FISH WELFARE: STRESS EVALUATION USING ALKALINE COMET ASSAY Malandrakis E.E., Kavouras M., Kassimatis D., Dadali O., Chatzipli C., Golomazou E., Exadactylos A., Panagiotaki P. Department of Ichthyology & Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Fytokou Str., 38446, Nea Ionia Magnesias, Hellas ABSTRACT The comet assay, which is also called the single cell gel assay (SCG), is lately used in order to detect and quantify DNA damage of single cells in micro-gel electrophoresis. It consists of the following steps: the preparation of cell suspension, the preparation of agarose gel in a microscope plate, cell lysis, unwinding of the DNA and microscopic analysis of the image processing system under fluorescence. Comet assay is used for the detection of DNA damage in various types of cells under stressful conditions. Stress induces the activation of enzymes such as endonucleases and topoisomerases (e.g., through elevation of intracellular free calcium) may give rise to strand breaks as will increases in the level of endogenous reactants such as superoxide radical and nitric oxide. Stress is of major importance in aquaculture production, and the lack of agreement about its quantification still continues. ΕΥΖΩΙΑ ΙΧΘΥΩΝ: ΕΚΤΙΜΗΣΗ ΣΤΡΕΣ ΜΕ ΤΗ ΧΡΗΣΗ ΤΗΣ ΜΕΘΟΔΟΥ ΤΟΥ ΚΟΜΗΤΗ ΣΕ ΑΛΚΑΛΙΚΕΣ ΣΥΝΘΗΚΕΣ Μαλανδράκης Ε.Ε., Κάβουρας Μ., Κασιμάτης Δ., Ντανταλή Ο., Χατζηπλή Κ., Γκολομάζου Ε., Εξαδάκτυλος Α., Παναγιωτάκη Π. Τμήμα Γεωπονίας Ιχθυολογίας και Υδάτινου Περιβάλλοντος, Σχολή Γεωπονικών Επιστημών, Πανεπιστήμιο Θεσσαλίας, Οδός Φυτόκου, 38446, Νέα Ιωνία Μαγνησίας, Ελλάς ΠΕΡΙΛΗΨΗ Η τεχνική του κομήτη, γνωστή και ως SCG, χρησιμοποιείται για την ανίχνευση και την ποσοτικοποίηση της βλάβης του DNA σε απομονωμένα κύτταρα, με την ηλεκτροφόρηση σε πηκτή αγαρόζης. Συνοπτικά αποτελείται από τα ακόλουθα βήματα: την προετοιμασία του κυτταρικού αιωρήματος, την επικάλυψη της αντικειμενοφόρου πλάκας με πηκτή αγαρόζης, την κυτταρική λύση, την αποπεριέλιξη του DNA, και την ανάλυση της εικόνας σε μικροσκόπιο φθορισμού. Η ανάλυση του κομήτη χρησιμοποιείται για την ανίχνευση της βλάβης του DNA σε διάφορους τύπους κυττάρων υπό συνθήκες στρες. Το στρες προκαλεί την ενεργοποίηση ενζύμων όπως οι ενδονουκλεάσες και οι τοποϊσομεράσες (μέσω της αύξησης του ενδοκυτταρικού Ca ++ ) οι οποίες αυξάνουν το ποσοστό κερματισμού του DNA. Αιτία της ενεργοποίησης των ενζύμων αυτών, αποτελεί η αύξηση των επιπέδων ενδογενών ενώσεων, όπως τα υπεροξείδια, οι ελεύθερες ρίζες και το μονοξείδιο του αζώτου. Το στρες των εντατικά εκτρεφόμενων ιχθύων είναι γεγονός υψίστης σημασίας στις υδατοεκτροφές και η ποσοτικοποίηση του αποτελεί αντικείμενο συζήτησης στη διεθνή βιβλιογραφία. 203
INTRODUCTION Animal welfare is by no means a reason for scientific debate, engaging aquaculture experts worldwide. The two major issues nowadays are the definition of animal welfare and its unbiased assessment. There is no direct approach of measuring welfare, nevertheless a wide range of physiological, biochemical and behavioral parameters are used for its evaluation (Ashley, 2007). Stress in rearing conditions is definitely an indicator of poor welfare. Among others, a technique to determine genotoxic effects induced by stressors is the comet assay (Figure 1). Comet assay or single cell gel electrophoresis (SCGE) is a rapid, robust and inexpensive method for detecting DNA strand breaks. The DNA damage can be a reflection, not only of direct strand breakage, but also of alkali-labile sites and of repair enzyme-mediated breakage. METHODS AND MATERIALS Cell extraction The liver is extracted from fish and placed in cold HBSS balanced solution (Mg ++, Ca ++ free) on ice. The tissue is injected with collagenase (CLS Type I) 0.04% for digestion. Afterwards the sample is incubated for 15 min and then posed in petri plate Figure 1 Flow diagram of the comet assay and sliced in small pieces. Both sample and solution, are transferred in a beaker and stirred for 30 min. The cell suspension is filtered through a sterilized bandage in a centrifuge tube, followed by centrifugation at 2000 rpm and the remaining pellet is resuspended in 10 ml PBS (phosphate buffer saline). Centrifugation and resuspension are 204
