In Comet Assay for Comparing with Various Mutagenicity Tests

[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 concentration and a high-sensitivity evaluation of Fig.1 DNA damage evaluation indices with Comet assay difference in DNA damage among individual Tail length(L),overall length of comet;Ratio, cells.On the other hand,the data evaluated ratio of damaged part to entile comet by ratio was obtained the smallest C.V.,and {luminance in[〓]area / total luminance(in it can be presumed that evaluation with [〓]part+[■] part)×100};Tail moment, largeness of damage{D ×ratio};Nuclear minimum difference among individual cells is diameter (R),diameter of nuclear;DNA achievable,unlike tail moment,tail length migration (L-R),degree of migrated DNA; and DNA migration.The results indicate that Shape factor(LJR),ratio of overall length to tail moment and ratio are effective indices for nuclear diameter;Tail intensity,area of the evaluation of DNA damage.We decided to damaged part[〓] 66 Japanese J.Wat.Treat.Biol.Vol.40 No.2

Table 1 Data of four indices at 5 μM MNNG treatment with E.gracilis cells(Mid- logarithmic phase)

Table 2 Dose-dependent increase of average at MNNG treatment in comet assay

Significance:*,p<0.05;**,p<0.01;***,p<0.001

use tail moment,which has been generally in the late-logarithmic phase is more uniform used in most cases and has been regarded than that in mid-logarithmic phase. more adequate to precisely describe a recorded DNA damage in some papers using Normalization of data for evaluation of tail moment12,13). DNA damage The comet assay results Besides,the dispersion caused by nucleus were statistically evaluated DNA damage to diameter differences should be suppressed at compare with control in each experiment. the control.Nuclear diameter was compared But,it is difficult to precisely compare between cells in two different logarithmic between different experiment results.

phases,i.e.,cells in mid-logarithmic phase Normalization of tail moment was attempted and those in late-logarithmic phase.This to facilitate comparison of the data obtained difference was used to suppress differences from each experiment.Then,the following among individual cells(Table 3).Between the formula based on the numeric values of the two kinds of cells,there was a difference of control was introduced. about 4μm in the maximum and minimum T=(Xi-μcontrol)/σcontrol(i=1,2,3,..., n) values,but there was not a difference in the where,Xi:measured value in each cell

average value.Also,the standard deviation control:average value in the control (S.D.)was 3.4μ m in mid-logarithmic phase σcontrol:standard deviation in the control and 2.3μm in late-logarithmic phase.lt was As a result,the average value and standard confirmed that cells in the late-logarithmic deviation in the control can be normalized to

phase display little variation,and variation 0 and 1,respectively.Therefore,the data among individual cells is suppressed in the from control cells show equivalent late-logarithmic phase. Furthermore,cell size distribution.We have previously evaluated DNA damage in MNNG,BAP(+S9,-S9), MMC and AMD, significant difference(p< Table 3 Comparison of nuclear diameter at different 0.001) was showed in each treatment growth phase with E.graciliscells concentration of MNNG and BAP3). But, when comparison of each data(ex.5,μM MNNG versus 5μ M BAP)was attempted,it was impossible to precisely compare by the difference between each control.However,in Fig.2,since the control in each experiment had the same average value(control=1)and standard deviation (control=0),the com- Normalization of DNA Damage Indices in Comet Assay 67

C oncentration(μM) Concentration(μM)

Concentration(μM) Concentration(μM)

Fig.2 Normalizationof tailmoment for evaluationof DNA damage induced by four chemicals(MNNG,BAP,MMC and AMD)with comet assay Significance=***,p<0.001

parison of data obtained under different Dichloromethane is highly volatile and might conditions was easier than that without data be vaporized.Chloroform is metabolized to conversion. In addition,each value can be generate primarily phosgene,which induces e xpressed without dimension.Normalization cross-linking of DNA15).It is also known that with reference to the control alleviated the formaldehyde causes the cross-linking of influence of individual cell difference and DNA16).Therefore,unwinding was disturbed simplified mutual comparison of experi- and fragmentation of DNA was suppressed, mental results.The normalization of DNA which is why DNA damage was not detected. damage evaluation indices proposed in this Next,using the seven chemicals measured study would be an effective and useful for so far,comet assay was compared with Ames extensive application of the comet assay. test,umu test and Rec assay for detection sensitivity.In comet assay,a concentration Comparison of sensitivityamong different showing a statistically significant difference mutagenicity tests In addition to the from the control was defined as positive.The tested chemicals(MNNG,BAP,MMC and results of comparison between comet assay AMD),the chemicals detected in en- and Ames test,umu test and Rec assay are vironmental water i.e.dichloromethane, shown in Table 4.The experimental results chloroform and formaldehyde,were evaluated using the plate method and pre-incubation (Fig.3).No significant difference in DNA method in Ames test were adopted,and damage was observed. Dichloromethane is TA98,TA100 and TA102 were selected as metabolized by glutathione-S-transferase object strainss6,16-18).Since treatment concen- (GST)causing a DNA single strand break14). tration in Ames test was indicated in weight However,because GST exists in S100 of mutagen in a plate,concentration of fraction,rather than S9 fraction,the comet mutagen(μM)was calculated as dose of assay failed to detect DNA damage. mutagen in 2.7 ml mixture. Formaldehyde 68 Japanese J.Wat.Treat.Biol.Vol.40 No.2

