Aquacult Int (2007) 15:163–168 DOI 10.1007/s10499-007-9073-6 SHORT COMMUNICATION

MS222 does not induce primary DNA damage in fish R. E. Barreto Æ A. M. M. C. Gontijo Æ R. O. Alves-de-Lima Æ V. C. Raymundi Æ D. Pinhal Æ V. A. V. Reyes Æ G. L. Volpato Æ D. M. F. Salvadori

Received: 28 June 2006 / Accepted: 3 January 2007 / Published online: 14 February 2007
Springer Science+Business Media B.V. 2007

Abstract The genotoxicity of the anaesthetic MS222 (tricaine) was analysed in fish under both in vivo and in vitro conditions. Based on results of the single cell gel/ Comet assay, MS222 had no direct genotoxic effect on the experimental fish, indicating that MS222 does not induce primary DNA damage. These results suggest that the use of this important anaesthetic in aquaculture can be considered to be safe in terms of genotoxicity. Keywords Anaesthetic tricaine (ethyl meta-aminobenzoate) Æ DNA damage Æ Methanesulfonate salt Æ MS222 Æ Single cell gel (SCG)/Comet assay Æ Stressful practices in aquaculture

Introduction The anaesthetization of fish during any painful or stressful procedure, such as toxicological biomonitoring, experimental procedures and aquacultural practices, contributes to an improvement in fish welfare. However, the anaesthesia itself may induce unknown side effects in the fish, such as cytotoxicity and DNA damage. Good R. E. Barreto and A. M. M. C. Gontijo contributed equally to the study. R. E. Barreto (&) Æ V. C. Raymundi Æ G. L. Volpato Departamento de Fisiologia, Instituto de Biocieˆncias, UNESP, Rubia˜o Jr. s/n, 18618-000 Botucatu, SP, Brazil e-mail: [email protected] A. M. M. C. Gontijo Unite´ de Biologie des Interactions Hoˆte-Parasite, Institut Pasteur, Paris, France R. O. Alves-de-Lima Æ D. Pinhal Æ V. A. V. Reyes Æ D. M. F. Salvadori TOXICAN – Nu´cleo de Avaliac¸a˜o Toxicogene´tica e Cancerı´gena, Departamento de Patologia, Faculdade de Medicina, UNESP, Botucatu, SP, Brazil

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scientific practice requires that these putative side effects in fish be studied prior to the routine use of an anaesthetic for the purpose of facilitating blood sampling during research projects and aquaculture. Tricaine – MS222 (ethyl meta-aminobenzoate or methanesulfonate salt; CAS: 886-86-2) – is one of the most important and commonly used anaesthetics on fish, and the only anaesthetic approved by the U.S. Food and Drugs Administration (FDA) for use on aquatic organisms (Kelsch and Shields 1996). In a previous study, we reported that the anaesthetic benzocaine is not genotoxic to fish (Gontijo et al. 2003). As benzocaine is structurally similar to MS222 (both are analogues of procaine), the expectation is that MS222 is likewise non-genotoxic for fish. However, another procaine analogue, carbisocaine, has been shown to have genotoxic activity (Siekel 1990). These results suggests that each analogue may have to be tested individually to determine its genotoxicity, or lack thereof. Consequently, the aim of this study was to evaluate the genotoxicity of the anaesthetic MS222 to the cichlid fish Nile tilapia, Oreochromis niloticus (L.) under in vitro and in vivo conditions. To this end, the erythrocytes of the experimental fish were assayed for genotoxic effects using the single cell gel (SCG)/Comet assay. Blood erythrocytes are standardly used as sentinel markers of genotoxic exposure in fish (Mitchelmore and Chipman 1998; Gontijo et al. 2003; Andrade et al. 2004). Nile tilapia was chosen because it is an important species in commercial aquaculture (Barreto et al. 2003), and there is well-established Comet assay methodology available (Gontijo et al. 2003). The SCG assay is a simple, quick, sensitive and costeffective technique for estimating primary DNA damage in environmental monitoring studies (Mitchelmore and Chipman 1998; Andrade et al. 2004) and has been employed to assess exposure to various DNA-damaging chemicals in other fish (Moretti et al. 1998; Andrade et al. 2004; Cestari et al. 2004; Ferraro et al. 2004). If MS222 is not genotoxic, painful procedures can be avoided by treating the fish with MS222 anaesthesia prior to the procedure, which is in line with the recommendations of ethical committees for animal experimentation stating that animals should be freed from pain, discomfort and distress (Gonyou 1994) and recently legitimately expanded to include fish (Chandroo et al. 2004; Conte 2004).