repeated for 2 additional times. All manipulations are carried out on ice, unless otherwise mentioned (Baksie and Fazier, 1990: Devaux et al., 1997: Μitchelmore and Chipman, 1998). Microscope slides preparation Microscope slides pre-treated with 40 μl of 0.5% NMP agarose prepared in phosphate-buffered saline (PBS) evenly spread are air-dried and cell suspension (20μl) mixed with 80 μl of low melting point agarose in PBS kept at 37 C is pipetted over the slides. Slides are covered and left on ice (Tice et al., 2000). Cell lysis and electrophoresis Slides with the stabilized cells are then rinsed in lyse solution (NaCl 2.5M, EDTA 100mM, Tris-Base 10mM, 100X Triton 1%, DMSO 10%v/v, ph 8) for 1 hour at 4 o C. All operations are conducted under dim light. The slides are placed in a horizontal gel electrophoresis tank containing freshly prepared cold electrophoresis buffer (0.075 mm EDTA and 300 mm NaOH, ph>12). The nuclei are incubated 20 min to facilitate DNA unwinding prior to electrophoresis at 25 V 300 ma for 15 min at 4 C. Electrophoresed slides are then soused in neutral buffer (TrisBase 0.4M, ph 7.5) for 15min in order to remove any remaining EDTA. The slides are stained with 50 μl ethidium bromide (20 μg ml 1 ) for 5 min, dipped in ice-cold water to remove the excess of ethidium bromide and covered with a cover slip (McKelvey-Martin et al., 1993). DNA damage quantification For each slide, 100 randomly chosen nuclei are analyzed using a fluorescence microscope with an excitation filter 515 560 nm and a barrier filter of 590 nm. A computerized image analysis system (CASP, open source) is employed (Figure 2). The tail moment (TM) is commonly used as the measure of DNA damage. A minimum of 3 biological replicates is taken for each treatment and from each replicate two SCGE slides are prepared. In total, 600 nuclei are analyzed per treatment. This technique permits quantification of DNA damage by evaluating the Tail Moment (TM) parameter of the comet assay. TM considers both tail length and fraction of DNA in the comet tail (Singh et al. 1988), is defined as DNA product in the tail, and is calculated according to the following formula: TM = (tail intensity/total comet intensity)x(tail center of gravity-head center), Where the percentage of DNA migrated into the tail (i.e., tail intensity/total comet intensity) is multiplied by the mean distance of migration in the tail (i.e., the distance between the tail center of gravity, which is the sum of tail positions divided by the number of points and the head center). 205
APPLICATIONS Figure 2 Image analysis using CASP This technique is applicable in various cell types of vertebrate and invertebrate aquatic species, following in vitro and in vivo exposures under laboratory conditions. It has been demonstrated that various isolated cells from aquatic species respond to a range of direct-acting (not requiring metabolic activation) and indirect acting agents. The fact that DNA strand breakage has also been caused by compounds that require metabolic activation, in various cells in vitro also reflects the metabolic activation capacity of the cell types employed. DNA strand breakage has also been seen in cells following various laboratory exposures of fish in vivo (Mitchelmore and