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1 CANCER PREVENTION RESEARCH ORIGINAL ARTICLE 유전독성시험법 in vivo Comet Assay 에대한국내시험기관간검증연구 1 동국대학교바이오시스템대학생명과학과및 Green Chemistry 환경의학연구소, 2 경희대학교의과대학기초의과학교실, 3 안전성평가연구소유전독성실, 4 서울대학교병원의생명연구원, 5 메드빌중앙연구소 GLP 센터, ( 주 ) 메드빌, 6 동국대학교일산병원가정의학과, 7 식품의약품안전청독성평가연구부 권지영 1,2 * ㆍ김지영 3 * ㆍ정문구 3 ㆍ김윤순 4 ㆍ제정환 4 ㆍ오상석 5 ㆍ홍은경 5 오상우 6 ㆍ김주환 7 ㆍ염영나 7 ㆍ손수정 7 ㆍ서영록 1,2 Inter-Laboratory Validation of in vivo Comet Assay in Korea Jee Young Kwon 1,2 *, Ji-Young Kim 3 *, Moon-Koo Chung 3, Yoon Sun Kim 4, Jeong-Hwan Che 4, Sang Suk Oh 5, Eun-Kyung Hong 5, Sang Woo Oh 6, Joo-Hwan Kim 7, Young-Na Yum 7, Soojung Sohn 7 and Young Rok Seo 1,2 1 Department of Life Science, Institute of Environmental Medicine for Green Chemistry, Dongguk University, Seoul , 2 Department of Biomedical Science, School of Medicine, Kyung Hee University, Seoul , 3 Laboratory of Genetic Toxicology, Korea Institute of Toxicology, KRICT, Daejeon , 4 Clinical Research Institute, Seoul National University Hospital (SNUH), Seoul , 5 Medvill Central Institute, GLP Center, Medvill Co., Ltd., Seoul , 6 Department of Family Medicine, Dongguk University International Hospital, Goyang , 7 Toxicology Evaluation and Research Department, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration, Chungcheongbuk-do , Korea The comet assay can be used to investigate the genotoxicity of various agents including industrial chemicals, biocides, agrochemicals and pharmaceuticals. Nowadays, international expert groups for comet assay have published recommendations describing standards which are aimed at establishing high quality protocols in order to obtain valid and reliable data. Despite that the international activity for validation of in vivo comet assay has been carried out, the distribution of protocol and interlaboratory validation has not yet conducted in Republic of Korea. Here, three laboratories participated in interlaboratory assessment for the test validity of the in vivo comet assay under the GLP (Good Laboratory Practice) system. Two genotoxic chemicals, N-methyl-N-nitrosourea (MNU) and ethyl methansulfonate (EMS),were applied as known and unknown chemicals, respectively, which were selected on the basis of the 3rd international validation study result. Among all GLP laboratories, the results showed a significant increase in DNA damage as % of tail DNA as dose-related manner in both liver and stomach tissues treated with MNU relative to negative control group. Similarly, EMS-treated group also showed significant incremental % of tail DNA as dose-dependent manner compared with negative control group in both tissues. The in vivo comet results from all GLP laboratories in Republic of Korea were obviously consistent with the 3rd international validation study. In conclusion, we confirmed the interlaboratory validity of in vivo comet assay using two genotoxic chemicals, which was assessed by three GLP laboratories in Republic 책임저자 : 서영록, , 서울시중구필동로 1 길 30 동국대학교바이오시스템대학생명과학과및 Green Chemistry 환경의학연구소 Tel: , Fax: seoyr@dongguk.edu 접수일 :2011 년 11 월 1 일, 1 차수정일 :2011 년 11 월 7 일, 2 차수정일 :2011 년 11 월 11 일, 게재승인일 :2011 년 11 월 16 일 * 저자들은본연구에동일하게기여하였음. Correspondence to:young Rok Seo Department of Life Science, Institute of Environmental Medicine for Green Chemistry, Dongguk University, 30, Pil-dong-ro 1-gil, Jung-gu, Seoul , Korea Tel: , Fax: seoyr@dongguk.edu 294