Materials and methods Juvenile Nile tilapia, Oreochromis niloticus (Linnaeus 1759), were acclimatized for about 2 months in a 1200-l tank (1 fish/20 l; holding density: approx. 1 g/l). During this period, there was a constant flow of dechlorinated water through the tank and the water was continuously aerated; the mean water temperature was maintained at 24C. The photoperiod extended from 06:00 to 18:00 hours. The fish were fed daily in excess with tropical fish chow (38% protein; Purina Ltda, Campinas, SP, Brazil). MS222 (ethyl meta-aminobenzoate or methanesulfonate salt; CAS 886-86-2) was purchased from Sigma (St. Louis, Mo.). The anaesthetic solution comprised a 1:1 ratio of MS222 and sodium bicarbonate (NaHCO3) in distilled water, and the fish were exposed to the appropriate dilution of MS222 by bath exposure. The SCG/Comet assay was carried out according to Tice et al. (2000) and was performed exactly as previously standardized for Nile tilapia blood erythrocytes (see Gontijo et al. 2003). Throughout this study, diluted and treated aliquots of peripheral blood erythrocytes were tested for viability using a carboxyfluorescein/ethidium

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bromide dual stain test (see Hartmann and Speit 1997). Viable cells appear greenfluorescent, whereas orange-stained nuclei indicate dead cells. At least 200 cells were counted per sample. Throughout the experiment, at least 74% of the cells were consistently viable. Nile tilapia erythrocytes were treated in vitro at room temperature with increasing concentrations of MS222 (0, 150, 300, and 600 mg/l) for 15 min or for 4 h. The experiments were performed with blood from three Nile tilapia per treatment. The blood was protected from light exposure throughout the experiments. Twenty-four Nile tilapia were housed in glass aquaria (28.0 · 11.4 · 19.6 cm) divided into four similar compartments (one fish/compartment; 6.0 · 11.4 · 19.6 cm) for 2 days prior to the experiment. The fish were then randomly designated into three groups (eight fish each): (1) fish were exposed to methyl methanesulfonate (MMS; 7.5 mg/l) for 24 h; (2) each fish was transported to a container with MS222 solution (150 mg/l) for anaesthetization; (3) each fish was handled as described for group 2, but was exposed to neither to MMS nor MS222 (control). Blood samples were collected immediately after anesthetization or handling, following which the fish were housed in new, but similar aquaria with clean water for 3 days, at which time the blood was sampled again. The criteria for anaesthesia efficacy is the total loss of equilibrium, with no observable reaction to handling (Munday and Wilson 1997). This corresponds to classical stage II anaesthesia (Stoskopf 1993) and to the handleable stage used by Gilderhus and Marking (1987). In the present study, efficient anaesthesia occurred at 3 min of bath exposure, but the fish were left exposed to the anaesthesia until the cessation of opercular beats (after approx. 15 min), primarily to satisfy all of the criteria of anaesthesia, such as all of those described by Bernier and Randall (1998). The standard length of the fish used in this experiment was approximately 7.0 cm, and body weight were approximately 20 g. Water temperature averaged 25.6C; dissolved oxygen was around 6 mg/l. Photoperiod was controlled by a timer from 06:00 to 18:00 hours and light was provided by artificial fluorescent illumination (daylight). The data presented herein had a normal distribution as determined by the Shapiro-Wilks test for normality. In vivo data were compared by ANOVA (completely randomized design), and post-hoc comparisons were carried out using the Newman-Keuls test when necessary. To assess the concentration-related effect on statistical significance in vitro, multiple pairwise comparisons were conducted between the control data and each dose using the Student’s t-test as the trend test with a corrected a-value (a/number of comparisons) (Singh et al. 1988). Data were considered to be statistically different when P < 0.05.