Concentration(μM) Concentration(μM)

Concentration(μM)

Fig.3 Evaluationof DNA damage induced by three chemicals(dichloromethane, chloroform and formaldehyde)with comet assay

Table 4 Comparison of sensitivitycomet assay with Ames test,umu testand Rec assay

-,Negative

and MMC,which were negative in comet mutagenicity has been confirmed.In the assay,were positive in Ames test.The plate method and pre-incubation method, detection concentrations are 620,μM at since dichloromethane is vaporized,the formaldehyde16)and 0.55,μM at MMC17).This results vary from one test to another.The difference signifies that the two chemicals act concentration of MNNG and BAP,which as cross-linking agents to suppress the were positive in both tests,were compared. unwinding of DNA.The reason that MNNG was 5.0μMin comet assay and 25 chloroform is negative in Ames test is μ Min Ames test18).BAP was 5.0μMin comet

presumably because phosgene is highly assay and 3.7μ M in Ames tests6).Therefore, reactive and most of it reacts with protein it can be concluded that comet assay is more and lipid in S9 before reacting with DNA. sensitive to MNNG than Ames test,but the Dichloromethane is also highly volatile, two tests are the same for BAP.The data therefore,the vapor method is usually from umu test in Table 3 are from a report by adopted for this substance,and its Nakamura et al.19).The detection concen- Normalization of DNA Damage Indices in Comet Assay 69

trations were 630μM at formaldehyde19),4.1 obtained results similar to those obtained by μ M at MNNG19),4.0μM atBAP19)and0.15 other methods.In addition,it was showed μ M at MMC19).As in the comparison with that comet assay is more sensitive than Ames Ames test,differences appear for form- test and Rec assay and has the same aldehyde and MMC,but the results are the detection sensitivityas umu test.In sum,tail same for the other chemicals.MNNG was 4.1 moment was the best DNA damage μ M and BAP has 4.0μM in umu test, evaluation index in comet assay,and confirming that comet assay has the same normalized tail moment was useful for detection sensitivity as umu test.The data of comparing data from different experiments. Rec assay in Table 3 are from a report of The detection characteristicand sensitivityof Matsui et al.20).The liquid method is used the the comet assay were clarified through CR50 value of Rec+ as a reference comparison with mutagenicity tests using concentration.It displays the capacity to various chemicals.In addition,it is expected evaluate an extremely broad range of that the normalized comet assay is applicable mutagens.However, MMC,formaldehyde to eco-toxicological evaluation of water and chloroform show positive in the liquid environment such as the landfillleachate and method,which uses the S-probit value as an the rubbish disposal plant. index.Detection concentrations were 0.073 μ M for MMC19),320μM for formaldehyde20 REFERENCES ) and 16000 μM for chloroform20).Con- centration for MNNG and BAP,which were 1) Singh N.P., McCoy M.T., Tice R.R., and