2 권지영외 11 인 : 유전독성시험법 in vivo Comet Assay 에대한국내시험기관간검증연구 295 of Korea. This study can strengthen in vivo comet assay to be suggested as an international standard protocol or guideline for genotoxicity test of various genotoxic agents. (Cancer Prev Res 16, , 2011) Key Words: In vivo comet assay, Interlaboratory validation, Good laboratory practice (GLP) system 서 Comet assay는단일세포젤전기영동 (SCGE, single cell gel electrophoresis) 으로불리며개별세포들의 DNA 손상정도를분석하고정량화하기위한섬세하고빠른기법으로알려져있다. 이러한이유로암연구분야에서화학예방요법 (chemoprevention) 유효성과유전독성 (genotoxicity) 평가를위해사용되고있는시험기법중의하나이다. 구체적인방법은 agarose gel 안의 DNA fragments가전기영동에의해이동하는정도를형광현미경으로관찰하여유전자손상정도를평가하는방식으로세포의핵부분을 head라하며 DNA fragments 또는 strands가이동하여끌림현상을보이는부분을 tail이라한다. 1) Comet assay는 1984 년 Őstling과 Johanson에의해처음개발되었으며초기에는 neutral condition에서전기영동을수행함으로서 double strand break를감지할수있는방법으로고안되었다. 2) 이후 Singh에의하여 ph 13 이상조건의 alkaline condition에서전기영동을하는시험법이개발되었으며 3) Olive에의해 ph 12.3 이상조건에서의실험법도제시되었다. 4) Neutral condition에서의시험법은 double strand break (DSB) 만을감지할수있는반면, alkaline condition에서의시험법은대부분의유전독성물질이야기할수있는 single strand break (SSB) 및 SSB 관련 incomplete excision repair sites, ALS (alkali-labile sites) 와같은유전자손상을감지할수있기에독성물질의유전독성을평가하는데널리사용되기시작하였다. 5) 특히, in vivo comet assay는다른 in vivo indicator test인 unscheduled DNA synthesis (UDS) test 및 alkaline elution test 와비교시많은장점을가지고있다. In vivo UDS test는간조직에서만수행할수있는반면 comet assay는다양한조직에서유전자손상평가가가능하며 SSB에서부터산화적염기손상에이르는넓은범위의유전자부위를감지할수있다. 6) 또다른시험법인 alkaline elution test는많은수의세포가필요한반면, comet assay는적은수의세포만으로도실험이가능하다는장점을가지고있기에 in vivo UDS test 및 alkaline elution test를대체할수있는시험법으로주목받고있다. 7) 론 In vivo comet assay를통해얻은결과는시험물질의유전독성유무및용량반응평가뿐아니라, 유전독성의분자적메카니즘의이해를돕는데도움을줄수있으며이러한 comet assay의장점은학술적인분야에서뿐아니라, 정책적인분야에서도그유용함을인정받아 7) 현재 IWGT (International Workgroup on Genotoxicity testing) 을중심으로시험법의검증연구가진행중에있다. 8) 하지만아직까지국내에는국제적검증연구에사용되고있는시험법의소개및보급이미흡한실정이며최신유전독성시험법의습득및검증연구가요구될것으로예상된다. 이에본연구에서는 in vivo comet assay의국내실험실간의검증연구를진행하고자하였다. 유전독성을일으키는것으로잘알려진물질들을사용하여국내 GLP 연구기관인안전성평가연구소, ( 주 ) 메드빌, 서울대학교병원의생명연구원에서국제적인검증연구에사용되는시험법에따라 in vivo comet assay를수행하여결과를얻었다. 본연구를통하여최신의유전독성기법을국내에보급및정착시키고자하였으며국내유전독성평가기반을강화하고자하였다. 재료및방법 1. 시험물질선정및시험물질농도설정 In vivo comet assay의실험실간검증연구를위해제3차 in vivo comet 국제검증연구 10) 에서양성판정이나온물질들중 N-methyl-N-nitrosourea (MNU) (Sigma, USA) 와 Ethyl methanesulfonate (EMS) (Sigma) 를시험물질로선정하였으며동일한시험물질들을각 GLP 연구기관으로배분하여검증연구에사용하였다 (Table 1). 음성대조군과양성대조군으로는각각 Saline (Green cross, Korea) 와 EMS (Sigma) 를사용하였다. MNU의시험물질농도는제3차 in vivo comet 국제검증연구에서사용된농도 (25, 50, 100 mg/kg/day, 2 회경구투여 ) 를사용하였으며, 신뢰성있는검증연구를위하여 EMS는맹검상태를취하여각실험실에서용량설정시험및 in vivo comet assay ( 시험물질 3회경구투여 ) 를수행하였다.

