfare issue, J. Anim. Sci. 72 (1994) 2171– 2177. [26] C.W. Theodorakis, S.J. D’Surney, L.R. Shugart, Detection of genotoxic insult as DNA strand breaks in fish blood cells by agarose gel electrophoresis, Environ. Toxicol. Chem. 13 (1994) 1023–1031. [27] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35 (2000) 206–221. [28] M. Klaude, S. Eriksson, J. Nygren, G. Ahnström, The comet assay: mechanisms and technical considerations, Mutat. Res. 363 (1996) 89–96. [29] M. Stoskopf, Anaesthesia, in: L. Brown (Ed.), Aquaculture for Veterinarians, Pergamon Press, Oxford, 1993, pp. 161–167. [30] P.A. Gilderhus, L.L. Marking, Comparative efficacy of 16 anesthetic chemicals on rainbow trout, North Am. J. Fish. Manag. 7 (1987) 288–292. [31] N.J. Bernier, D.J. Randall, Carbon dioxide anesthesia in rainbow trout: effects of hypercapnic level and stress on induction and recovery from anesthetic treatment, J. Fish Biol. 52 (1998) 621–637. [32] D.P. Lovell, G. Thomas, R. Dubow, Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies, Teratog. Carcinog. Mutagen. 19 (1999) 109–119. [33] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. [34] FAO, Aquaculture production statistics 1987–1996, FAO Fisheries Circular 815, Revision 10, 1998, 197 pp. [35] J.R. Meinertz, G.R. Stehly, W.H. Gingerich, Pharmacokinetics of benzocaine in rainbow trout (Oncorhynchus mykiss) after intraarterial dosing, Aquaculture 148 (1996) 39–48. [36] P. Siekel, A genotoxicological study of the new local anesthetic carbamate derivates carbisocaine, heptacaine, and pentacaine, J. Appl. Toxicol. 10 (1999) 239–243. [37] J. Leuschner, D. Leblanc, Studies on the toxicological profile of the local anesthetic articaine, Arzneimittelforschung 49 (1999) 126–132. [38] R.C. Yu, T.C. Lee, J.H. Wand, Genetic toxicity of cocaine, Carcinogenesis 20 (1999) 1193–1199. [39] D.M.F. Salvadori, L.F. Barbisan, A.P. Bazo, E.Q. de Santana, R. Denadai, S.V. de Oliveira, L.R. Ribeiro, J.L.V. de Camargo, Cocaine mutagenicity and hepatocarcinogenicity evaluations in rodents, Terat. Carcin. Mutagen. 18 (1998) 199–208. [40] D.W. Kuehl, J. Serrano, S. Naumann, Identification of potentially mutagenic contaminants in the aquatic environment by liquid chromatographic-thermospray mass spectrometric characterization of in vitro DNA adducts, J. Chromatogr. A 684 (1994) 1113–1119. [41] D.E. Nacci, S. Cayula, E. Jackim, Detection of DNA damage in individual cells from marine organisms using the single cell gel assay, Aquat. Toxicol. 35 (1996) 197–210.
Anesthesia of fish with benzocaine does not interfere with comet assay results Álisson Marques de Miranda Cabral Gontijo a , Rodrigo Egydio Barreto b , Günter Speit c , Victor Alexis Valenzuela Reyes a , Gilson Luiz Volpato b , Daisy Maria Favero Salvadori a,∗ b
a Departamento de Patologia, Faculdade de Medicina, UNESP, Rubião Jr. s/n 18618-000, Botucatu, SP, Brazil Laboratory of Animal Physiology and Behavior, Departamento de Fisiologia, Instituto de Biociˆencias, Research Center on Animal Welfare—RECAW, CAUNESP, UNESP, Rubião Jr. s/n 18618-000, Botucatu, SP, Brazil c Universitätsklinikum Ulm, Abteilung Humangenetik, D-89070 Ulm, Germany
Received 7 March 2002; received in revised form 15 October 2002; accepted 23 October 2002
Abstract Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80 mg/l for ∼10 min). Furthermore, benzocaine (80–600 mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48 h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Anesthetic benzocaine (ethyl p-aminobenzoate); Single cell gel (SCG)/comet assay; DNA damage; Fish; Nile tilapia; Animal welfare
1. Introduction Aquatic organisms are extensively used in environmental monitoring [1–9]. In fish, blood erythrocytes are mainly used as sentinel markers of genotoxic ex∗ Corresponding author. Tel.: +55-14-68228255; fax: +55-14-68212346. E-mail address: [email protected] (D.M. Favero Salvadori).