positive in both tests,were 1200μ M20)and Schneider E.L.: A simple technique for 2800 μ M20),respectively.This index was quantitation of low levels of DNA intended not for DNA damage alone but for a damage in individual cells, Exp. Cell. total evaluation of DNA damage and Res., 175, 184-191 (1988) cytotoxicity for Bacillus subtilis.Rec assay is 2) Iwahori K., Miyata N., Umeda Y., Aoyama useful for detection of mutagenicity in a wide K., Shimoi K., and Kinae N.: Applicability range,but the mutagenicity of a chemical of environmental microorganisms to that is positive in this test might not be fully alkaline single cell gel electrophoresis represented21). (comet) assay, Jpn. J. Water Treat. Biol., 35, 261-270 (1999) (in Japanese) 3) Aoyama K., Iwahori K., and Miyata N.: CONCLUSIONS Application of Euglena gracilis cells to We examined the characteristics and comet assay: evaluation of DNA damage usefulness of DNA damage evaluation indices and repair, Mutat. Res., 538, 155-162 in comet assay, and compared its detection (2003) sensitivityof this assay with that of other 4) McCarthy P.J.,Sweetmann S.F., McKenn mutagenicity tests.Of the evaluation indices, P.G., and McKelvey-Martin V.J.: Evalua- tail moment provided a high-sensitivity tion of manual and image analysis evaluation of difference among individual quanti-fication of DNA damage in the cells,and its normalization based on the alkaline comet assay, Mutagenesis, 12, control alleviated the influence of the above 209-214 (1997) differences and simplified comparison of 5) Ashby J., Tinwell H., and Lefevre P.A.: experimental results.Based on those results, The single cell gel electrophoresisassay it was indicated that use of the normalized for induced DNA damage (comet assay): tail moment is the most effectiveof the DNA measurement of tail length and moment, damage evaluation indices used in comet Mutagenesis, 10, 85-90 (1995) assay.When comet assay was compared to 6) Ames B.N., Mccann J.,and EYamasaki E.: Ames test,umu test and Rec assay, we found Methods for detecting carcinogens and that comet assay does not have the capacity mutagens with the salmonella/ to detect DNA damage caused by cross- mammalian-microsome mutagenicity linking agents,but for other chemicals it test,Mutat. Res., 31, 347-363 (1975) 70 Japanese J. Wat. Treat. Biol. Vol.40 No.2

7) Oda Y., Nakamura S., Oki I., Kato T., and salmonella,Environ. Mol. Mutagen., 30, Shinagawa H.: Evaluation of the new 440-447 (1997)

system (umu-test) for the detection of 15) Rosenthal S.L.: A review of the environmental mutagens and carcin mutagenicity of chloroform, Environ. ogens, Mutat. Res., 147, 219-229 (1985) Mol. Mutagen., 10,211-226 (1987) 8) Kada T., Tutikawa K., and Sadaie Y.: In 16) O'Donovan M.R. and Mee C.D.: Form- vitro and host-mediated“rec-assay”proce aldehyde is a bacterialmutagen in a - dures for screening chemical mutagens; range of Salmonella and Escherichia and phloxine, a mutagenic red dye indicatorstrains, Mutagenesis, 8, 577- detected, Mutat. Res., 16, 165-174 (1972) 581 (1993) 9) Vahl H.H.,Kaebe L., and Westendorf J.: 17) Maron D.M. and Ames B.N.: Revised Genotoxicity assessment of suspended methods for the Salmonella mutagenicity particulate matter in the Elbe river: test,Mutat. Res.,113, 173-215 (1983) comparison of Salmonella microsome 18) Diehl M. and Fort F.: Spiral Salmonella test, arabinose resistance test, and umu assay: Validation against the standard -test, Mutat. Res., 394, 81-93 (1997) pour-plate assay, Environ. Mol. Muta 10) Hutner S.H., Zahalsky A.C., Aaronson S., gen.,27, 227-236 (1996) Baker H., and Frank O.: Culture media 19) Nakamura S.,Oda Y., Shimada T., Oki I., for Euglena gracilis, in: D.M. Prescott and Sugimoto K.: SOS-inducing activityof (Ed.), Methods in Cell Physiology, vol.2, chemical carcinogens and mutagens in Academic Press, New York, 217-228 Salmonella typhimurium TA1535/pSK (1996) 1002: examination with 151 chemicals, 11) Tennant J.R.: Evaluation of the trypan Mutat. Res.,192, 239-246 (1987) blue technique for determination of cell 20) Matsui S.,Yamamoto R., and Yamada H.: viability, Transplantation, 2, 685-694 The Bacillus subtilis/microsomerec

(1961) -assay for the detectionof DNA damaging 12) Helbig R. and Speit G.:DNA effects in sub-stances which may occur in repair-deficient V79 Chinese hamster chlorinatedand ozonated waters,Water cells studied with the comet assay, Sci.Technol., 21, 875-887 (1989) Mutat. Res., 377, 279-286 (1997) 21) Matsui S., Tokugawa M., and Koide Y.: 13) Hartmann A.and Speit G.: The Evaluation of results of the Bacillus contribution of cytotoxicity of DNA- subtilisrec-assay liquid-cultivation meth effects in the single cell gel test(comet od by introductionof Probit theory and

assay), Toxicol. Lett., 90, 183-188 (1997) Target and Hit theory, Jpn. J. Water 14) DeMarini D.M., Shelton M.L., Warren Pollut. Res., 35, 172-180 (1985) (in S.H., Ross T.M., Shim J.Y., Richard A.M., Japanese) and Pegram R.A.: Glutathione S (Submitted 2004.3.16) - transferase-mediated induction of GC (Accepted2004.5.11) AT transitions by halomethanes in