3 296 Cancer Prevention Research Vol. 16, No. 4, In vivo comet assay 1) 실험동물및시험물질처리 : 국제적검증연구에서사용된시험법 9) 에따라 in vivo comet assay를수행하였다. Rat:Crl:CD (SD) 6주령 (Orient Bio Inc., South Korea) 을입수한후, 7일간의검역및순화기간을거쳐군분리하였다. 각그룹당 5마리의시험동물을사용하였으며시험물질은 1일 1회투여하는방식으로 MNU는 2회 (0 hr, 21 hr), 시험물질 EMS는 3회 (0 hr, 24 hr, 45 hr) 경구투여하였다. 양성대조군은 EMS 200 mg/kg/day를사용하였으 Table 1. Tested chemicals for interlaboratory validation of in vivo comet assay Chemical Structure CAS No. N-methyl-N-nitrosourea (MNU) Ethyl methansulfonate (EMS) O H 3C N NH 2 NO O H 3C-S-O CH 3 O Table 3. (A) Clinical signs and mortalities of male rats in Medvill Co., Ltd Dose (B) Body weights of male rats in Medvill Co., Ltd Chemical treated Body weights (Mean±S.D., g) b at Dose the time of dosing (No. of animal) 1st 2nd Vehicle ±7.4 (5) 266.6±5.7 (5) Test chemical ±6.7 (5) 259.8±7.2 (5) (Low dose) Test chemical ±6.7 (5) 255.0±4.9 (5) (Mid. dose) Test chemical (High dose) ±6.7 (5) 247.6±4.4 (5) Positive control ±6.4 (5) 237.8±8.9 (5) Test chemical: N-methyl-N-nitrosourea (MNU), Vehicle: Saline, Positive control: Ethyl methanesulfonate (EMS). a Number of animals with clinical sign/total number of animals observed, b Bartlett test's and ANOVA test. Table 2. (A) Clinical signs and mortalities of male rats in KIT Dose (B) Body weights of male rats in KIT Chemical treated Dose Body weights (Mean±S.D., g) b at the time of dosing (No. of animal) 1st 2nd Vehicle ±6.23 (5) 270.9±7.23 (5) Test chemical ±5.03 (5) 242.0±5.34 (5) (Low dose) Test chemical ±5.16 (5) 256.9±6.46 (5) (Mid. dose) Test chemical (High dose) ±9.28 (5) 257.4±13.77 (5) Positive control ±5.80 (5) 260.5±6.22 (5) Test chemical: N-methyl-N-nitrosourea (MNU), Vehicle: Saline, Positive control: Ethyl methanesulfonate (EMS). a Number of animals with clinical sign/total number of animals observed, b Bartlett test's and ANOVA test. Table 4. (A) Clinical signs and mortalities of male rats in SNUH Dose (B) Body weights of male rats in SNUH Chemical treated Body weights (Mean±S.D., g) b at Dose the time of dosing (No. of animal) 1st 2nd Vehicle ±19.10 (5) 267.7±24.18 (5) Test chemical ±11.17 (5) 257.5±10.58 (5) (Low dose) Test chemical ±10.84 (5) 255.1±12.10 (5) (Mid. dose) Test chemical (High dose) ±6.82 (5) 255.6±6.69 (5) Positive control ±9.03 (5) 248.0±6.27 (5) Test chemical: N-methyl-N-nitrosourea (MNU), Vehicle: Saline, Positive control: Ethyl methanesulfonate (EMS) a Number of animals with clinical sign/total number of animals observed, b Bartlett test's and ANOVA test.

권지영외 11 인 : 유전독성시험법 in vivo Comet Assay 에대한국내시험기관간검증연구 297 며 2회 (0 hr, 21 hr) 경구투여하였다. 시험물질및양성대조군최종투여 3시간후, CO 2 gas를이용하여안락사하였다. 2) 단일세포분리 : 각동물로부터적출한간과위조직에서단일세포를분리하여 comet slide를제작하였다.

