63 [Japanese Journal of Water Treatment Biology Vol.40 No.2 63-70 2004] Normalization of DNA Damage Indices in Comet Assay for Comparing with Various Mutagenicity Tests KEISUKE IWAHORI1*, NAOYUKI MIYATA1, YASUHIRO YAMAMOTO2, DAIJIRO SONE2, and KOTARO AOYAMA3 1Institute for Environmental Sciences ,University of Shizuoka 2Graduate School of Nutritional and Environmental Sciences , University of Shizuoka /52-1, Yada, Shizuoka 422-8526, Japan 3Hitachi Plant Engineering and Construction Co .,Ltd. /537, Kamihongo, Matsudo, Chiba 271-0064, Japan Abstract The comet assay has been widely used in genetic toxicology, environmental biomonitoring and clinicalinvestigations. However, many indicesare typicallyused to evaluate degree of DNA damage in comet assay and various bioassays have also been employed for the mutagenicity test of environmental waters. In this study, the DNA damage evaluation characteristicsof each index were examined and the numerical data obtained were normalized on the basis of untreated controls to compare with data obtained under different experiment conditions. For confirmation of detection characteristicsand sensitivity,comet assay were compared with Ames test,umu test and Rec assay. Among the comet assay indices,tail moment most effectivelyrepresented the sensitivitiesof individual cells and dose-dependent DNA damage was detected. By normalization of tailmoment, comparison of experimental resultsin differentconditions was simplified.The comet assay did not have the capacity to detect DNA cross-linking agent. The comparison of data revealed that mutagenicity detectionsensitivity for comet assay is greater than Ames testand Rec assay and as high as that of umu test. Key words: comet assay; tail moment, normalization, mutagenicity test other mutagens3).Given the ease of culturing INTRODUCTION and handling E.gracilis cells as well as its The alkaline single cell gel electrophoresis sensitivity,comet assay using E. graciliscells assay (comet assay),established by Singh et is undoubtedly useful tool for testing the al.1)isa sensitive,simple and rapid technique genotoxicity of chemicals and for assessing of and widely used for detecting DNA damage genotoxic potential in natural environments. in cellsexposed in vitroor in vivo to a variety We have used tail length and tail moment, of physical and chemical agents.Since it is the most popular indices for evaluating possible to subcultivate and evaluate chronic degree of DNA damage.However,a number influence of mutagens,we examined of indices have been used to evaluate degree conditions for using unicellular green alga of DNA damage in comet assay and the Euglena gracilis as environmental micro- relations between various indices and their organism in comet assay2)and verified that dominance have been investigated.McCarthy this technique can be used to evaluate DNA et al.4)evaluated DNA damage using head damage and repair caused by radiation and diameter,comet length,comet length/head *Corresponding Author 64Japanese J.Wat.Treat.Biol.Vol.40 No.2 diameter,area,%tail DNA,tail length,head Cells were grown at 25℃ under 12h extent/2,tail moment and tail length/head dark/light conditions (0.9 lx) in Hutner radius(IJH)in the image analysis comet medium10).It contained(per liter)20 mg of system,and stated that%tail DNA,tail KH2PO4,25 mg of MgSO4・7H2O,400 mg of moment and L/H are good parameters for sodium acetate,40 mg of potassium citrate, displaying the expected trends and are in 600mg of polypeptone,400 mg of yeast agreement with the results obtained from extract,0.5μg of vitamin B12 and 0.4 mg of manual analysis.Ashby et al.5)used tail thiamine HCl(pH 6.4).Cells were harvested length,%DNA tail and tail moment,and by centrifugation(9000 × g,5min)and reported that they all correlated well.In spite washed once with phosphate-buffered saline of its wide applications and increasing (PBS).Cells were grown to mid-logarithmic popularity,no standard indices have been phase(3days) and late-logarithmic phase(6 developed for detecting and comparing with days). the response of cells to different agents. Besides,in vitro tests such as comet assay, Cell treatment E.gracilis was re- Ames test,umu test and Rec assay,are suspended in PBS at 4×105 cells/ml and widely used for measuring the potential incubated with chemicals. The chemicals(1- mutagenicity of water environment.The methyl-3-nitro-1-nitrosoguanidine(MNNG), Ames test is the mutagenicity test that benzo(a)pyrene(BAP,98%pure), mitomycin counts the colonies formed by mutants of C(MMC),actinomycin D(AMD),formal- Salmonella typhimurium selected for dehyde,dichloromethane and chloroform sensitivity and specificity for reversion from a were obtained from Wako Pure Chemical histidine requirement back to prototrophy by