posure [7]. Usually, the blood for genotoxicity assessment is collected by methods without previous anesthesia (e.g. cardiac or caudal vein puncture), which are potential discomfort and pain inducers. Although pain perception and analgesia in non-malmmalian vertebrates have not been studied adequately, anatomic, behavioral, physiologic, and biochemical studies indicate the probability of pain perception in non-mammalian vertebrates analogous to that in mammals and support
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 2 7 6 - 0
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the use of analgesia and anesthetic for invasive or painful procedures [10]. Several studies have provided evidence that na¨ıve fish may recognize by visual cues potentially harmful agents (e.g. predator threat) and trigger stress response to ‘fight or flight’ [11–14], although others claim that awareness of fear is impossible for fishes due to the absence of cerebral cortical structures crucial for the experience of fear, which are also necessary for pain perception [15]. Nevertheless, there is a general agreement that fish display robust, unconscious, neuroendocrine, and physiological stress responses to noxious stimuli, making avoidance of potentially injurious stress responses an important issue in considerations about fish welfare [15]. Thus, anesthesia of fish during aquatic biomonitoring or laboratory studies would contribute to improve fish welfare. However, the anesthetic itself might induce primary DNA damage, interfere with other genotoxic effects, and thus confound experimental results. In the present study, we evaluated the in vivo and in vitro genotoxicity of the anesthetic benzocaine (ethyl p-aminobenzoate; CAS 94-09-7) by the single cell gel (SCG)/comet assay with erythrocytes of the cichlid fish Nile tilapia, Oreochromis niloticus. We further investigated possible in vitro interactions with two known mutagens: the alkylating agent methyl methanesulfonate (MMS) and the oxidatively damaging agent hydrogen peroxide (H2 O2 ). The comet assay is a simple, quick, sensitive, and cost-effective technique to estimate global DNA damage in environmental monitoring [8,16], and has already been employed to assess exposure to various DNA-damaging chemicals in other fish [17,18]. Benzocaine was chosen due to its common use in fish management [19–23], usually efficiently applied by bath exposure [24]. If benzocaine is not genotoxic and does not interact with mutagens, blood collection for genotoxicity assessment would be possible with previous anesthesia, since ethical committees for animal experimentation recommend that animals should be freed from pain, discomfort, and distress [25]. 2. Materials and methods 2.1. Fish Adult Nile tilapia, Oreochromis niloticus (Linnaeus, 1759), were acclimatized for about 5 months in a
1200 l tank (one fish/4 l). During this period, the water temperature averaged 24 ◦ C, with continuous aeration through a biological filter, and the photoperiod was set up from 06:00 to 18:00 h. Ad libitum feeding was provided. 2.2. Anesthetic Benzocaine (ethyl p-aminobenzoate; CAS 94-09-7) was purchased form Sigma. A stock solution was prepared by the dilution of 6 g in 500 ml of ethanol. 2.3. Comet assay standardization for the Nile tilapia erythrocytes In fish, 97% of total blood cells are erythrocytes [26]. Therefore, no cell separation method was performed and whole blood cells are referred to as erythrocytes from hereafter. In this study, all whole blood samples (500 1) were collected with a heparinized syringe by cardiac puncture, immediately light protected, kept on ice, diluted 1:10 in ice-cold PBS, and processed by the comet assay within 30 min. The comet assay was conducted according to published recommendations [27]. However, due to unique characteristics of the material investigated, modifications were included, which are described below. In order to set the comet assay for Nile tilapia erythrocytes, cell suspension aliquots were treated on ice with H2 O2 (0, 2, and 10 M) for 5 min, and evaluated for a dose-response using different protocols. For each sample, 0.5 l of cell suspension were added to 70 l of 0.5% low melting point agarose, layered onto a slide pre-coated with 1.5% normal melting point agarose [28], and covered with a coverslip. After brief agarose solidification on ice, the coverslip was carefully removed and slides were immersed into lysis solution (2.5 M NaCl2 , 100 mM Na2 EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate with 1% Triton X-100, and 10% DMSO added just before use) for at least 1 h, at 4 ◦ C. Afterwards, slides were washed in ice-cold PBS for 5 min in order to remove excess of salt and detergents, left in electrophoresis buffer (0.3 mM NaOH, 1 mM EDTA, pH > 13) for different periods (5–40 min) for DNA unwinding, and electrophoresed in the same buffer for different periods (0.66 V/cm for 5–40 min). Following electrophoresis, slides were neutralized in 400 mM Tris–HCl (pH 7.5),