위조직의경우, 전위 (forestomach) 를제거하고 glandular stomach에서 scrapper로긁어위점막 (gastric mucosa) 을제거한후, 위상피세포 (stomach epithelia) 를조심스럽게긁어단일세포를방출시킨후, 얼음에 30초간 Fig. 1.

For each animal of treatment, % tail DNA of 100 randomly selected cells were analyzed using Komet 5.0 software.

05, two-sided and Linear trend at p<0.05, two-sided). Positive control also showed significantly increased DNA damage compared with the negative control in all laboratories (Student's t-test at p<0.

4 권지영외 11 인 : 유전독성시험법 in vivo Comet Assay 에대한국내시험기관간검증연구 297 며 2회 (0 hr, 21 hr) 경구투여하였다. 시험물질및양성대조군최종투여 3시간후, CO 2 gas를이용하여안락사하였다. 2) 단일세포분리 : 각동물로부터적출한간과위조직에서단일세포를분리하여 comet slide를제작하였다. 간조직의경우, 동물의복부대정맥과동맥을절단하여방혈을실시하고좌엽을적당한크기로자른후, 차가운 mincing buffer (20 mm EDTA와 10% DMSO를포함하는 HBSS) 에담궈잔혈을제거하고가위로세절한후, 얼음에 30초간방치하였다가상층액을취하여단일세포를얻었다. 위조직의경우, 전위 (forestomach) 를제거하고 glandular stomach에서 scrapper로긁어위점막 (gastric mucosa) 을제거한후, 위상피세포 (stomach epithelia) 를조심스럽게긁어단일세포를방출시킨후, 얼음에 30초간 Fig. 1. (A) N-methyl-N-nitrosourea (MNU)-induced DNA damage in rat liver tissue was measured by in vivo comet assay. For each animal of treatment, % tail DNA of 100 randomly selected cells were analyzed using Komet 5.0 software. Treatment groups showed significantly differences of % tail DNA as dose-dependent manner, relative to negative control group in all laboratories (Dunnett's test at p<0.05, two-sided and Linear trend at p<0.05, two-sided). Positive control also showed significantly increased DNA damage compared with the negative control in all laboratories (Student's t-test at p<0.025, one-sided). (B) Representative comet images of cells from liver tissue treated with increasing dose of MNU in comparison to the negative and the positive controls were taken by fluorescence microscope at 200X magnification. (C) DNA damage induced by MNU was measured using in vivo comet assay in rat stomach tissue. For measurement of DNA damage, total 100 randomly selected cells in each animal were scored by Komet 5.0 software. Treatment groups showed significantly differences of % tail DNA as dose-related manner, relative to negative control group in all laboratories (Dunnett's test at p<0.05, two-sided and Linear trend at p<0.05, two-sided). Positive control also showed significantly augmented DNA damage compared with the negative control in all laboratories (Student's t-test at p<0.025, one-sided). (D) Representative image comet images of cells from stomach tissue treated with increasing dose of MNU in comparison to the negative and the positive controls were taken with fluorescence microscope at 200 magnification.