Results Treatment of fish erythrocytes with MS222 alone (0–600 mg/l) did not induce an increased level of DNA migration, as determined by the SCG assay, for both the 15 min and 4 h exposure time-points (Fig. 1). The results of the in vivo experiments are summarized in Fig. 2. In the first blood sample measurement, the group exposed to MMS (positive control) had a higher level of DNA damage than the control and MS222-treated (i.e. anaesthetized) group. The DNA migration of these two last groups was statistically similar. In the

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T a il mo m e n t

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Fig. 1 In vitro assessment of concentration-related genotoxic effect of MS222 in Nile tilapia (Oreochromis niloticus L.). No statistical difference was found among the tested doses for both exposure intervals. Multiple pairwise comparisons were conducted between the control data and each dose using the Student’s t-test as the trend test with a corrected a-value (a/number of comparisons). The a-value was 0.05

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Fig. 2 In vivo assessment of genotoxic effect of MS222 in Nile tilapia. The asterisk (*) denotes a statistical difference between the MMS group and the control and MS222 groups for both timepoints (ANOVA; P < 0.05)

second blood measurement, DNA migration in the MMS-exposed group was again higher than the control and MS222-treated group, whereas there was no statistically significant difference between the two last groups.

Discussion In the present study, there was an absence of a direct genotoxic activity induced by MS222 (tricaine, an analogue of procaine) in Nile tilapia under both the in vivo and in vitro conditions. The blood erythrocytes did not show increased DNA damage following the in vivo bath exposure of the fish to MS222. Similarly, in vitro treatments of erythrocytes with increasing doses of MS222 did not induce a significant increase of DNA damage at exposure times of 15 min and 4 h. Consequently, the data from the in vitro and in vivo experiments are in agreement. The alkaline version of the SCG assay used here is sensitive to a wide variety of DNA lesions, including

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DNA strand breaks, alkali-labile adducts (abasic sites, among others) and incomplete repair sites. In addition, no cytotoxicity was found following in vivo or in vitro MS222 exposure. Yoshimura et al. (1981) found no MS222-induced mutagenicity in tests using bacteria. Other procaine analogues, all typically used as local anaesthetics in humans, have been tested for genotoxicity and mutagenicity in different organisms. There has been no evidence of articaine being genotoxic to mammals (Leuschner and Leblanc 1999) or of heptacaine and pentacaine being genotoxic to some bacteria and Drosophila melanogaster (Siekel 1990). Cocaine, which is also similar in structure and in function to MS222, was found only to be a weak clastogen in vitro (Yu et al. 1999) and not to be mutagenic or carcinogenic in rodents (Salvadori et al. 1998). In addition, the anaesthetic benzocaine was found not to be genotoxic in Nile tilapia fish (Gontijo et al. 2003). On the other hand, Siekel (1990) reported a genotoxic effect of carbisocaine in Euglena gracilis, but not in Salmonella typhimurium, Escherichia coli and D. melanogaster. These reports on procaine analogues actually indicate the absence of any relevant genotoxic effects of this group of compounds. We therefore suggest that MS222 is a safe anaesthetic for use on fish to be used in painful or stressful practices inherent to some experimental procedures and aquacultural management. However, the results of this study do not exclude the necessity for further investigations on possible co-genotoxic effect of MS222 with other genotoxins. Acknowledgements The authors thank Mr. A.C.B. Tardivo for assistance during the experiments.

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