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fixed in absolute ethanol (both for 5 min), and stored until analysis. All of these steps were carried out under dim indirect light. The dried microscope slides were stained with ethidium bromide (20 g/ml in dH2 O; 50 l per slide), covered with a coverslip, and immediately analyzed at 400× magnification in a fluorescence microscope under green light. Analysis was performed by image analysis (Comet Assay 2.2, Perceptive Instruments, Suffolk, UK), where the mean tail moment (product of tail DNA/total DNA by the tail center of gravity) tail migration (distance from the end of the head to the end of the tail), and tail intensity (percentage DNA in tail) of 50 cells per sample were determined. Different periods of electrophoresis were compared by ANOVA, and post hoc compared by the Tukey HSD test in order to set the comet assay parameters to where a significant dose response to H2 O2 in the Nile tilapia erythrocytes was observed. Throughout this study, diluted and treated aliquots were tested for viability by trypan blue exclusion, and constantly >90% of cells excluded trypan. 2.4. In vitro treatments The comet assay was carried out as described above, however erythrocytes were treated in vitro with increasing concentrations of benzocaine (80, 160, and 600 mg/l) for 10 min, at room temperature. In further experiments, cells were treated with increasing concentrations of the alkylating agent MMS (0.001–0.1 M in PBS; for 10 min, at 37 ◦ C), or increasing concentrations of H2 O2 (1–100 M for 5 min on ice) in the presence or absence of benzocaine (80 mg/l). MMS, H2 O2 , and benzocaine experiments were performed with blood of three Nile tilapias each. 2.5. In vivo study design Twelve Nile tilapia were isolated one per glass aquarium (28 cm × 11.4 cm × 19.6 cm) and were randomly designated into four experimental groups (three fish each), two of which where exposed to MMS (7.5 mg/l) during 24 h. Of these, one was transported to an anesthesia tank containing benzocaine solution (80 mg/l). According to Munday and Wilson [23], the criteria for benzocaine efficacy was of total loss of equilibrium, with no visible response to
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handling which is equivalent to the classical stage II anesthesia [29], and the handleable stage used by Gilderhus and Marking [30]. In this study, efficient anesthesia occurred typically at 3 min of bath exposure, albeit the fish were maintained exposed until cessation of opercula movements (after ∼10 min). This was particularly carried out to satisfy all criteria of anesthesia, such as complete anesthesia, e.g. [31]. The three remaining groups served as controls for the experiment: one was only handled with no exposure to MMS or to benzocaine; another was only exposed to benzocaine, and the last was only exposed to MMS. Blood samples were collected by cardiac puncture immediately after anesthesia or handling and processed by the comet assay, as described above. In a second experiment, the in vivo time-course of a potential genotoxic effect of benzocaine alone was studied in six Nile tilapias (one fish per aquarium: 40 cm × 25 × 20 cm cm). These fish were randomly designated out of a group of 12 fish selected by size and placed in the experimental aquaria. The remaining six fish served as controls. All fish were fed ad libitum every day. Before being transported to the anesthesia tank containing benzocaine solution (80 mg/l), a blood sample was collected by cardiac puncture from each fish in order to obtain basal DNA damage levels of each group. After anesthesia, the animals were immediately returned to experimental aquaria for recovery, which occurred after 4.19 ± 0.12 min. Post-anesthesia blood samples of each fish were collected after 30 min, 2, 8, 24 and 48 h. As controls, blood samples were collected from the other six fish with no anesthesia, at the same time intervals, with exactly the same handling procedure. The comet assay was conducted as described above. In the first experiment, the standard length of the fish ranged from 16.7 to 18.3 cm, and body weight from 166.7 to 196.8 g; in the second, standard length ranged from 9.4 to 11.3 cm, body weight from 28.03 to 50.31 g. During the experimental period, aquaria were provided with continuous aeration through a biological filter. Overall water conditions were as follows: temperature averaged 25.6 ± 0.8 ◦ C; pH 6.6–6.8; nitrite and ammonia were lower than 0.5 and 0.01 mg/l, respectively; and dissolved oxygen ranged from 5.84 to 6.13 mg/l. Photoperiod was regulated by a timer
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Table 1 Comet assay standardization for the Nile tilapia erythrocytes exposed to H2 O2 in vitro
from 06:00 to 18:00 h, with artificial illumination (a fluorescent light, 590 lx).