5 298 Cancer Prevention Research Vol. 16, No. 4, 2011 방치하였다가상층액을취하여단일세포를얻었다. 3) Comet 슬라이드제작및세포용해 : 단일세포현탁액을 0.5% low-melting agarose (Invitrogen, USA) 와 1:9의비율로섞은후, comet 슬라이드 (Trevigen, USA) 의 hole에점적하고 30분간상온에서보관했다. 충분히굳은슬라이드는차가운 lysis buffer [2.5 M NaCl, 100 mm Na 2EDTA, 10 mm Tris-base, 10% DMSO, 1% Triton-X (ph 10)] 에담궈냉장상태로밤샘보관했다. 4) 전기영동, 중화, 탈수및염색 : 슬라이드를증류수로세척한후, 4 o C의 alkaline solution [0.3 M NaOH, 1 mm EDTA (ph>13)] 에슬라이드를담궈 20분간 unwinding을실시하고 0.7 V/cm로 30분간전기영동을실시했다. 전기영동후, neutralization solution [0.4 M Tris-base (ph 7.5)] 에 5분간, 100% ethanol에서 5분간방치하고실온에서충분히건조시켰다. SYBR Gold 용액 (Invitrogen) 으로 10분간염색하고수세, 건조하였다. 5) 검경 : 각동물로부터제작된검체는코드화한후, 200배배율형광현미경을이용하여검경을실시하였다. 각검체당 100개 (50개/hole) 의세포에대하여유전자손상정도를 Komet 5.0 프로그램 (Andor technology, UK) 을이용하여측정하였으며 % tail DNA (% DNA in tail) 를사용하여통계학적분석을실시하였다. 6) 통계처리 : 결과의판정은 % tail DNA (% DNA in tail) 에대해 Dunnett's test (two-sided, p<0.05) 와 Linear trend test (two-sided, p<0.05) 적용하여양성또는음성을판정하였으며양성대조군은 Student's t-test (one-sided, p<0.025) 적용시음성 ( 부형제 ) 대조군과비교하여통계학적으로유의성있는증가여부를판단하였다. 결과및고찰 1. N-methyl-N-nitrosourea (MNU) 을이용한 in vivo comet assay 결과국내 GLP 기관간의 in vivo comet assay 검증연구를위하여제3차 in vivo comet 국제검증연구에서시험물질로사용하였던 MNU를 10) 본연구에서사용하였으며결과의비교분석을위하여국제검증연구에서사용한농도 (25, 50, 100 mg/kg/day, 2회경구투여 ) 로 in vivo comet assay를수행하였다. 시험물질을투여한모든동물에서특이한증상이관찰되지않았으며투여군간의체중비교에서도 MNU를투여한모든군및양성대조물질투여군에서음성대조군에비해통계학적으로유의한변화가관찰되지않았다. 이는국내 GLP 3 기관에서모두동일한결과가나타났다 (Table 2 4). MNU를투여한동물의간조직에서의유전자손상정도를평가한결과, Fig. 1A, B와같이 GLP 3 기관에서모두양성대조군을포함한시험물질투여군에서음성대조군에비해 % tail DNA가통계학적으로유의성있게증가한것으로나타났다. MNU를투여한동물의위조직에서도음성대조군에비하여양성대조군을포함한시험물질투여군에서통계학적으로유의한차이를나타내며유전자손상이증가하였음을확인할수있었다 (Fig. 1C, D). 이러한연구결과는제3차 in Table 5. (A) Clinical signs and mortalities of male rats in KIT Dose Day 2 0/5 0/5 0/5 0/5 0/5 (B) Body weights of male rats in KIT Chemical treated Dose Body weights (Mean±S.D., g) b at the time of dosing (No. of animal) 1st 2nd 3rd Vehicle ±11.50 (5) 270.2±10.08 (5) 280.6±11.65 (5) Test chemical (Low dose) ±9.56 (5) 271.8±9.23 (5) 280.3±9.66 (5) Test chemical (Mid. dose) ±9.69 (5) 269.2±8.74 (5) 270.2±11.93 (5) Test chemical (High dose) ±12.50 (5) 260.2±10.13 (5) 254.9±9.71 (5) Positive control ±9.70 (5) 274.3±10.62 (5) 263.0±13.78 (5) Test chemical: Ethyl methanesulfonate (EMS), Vehicle: Saline, Positive control: Ethyl methanesulfonate (EMS). a Number of animals with clinical sign/total number of animals observed, b Bartlett test's and ANOVA test.