Chipman, 1998b). The fact that in vivo exposures have led to an increase in strand breakage (as detected by a range of techniques) is important for the use of this parameter as part of a biomonitoring strategy. Clearly, sufficient concentrations of chemical (or in many cases reactive metabolite), even from single doses or from spiked sediment exposures, can reach the cells under study. The DNA damage produced from a range of compounds can be manifested as strand breakage, and is sufficiently persistent to be retained during cell isolation. The biochemical and physical factors that can influence genotoxicity in different individuals have been discussed by Zaleski et al (1991). In particular, metabolizing enzymes that are involved in both metabolic activation and detoxification are known to vary dramatically in response to environmental conditions in fish. Important influences are temperature, stress, diet, reproductive activity and the presence of inhibitors and inducing agents. 206
In conclusion the comet assay is a sensitive, rapid and economic technique for the detection of strand breakage, which is ideally suited as a non-specific biomarker of genotoxicity in fish and other aquatic species. It has been used successfully to reflect a range of in vivo exposures in a range of species. However, there is now a need for more comprehensive field studies supported by adequate information on chemical exposures. There is also a potential to exploit the assay for more detailed information on cellspecific effects, inter-individual variability, and on the persistence of lesions that can be expressed as strand breakage under alkali conditions. Finally there are many studies demonstrating an association between DNA strand damage and various biological genotoxic factors present in the field. Comet assay is a pivotal method in order to measure DNA damage as a contributor to irreversible toxicity, to detect characteristic rates of mutation and to estimate repair enzyme-mediated breakage. Further more it is a rapid and sensitive tool, to evaluate genotoxic effects related with stressful conditions. REFERENCES Ashley P.J., (2007). Fish welfare: Current issues in aquaculture. Applied Animal Behavior Science, 104:199 235 Baksi S.M. and J.M. Frazier, (1990). Isolated hepatocytes model systems for toxicology research. Aquatic Toxicology, 16:229 259. Devaux A., M. Pesonens, and G. Monod, (1997). Alkaline comet assay in rainbow trout hepatocytes. Toxycology in Vitro, 11:71-79. Lee R.F. and S. Steinert, (2003). Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutation Research, 544:43-64 McKelvey-Martin V.J., M.H.L. Green, P. Schmezer, B.L. Pool-Zobel, M.P. De Meo and A. Collins, (1993). The single cell gel electrophoresis assay (comet assay): A European review. Mutation Research, 288:47-63. Mitchelmore C.L. and J.K. Chipman, (1998a). DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutation Research, 399:135 147 Mitchelmore C.L. and J.K. Chipman, (1998b). Detection of DNA strand breaks in brown trout (Salmo trutta) hepatocytes and blood cells using the single cell gel electrophoresis (comet) assay. Aquatic Toxicology, 41: 161-182. Singh N.P., M.T. McCoy, R.R. Tice, and E.L. Schneider, (1988). A simple technique for quantification of low levels of DNA damage in individual cells. Experimental Cell Research, 175:184 191 Speit G. and A. Hartmann, (1995). The contribution of excision repair to the DNA-effects seen in the alkaline single cell gel test (comet assay). Mutagenesis, 10:555 559 Zaleski J., R.A. Steward, and H.C. Sikka, (1991). Metabolism of benzo[a]pyrene and ( )-transbenzo[a]pyrene-7,8-dihydrodiol by freshly isolated hepatocytes from mirror carp. Carcinogenesis, 12: 167 174. 207