H2 O2 (M)
2.6. Statistical analysis
0 2 10
Electrophoresis time 5 min
10 min
20 min
3.06 ± 1.48 a 1.48 ± 1.22 a 5.23 ± 2.31 b
2.24 ± 1.07 a 4.72 ± 1.56 b 12.31 ± 5.45 c
2.55 ± 1.16 a 9.35 ± 2.65 b 11.12 ± 6.43 b
Mean (±S.D.) tail moments that do not share a same letter in an electrophoresis time interval are statistically different (P < 0.05).
Table 2 Comet assay of Nile tilapia blood erythrocytes exposed to benzocaine in vitro for 10 min DNA migration (mean ± S.D.)
Benzocaine (mg/l)
0 80 160 600 Trend test a b
Tail momenta
Tail intensitya
0.74 ± 1.17 ± 1.12 ± 1.04 ± N.S.b
6.90 9.25 9.17 9.31 N.S.
0.21 0.45 0.28 0.13
± ± ± ±
Statistical analysis was performed as recommended by Lovell et al. [32]. Namely, for the in vivo experiment, the mean values were statistically analyzed by ANOVA, and when necessary were post hoc compared by the Tukey HSD test. The possibility to look for single-cell distribution is one the biggest advantages of the comet assay. To gain insight into the dispersion of comets within each fish, we calculated the coefficient of variance (CV; S.D./mean tail moment). This way, each fish had a CV value indicating if the migration pattern was homogenous or heterogeneous. Except were mentioned, mean values were considered
0.83 3.17 1.53 0.89
Data of three independent repeats. N.S.: not significant.
Table 3 Comet assay with Nile tilapia blood erythrocytes exposed to MMS or H2 O2 in vitro in the presence or absence of the anesthetic benzocaine Agent
DNA migration (tail moment)a Without benzocaine
MMS (M) 0 0.001 0.01 0.1 H2 O2 (M) 0 1 10
0.96 1.16 2.57 11.23
± ± ± ±
0.12 0.24 0.35c 1.68c
1.42 ± 1.31 4.45 ± 1.01c 9.38 ± 1.23c
P-value
With benzocaine (80 mg/l) 1.07 1.24 4.65 8.82
± ± ± ±
0.48 0.07 3.68d 0.27c
2.05 ± 1.43 3.27 ± 1.81d 6.61 ± 1.35c
N.S.b N.S. N.S. N.S. N.S. N.S. N.S.
Data is represented as mean ± S.D. of three independent experiments. b Comparisons between aliquots exposed or not to (80 mg/l) of benzocaine (N.S.: not significant, P > 0.05). c P < 0.01. d P < 0.05. a
Fig. 1. In vivo effects on DNA migration (depicted by the tail moment) of fish erythrocytes caused by anesthesia with benzocaine (80 mg/l) of fish (n = 3 per treatment) exposed or not to MMS (7.5 mg/l) (A). Mean coefficient of variance (CV = S.D./mean of 50 comets per animal) of the data depicted in (A). Note that reduced CVs have been associated to genotoxicity in fish erythrocytes (B). Mean values (±S.D.) that do not share the same letter are statistically different (P < 0.03; ANOVA).
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statistically significant at an α level of 0.05. To determine the concentration-related effect significance in vitro, multiple pair-wise comparisons were conducted between the control data and each dose using the Student’s t test as the trend test with a corrected α-value [α/number of comparisons] [33]. Data of benzocaine simultaneously treated aliquots were compared with controls by ANOVA.
3. Results 3.1. Standardization of the comet assay for fish erythrocytes After 20 min of resting in alkaline buffer, erythrocyte nuclei were fully unwound, with no nuclear core detectable. Electrophoresis for 10 min led to a clear and concentration-related effect on DNA migration after treatment with H2 O2 in contrast to the shorter or longer electrophoresis times (Table 1). After 40 min of electrophoresis, samples were unscorable
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with extensive tails that overlaid each other. Therefore, all comet assays were conducted with 20 min alkaline DNA unwinding and 10 min for electrophoresis. 3.2. In vitro effects Treatment of fish erythrocytes with benzocaine alone (80–600 mg/l) did not induce genotoxic effects in the comet assay. Although the values found after benzocaine treatment were somewhat higher than the controls, these differences were not statistically significant (P > 0.20), and did not indicate any concentration–effect relationship (Table 2). Fish blood erythrocytes were further assayed together with MMS or H2 O2 to look for possible interactions with the anesthetic benzocaine. A clear concentration-related effect was observed for both DNA-damaging agents (P < 0.05), and there was no significant difference in the presence of the anesthetic (P > 0.10) (Table 3). DNA damage was unscorable at the highest concentration of H2 O2 (100 M) tested because of comets
Fig. 2. DNA migration (tail moment) from erythrocytes of Nile tilapia anesthetized by bath exposure (∼10 min) to benzocaine (80 mg/l). Control (䉬) and anesthetized groups (䉫) (n = 6, each); P = 0.72; ANOVA (A). Percentage of comets with high levels of DNA damage in blood erythrocytes of Nile tilapia (mean ± S.E.; n = 6) anesthetized with benzocaine. Gray columns (control group); black columns (group exposed to benzocaine (80 mg/l); P = 0.33; ANOVA) (B).
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with virtually all DNA in the tail, which overlaid each other. This was regardless of benzocaine co-treatment. Therefore, this concentration was not included into the statistical analysis. 3.3. In vivo effects The 24 h bath exposure to MMS induced significant DNA migration (P < 0.03) in fish erythrocytes. This effect was not altered by the subsequent exposure to benzocaine (P > 0.40; Fig. 1A). Moreover, in fish not exposed to MMS, anesthetization with benzocaine did not induce DNA migration in comparison with fish not anesthetized (P > 0.30; Fig. 1A). Significantly reduced CVs were observed only for the groups exposed to MMS (Fig. 1B). In the time-course experiment, no significant difference was observed for DNA migration (P = 0.72) in anesthetized Nile tilapia erythrocytes when compared with controls throughout the sampling period of 48 h (Fig. 2A). Also, the percentage of comets with high levels of damage did not differ in fish exposed to benzocaine (P > 0.30) (Fig. 2B). The distribution of the migration of comets was very heterogeneous in vivo. As a reduction in comet migration heterogeneity may be indicative of an effect, we therefore compared the mean of individual CVs per sampling time for both controls and anesthetized fish and found high, albeit not significantly different CVs (P = 0.45) during the sampling period (Fig. 3).
Fig. 3. Tail moment distribution, depicted by the mean±S.D. of the coefficient of variance (CV = S.D./mean of 50 comets per animal) of erythrocytes of six Nile tilapia per group. Gray columns (control group); black columns (group exposed to benzocaine (80 mg/l); P = 0.45).
4. Discussion We have standardized the comet assay for whole blood erythrocytes of the cichlid fish Nile tilapia, Oreochromis niloticus, which is the most cultivated cichlid in the world [34]. We have also provided evidence of no DNA-damaging effect of the anesthetic benzocaine (an analog of procaine) on these cells. In vitro treatments of erythrocytes with increasing concentrations of benzocaine did not produce a significant induction of DNA damage. It is important to notice that the alkaline version of the comet assay used here is sensitive for a wide variety of DNA lesions. Among them are DNA strand breaks, alkali-labile lesions including abasic sites, and incomplete-repair sites. According to the proposed in vitro comet assay testing guideline [27], cells should be exposed for 3–6 h and the use of different exposure duration should be justified whenever negative data are obtained. Fish are usually considered anesthetized at 3 min of bath exposure. Moreover, it is known that plasma clearance of benzocaine is extremely rapid, being eliminated in its majority within the first 20 min after an intra-arterial bolus administration [35]. Thus, longer in vitro exposure periods would not be biologically relevant. To our knowledge, there is no published study that addressed the genotoxicity of benzocaine. Nevertheless, other analogs of procaine, all typically used as local anesthetics in humans, have been tested for genotoxicity and mutagenicity in different organisms such as bacteria, Drosophila melanogaster, and mammals, with no clear genotoxic effect [36,37]. Cocaine, also similar in structure and in function to benzocaine, was itself at best a weak clastogen in vitro [38], and was found not to be mutagenic or carcinogenic in rodents [39]. Thus, these reports on procaine analogs do not suggest relevant genotoxic effects of this group of compounds. Clear concentration-related effects were obtained for DNA damage in erythocytes exposed in vitro to two known genotoxins. Alkylating agents are expected to be the most potent and abundant chemical DNA-damaging agents found in our environment [40]. In this study, MMS served as a model for alkylation damage. DNA damage induced by MMS in erythrocytes is not altered by the simultaneous treatment with the anesthetic benzocaine in vitro. Furthermore, as a model for oxidative damage, which can happen endogenously or exogenously, H2 O2 was applied and
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there was also no evidence for an interaction with benzocaine. Correspondingly, in the in vivo tests, blood erythrocytes did not reveal increased DNA damage following benzocaine bath exposure. Likewise, DNA damage induced by MMS was not altered by anesthesia prior to sampling. Moreover, benzocaine anesthesia does not seem to have a late effect on DNA migration of blood erythrocytes, as evidenced by a 48 h follow-up. In this experiment, we observed substantial dispersion in the distribution of comet migration within each fish, as depicted by the individual CV. This has been reported for other fish and seems to be a feature not related to the technique [41]. In several studies, lower withinand between-animal variability (i.e. intra- and interindividual variability, respectively) were observed in fish exposed to chemical DNA-damaging agents, in comparison with controls [8,9,16]. Thus, apart from inducing DNA migration, MMS also homogenizes comet distribution, whereas benzocaine neither affects comet migration nor distribution. Exposure to MMS also decreased between-animal variability from ∼70 to <20% (Fig. 1A; S.D./mean), while previous anesthesia did not effect this variability. Besides, along the time-course experiment, the comet distribution was not significantly affected, corroborating the absence of an effect by benzocaine exposure. According to published studies, the within- and between-animal variability would be different in exposed groups, if there were an effect. In the literature, concentrations of benzocaine used for anesthesia by bath exposure commonly range from 20 to 100 mg/l and vary according to fish species e.g. [19–22]. The concentration tested herein was efficient at 3 min of bath exposure for the Nile tilapia. Fish were exposed for up to 10 min to 80 mg/l of benzocaine, a rather high concentration for other fish. No increase in basal DNA damage was observed after this prolonged and high-dose exposure. The exposure of Nile tilapia to higher concentrations, as 160 or 320 mg/l, might reduce the time for anesthesia and opercular cessation to approximately 3 and 1.5 min, respectively. However, under these concentrations, the fish are in high risk for cardiac arrest (data not shown). Even though higher concentrations were not tested in vivo, we consider that further testing would not be informative, and suggest the concentration tested herein to be safe for anesthesia of fish when using blood erythrocytes for
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genotoxicity monitoring. This view is also supported by our in vitro findings that did not indicate any significant increase in DNA damage in blood erythrocytes exposed to extremely high doses of benzocaine (up to 600 mg/l) for 10 min. Finally, it is valuable to notice that no cytotoxicity was evinced by benzocaine exposure in vivo or in vitro. In conclusion, based on the in vivo and in vitro experiments, which demonstrate that the anesthetic benzocaine does not have a genotoxic or co-genotoxic effect, we conclude that the use of benzocaine is safe and can be recommended for comet assay studies with Nile tilapia blood erythrocytes. The use of anesthetized fish contributes to the amelioration of animal welfare, because it avoids unnecessary suffering.
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