6 권지영외 11 인 : 유전독성시험법 in vivo Comet Assay 에대한국내시험기관간검증연구 299 vivo comet 국제검증연구에서도출된결과와동일한경향성을보였다. 10) 간조직보다위조직에서유전자손상이크게나타난것은위조직으로시험물질이직접투여되었기때문에이로인한유전자손상이큰것으로해석되어진다. 2. Ethyl methansulfonate (EMS) 를이용한 in vivo comet assay 결과국내 GLP 실험실간의 in vivo comet assay 검증시험을위하여선정된시험물질 EMS는유전독성을일으키는것으로잘알려졌으며 11) 제3차 in vivo comet 국제검증연구에서시험물질로사용된물질이다. 10) 각기관별용량설정시험을수행하였으며참고문헌 9) 에따라 1일 1회 3일 Table 6. (A) Clinical signs and mortalities of male rats in Medvill Co., Ltd Dose Day 2 0/5 0/5 0/5 0/5 0/5 (B) Body weights of male rats in Medvill Co., Ltd Chemical treated Dose Body weights (Mean±S.D., g) b at the time of dosing (No. of animal) 1st 2nd 3rd Vehicle ±7.2 (5) 267.7±7.5 (5) 283.3±7.9 (5) Test chemical (Low dose) ±5.8 (5) 254.8±8.9 (5) 250.7±3.8 (5) Test chemical (Mid. dose) ±6.9 (5) 260.0±7.9 (5) 267.1±8.7 (5) Test chemical (High dose) ±6.9 (5) 263.8±8.4 (5) 277.6±9.0 (5) Positive control ±8.1 (5) 265.3±9.1 (5) 239.8±10.2 (5) Test chemical: Ethyl methanesulfonate (EMS), Vehicle: Saline, Positive control: Ethyl methanesulfonate (EMS). a Number of animals with clinical sign/total number of animals observed, b Bartlett test's and ANOVA test. Table 7. (A) Clinical signs and mortalities of male rats in SNUH Dose Day 2 0/5 0/5 0/5 0/5 0/5 (B) Body weights of male rats in SNUH Chemical treated Dose Body weights (Mean±S.D., g) b at the time of dosing (No. of animal) 1st 2nd 3rd Vehicle ±13.32 (5) 280.6±14.20 (5) 293.4±14.81 (5) Test chemical (Low dose) ±9.84 (5) 273.1±10.81 (5) 283.6±10.10 (5) Test chemical (Mid. dose) ±8.62 (5) 272.7±11.65 (5) 280.7±12.12 (5) Test chemical (High dose) ±6.82 (5) 259.3±6.43 (5) 255.1±3.79 (5) Positive control ±10.96 (5) 275.5±11.72 (5) 270.6±12.45 (5) Test chemical: Ethyl methanesulfonate (EMS), Vehicle: Saline, Positive control: Ethyl methanesulfonate (EMS). a Number of animals with clinical sign/total number of animals observed, b Bartlett test's and ANOVA test.

300 Cancer Prevention Research Vol. 16, No. 4, 2011 간경구투여하여사망동물및일반증상을관찰한결과, 31.25, 62.

본시험에서시험물질을투여한모든동물에서특이한증상이관찰되지않았으며투여군간의체중비교에서도 EMS를투여한모든군에서음성대조군에비해통계학적으로유의한변화는관찰되지않았다 (Table 5 7).

2A, B) 에서음성대조군에비해 % tail DNA가증가한것으로나타났으며이는통계학적으로유의한차이를보였다. 이러한양상은위조직 (Fig. 2C, D) 에서도동일하게나타났다.

(A) Ethyl methanesulfonate (EMS)-induced DNA damage in rat liver tissue was measured by in vivo comet

For each animal of treatment, % tail DNA of 100 randomly selected cells were analyzed using Komet 5.

Treatment groups showed significantly incremental % tail DNA as dose-dependent manner, relative to

all laboratories (Student's t-test at p<0.025, one-sided).

comparison to the negative and the positive controls were taken by fluorescence microscope at 200X

(C) DNA damage induced by EMS was measured using in vivo comet assay in rat stomach tissue.