Industries(Osaka, Japan).These chemicals a variety of mutagenss6).The umu test were added to the suspension of E. gracilis eValUateS mUtageniCity by meaSUring β- cells to produce a final concentration of 100 galactosidase activity controlled by the umu μ Mor less.A cofactor-supplemented S9 from regulatory region7).The Rec assay examines induced rat liver(S-9/cofactor A set;Oriental the increased lethality of radiation of certain Yeast Co.,Osaka,Japan)was coinjected to chemicals on non-recombining(Rec-)versus metabolically activated BAP,dichloro- recombining(Rec+)bacteria8).However,many methane and chloroform.The cell treatment papers evaluated mutagenicity by only one was conducted under dark conditions to avoid method and few papers have described the activation of the compounds by light,and the data as compared with detection charac- treatment time was 2 to 3 hours.Cells teristics and sensitivity9). viability during this treatment time was Since the results of comet assay are measured by the dye-exclusion test11)and a influenced by the dispersion of control values value over 90%was confirmed. in each experiment unlike mutagenicity test, normalization of DNA damage indices has Comet assay Comet assay with E. been investigated on the basis of the control gracilis was conducted according to the in this study.Detection characteristics and method of Iwahori et al.2). Treated cells were sensitivity of typical mutagenicity tests collected and embedded in a layer of 0.5%low- (Ames test, umu test and Rec assay)have melting agarose sandwiched between a been compared with the normalized comet bottom and top layer of 0.5%normal-melting assay,thereby verifying its availability. agarose on a fully frosted microscopic slide. The slides were placed in a lysis solution containing 300 mM NaOH,30 mM Na2EDTA MATERIALS AND METHODS and 0.01%SDS for 5 min and then in an electrophoresis buffer(300 mM NaOH plus Organism E.gracilis strain NIES-49 1 mM Na2EDTA)for 20 min at 4℃to allow was obtained from the Microbial Culture unwinding of DNA.Electrophoresis was Collection,National Institute for conducted with the same buffer at 4℃ for 20 Environmental Studies (Tsukuba,Japan). min at 25 V and 300 mA.After the gel was Normalization of DNA Damage Indices in Comet Assay 65 neutralized by immersing it twice in 400 mM GraphPad Prism software (GraphPad Tris-HC1(pH 7.5)for 5 min,the DNA Software Inc.,San Diego,CA).When molecules were stained with ethidium significant differences (p <0.05) appeared, bromide. Donnett's multiple comparison test was used The migrated DNA(comet)images were to isolate the group(s)apart from the control observed by epi-fluorescence microscopy group. using Olympus BX60 equipped with a CCD camera,and DNA damage was evaluated RESULTS AND DISCUSSION with an automatic image analyzing system (Keio Electronics Co.,Osaka,Japan).The evaluation indices used in this study are Comparison of DNA damage evaluation shown in Fig.1.Data were obtained from indices The indices we used for about 30 comets per sample and represented comparison in this study are tail moment, by box and whiskers plots as follows: tail length,ratio, DNA migration,nuclear measured values for a tested compound were diameter,shape factor and tail intensity, shown by a box that including 50%of data. which could be measured and evaluated by The top and bottom of the box marked the automatic analysis software.The results for 25th and 75th percentiles, respectively,and DNA damage induced by MNNG treatment the inner line marked the median value;the using mid-logarithmic phase E.gracilis cells whiskers marked the 10th and 90th are shown in Table l and Table 2. Tail percentile value.Average values were moment appeared to dose-dependently represented by closed squares.To determine increase at MNNG and a significant signsficantdifferences among the treatment difference was detected at more than 5μM. groups and control(not treated)group,data Based on the calculations of the tail length were assessed by one-way ANOVA using and DNA migration (Fig.1),the results o btained for the two indices ,were equivalent and a significant difference from the control was detected at more than 1μM.However, since tail length and DNA migration evaluated only length of nuclear diameter and tail,the DNA-chemical interaction is not adequately represented.When the DNA damage was evaluated using the ratio, a significant difference was detected at more than 0.5μM appeared to dose-dependently increase at MNNG.With other indices, a significant difference was detected only at high concentrations,or no concentration- dependent DNA damage was detected(data not shown).Among the four indices,tail moment showed the largest coefficient of Centroid of nuclear Centroid of tail variation(C.V.)at each treatment concen-