7 300 Cancer Prevention Research Vol. 16, No. 4, 2011 간경구투여하여사망동물및일반증상을관찰한결과, 31.25, 62.5, 125 mg/kg/day를본시험의농도군으로설정할수있었다 (data not shown). 본시험에서시험물질을투여한모든동물에서특이한증상이관찰되지않았으며투여군간의체중비교에서도 EMS를투여한모든군에서음성대조군에비해통계학적으로유의한변화는관찰되지않았다 (Table 5 7). In vivo comet assay 결과, 양성 대조군을포함한시험물질투여군의간조직 (Fig. 2A, B) 에서음성대조군에비해 % tail DNA가증가한것으로나타났으며이는통계학적으로유의한차이를보였다. 이러한양상은위조직 (Fig. 2C, D) 에서도동일하게나타났다. 이는국내 GLP 실험실간의연구결과가유사함을 보였으며뿐만아니라, 제 3 차국제검증연구에서도출된결과와도동일한경향을보여줌으로서 10) 국내 GLP Fig. 2. (A) Ethyl methanesulfonate (EMS)-induced DNA damage in rat liver tissue was measured by in vivo comet assay. For each animal of treatment, % tail DNA of 100 randomly selected cells were analyzed using Komet 5.0 software. Treatment groups showed significantly incremental % tail DNA as dose-dependent manner, relative to negative control group in all laboratories (Dunnett's test at p<0.05, two-sided and Linear trend at p<0.05, two-sided). Positive control also showed significantly incremented DNA damage compared with the negative control in all laboratories (Student's t-test at p<0.025, one-sided). (B) Representative comet images of cells from stomach tissue treated with increasing dose of EMS in comparison to the negative and the positive controls were taken by fluorescence microscope at 200X magnification. (C) DNA damage induced by EMS was measured using in vivo comet assay in rat stomach tissue. For measurement of DNA damage, total 100 randomly selected cells in each animal were scored by Komet 5.0 software. Treatment groups showed significantly incremental % tail DNA as dose-dependent fashion, relative to negative control group in all laboratories (Dunnett's test at p<0.05, two-sided and Linear trend at p<0.05, two-sided). Positive control also showed significantly augmented DNA damage compared with the negative control in all laboratories (Student's t-test at p<0.025, one-sided). (D) Representative comet images of cells from stomach tissue treated with increasing dose of EMS in comparison to the negative and the positive controls were taken with fluorescence microscope at 200 magnification.

8 권지영외 11 인 : 유전독성시험법 in vivo Comet Assay 에대한국내시험기관간검증연구 301 실험실에서최신의 in vivo comet assay 시험법검증이잘 이루어졌음을나타낸다. 결 본연구는현재국제적인가이드라인확립작업이진 행되고있는최신 in vivo comet assay 법의국내 GLP 실험 실간의검증연구로서안전성평가연구소, ( 주 ) 메드빌, 서 울대학교병원의생명연구원에서유전독성유발물질인 MNU와 EMS를시험물질로선정하여 in vivo comet assay를수행하였다. 그결과, 검증연구에참여한 3기관에서두종류의시험물질모두음성대조군에비하여통계학적으로유의한변화를나타내었으며이는제3차 in vivo comet 국제검증연구에서발표한결과와도일치하는것을확인함으로써최신유전독성시험법인 in vivo comet assay의국내실험실간의검증연구가성공적으로수행되었다고여겨진다. 뿐만아니라, 본연구는암예방을위한발암성예측및다양한화학물질의유전독성평가기반마련에있어중요한국제적으로공인된시험법을국내 GLP 실험실을중심으로검증연구를함으로써국내유전독성평가연구발전에기여하였다고사료된다. 론 감사의글 본연구는 2010년도식품의약품안전청용역연구개발과제의연구개발비지원 (10182독관리509) 에의해수행되었으며이에감사드립니다. 참고문헌 1)Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR. 4th International Comet Assay Workshop. Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop. Mutagenesis 18, 45-51, ) Őstling O, Johanson KJ. Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123, , ) Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175, , ) Olive PL, Banath JP, Durand RE. Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells. J Natl Cancer Inst 82, , ) Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35, , ) Purschke M, Jacobi H, Witte I. Differences in genotoxicity of H 2O 2 and tetrachlorohydroquinone in human fibroblasts. Mutat Res 513, , ) Brendler-Schwaab S, Hartmann A, Pfuhler S, Speit G. The in vivo comet assay: use and status in genotoxicity testing. Mutagenesis 20, , ) Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A; In Vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing. Fourth International workgroup on genotoxicity testing: results of the in vivo comet assay workgroup. Mutat Res 627, 31-35, ) The Validation Management Team (VMT). International validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens (version 14). pp 1-14, ) Uno Y. The JaCVAM-sponsored international in vivo comet assay validation study. In: Proceedings of 8th International comet assay workshop, pp 14, ) Gocke E, Bűrgin H, Műller L, Pfister T. Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate. Toxicol Lett 190, , 2009.

012임수진

012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar