기술논문 저선량율방사선조사한 BALB/c 마우스에서의영향평가 김성대 *, 공은지 *, 배민지 *, 양광모 *,, 김중선 * * 동남권원자력의학원, 한국원자력의학원 2012 년 6 월 20 일접수 / 2012 년 7 월 13 일 1 차수정 / 2012 년 7 월 14 일채택 본연구는저선량율방사선에의한급성독성을조사하기위해 BALB/c 마우스에게낮은선량율에방사선을만성노출후일반증상과체중, 장기중량, 부검소견, 혈액및뇨검사를통해독성유무를관찰하였다. 각각암수 20 마리마우스는각 5 마리씩 4 군으로분류하고, 3.49 mgy h -1 의저선량율방사선을정상대조군, 0.02, 0.2, 2 Gy 를각각 5.7 시간, 2.4 일, 24 일간지속적으로노출시키고조사후 1 일에마우스를희생하여독성을평가하였다. 저선량율방사선은최고선량의 (2 Gy) 노출에도사망률, 임상증상, 체중, 사료와음수섭취량, 뇨검사, 혈청생화학에서독성이관찰되지않았다. 하지만고환, 난소, 자궁을포함한생식장기는방사선량의존적으로장기의무게가감소되었으나, 다른장기의무게변화는관찰되지않았다. 모든방사선조사군에서혈액학적인부작용은관찰되지않았고, 단지호중구수가노출선량에의존적으로증가하였다. 이번실험결과마우스에서저선량율방사선노출은최고선량인 2 Gy 의노출에다른부작용은관찰되지않았으나, 암컷수컷의생식장기에무게감소가관찰되었다. 중심어 : 저선량율방사선, 독성평가, 마우스, 고환, 난소 1. 서론 1) 최근일본대지진으로인한후쿠시마원자력발전소폭발사고는일본뿐아니라전세계적으로방사선노출에대한관심과우려를증가시켰고, 우리나라역시방사선노출에대한불안감이커진상황이다. 방사선이생체에미치는영향은총방사선조사총량뿐만아니라조사선량율이중요하다 [1]. 하지만그동안방사선보호정책및관련연구는높은선량율의방사선노출을전제로한연구였으며, 저선량율방사선노출에관한연구는미흡하였다. 기존의고선량율방사선에의한세포나동물을이용한연구결과는저선량율방사선의영역으로적용하여해석하지못했다 [2]. 최근일부연구에서저선량율방사선의노출은손상된 DNA 의복구를유도하고 [3], 면역증진 [4] 및마우스에서의수명연장을 [5] 유도하는것으로보고된바있다. 하지만일부연구결과에서는저선량율방사선의노출이고선량율방사선노출보다더많은세포사멸을유도하여생체에유해할수있다는보고도있다 [6]. 이러한저선량율방사선영향연구의경우연구목적이외의기타실험적인영향에따라실험결과에큰영향을미치게되는데, 부산동남권원자력의학원에구축된저선량방사선조사 SPF 동물시설은차폐된공간에질병및오염이없는환경에서저선량방사선에의한동물의영향 책임저자 : 김중선, jskim@dirams.re.kr 부산광역시기장군장안읍좌동길 40, 동남권원자력의학원 연구를가능하게하였으며, 연구목적이외의기타실험적인영향을최소화할수있도록시설을갖추었다 (Fig. 1). 기존의방사선연구는높은선량율에따른독성에관한연구가주를이루었으나, 이번연구에서는낮은선량율의방사선노출에따른마우스에서의다양한독성평가를통해독성유무를확인하고자하였다. 저선량방사선및저선량율방사선기준은 United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCER) 2000 에서보고된 6 mgy h -1 이었다 [6]. 본연구는저선량율방사선노출에대한안전성평가의일환으로, 이번연구에서는 3.49 mgy h -1 의선량율로 0.02, 0.2, 2 Gy 의방사선을 BALB/c 마우스에노출시키고식품의약품안전청의의약품등의독성시험기준 (1999 년 ) 에준하여실험을수행하였다 [8]. 2. 재료및방법 실험동물은 7 주령의수컷과암컷 BALB/c 마우스 ( 중앙실험동물, 서울 ) 를사용하였다. 실험동물은온도 23±2, 상대습도 50±5%, 조명시간은 12 시간 ( 오전 8 시점등 - 오후 8 시소등 ), 공기순화는시간당 13-18 번순환, 조도 200~300lux 로설정된시설에서수행하였다. 순화기간을거쳐 polycarbonate 사육상자에 5 마리씩수용하였고실험동물용고형사료 ( 중앙실험동물, 서울 ) 와정수장치를 JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012 159
Fig. 1. Low dose rate irradiation equipment in Dongnam Institute of Radiological & Medical Sciences. Table. 1. Clinical Sign & Mortality for 3 Weeks. Dose Final mortality Clinical signs (Gy) Male Female Male Female 0 0/10 0/10 - - 0.02 0/10 0/10 - - 0.2 0/10 0/10 - - 2 0/10 0/10 - - treatment groups were placed on shelves in the same facility and were shielded from the radiation. Values are expressed as the number of dead animal/total number of animal. - : No clinical signs. 통과한수도수를이용하여자유급식하게하였다. 실험군은정상대조군, 0.02, 0.2, 2 Gy 조사군을암수각각 5 마리의마우스를사용하였다. 모든동물실험은 Institute of Laboratory Animal Resource 의 Guide for the Care and Use of Laboratory Animal (1996, USA) 에준하여취급하였으며동물실험은동남권원자력의학원동물실험윤리위원회 (Institutional Animal Care and Use Committee) 의승인하에수행되었다. 저선량율방사선조사는 SPF 시설내에설치된 137 Cs source (185 GBq) 를사용하여선원에서 2 m 거리에서 3.49 mgy h -1 의선량율로 0.02 (5.7 시간 ), 0.2 (2.4 일 ), 2 (24 일 ) Gy 을케이지에마우스를넣고전신조사하였다 (Fig. 1.). 모든방사선조사를마치고하루동안대사케이지에서대사량을측정하고독성을평가하였다. 방사선조사는하루 24 시간동안노출되게하며다만일주일에 2 시간은케이지교환및사료와음수급여등을목적으로조사를정지하였다. 대조군의경우모든처치는방사선조사군과일치하며다만방사선으로부터차폐하여사육하였다. 매주 1 회에걸쳐음수량과사료섭취량을대사케이지를활용하여측정하였다. 부검하루전날에는대사케이지를활용하여뇨를채취하였으며채취한뇨를이용하여 bilirubuin, urobilinogen, nitrite, ph, 비중, 백혈구, 케톤, 잠혈, 단백질, 당을평가하였다. 최종방사선조사가끝이나고다음날마우스를희생하였으며체중및장기 ( 간, 비장, 심장, 신장, 폐, 고환, 난소, 자궁, 뇌 ) 무게를측정하였다. 부검에서채취한혈액을이용하여일부는혈액생리학검사를실시하여, 백혈구, 호중구, 림프구, 단핵구, 호염기구, 적혈구, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red cell distribution width (RDW), 혈소판의변화를동물용혈액자동분석기 (ADIVIA 2120, Siemens, Germany) 를이용하여분석하였다. 더불어혈액내의 total protein, total cholesterol, creatinine, inorganic phosphorus, total bilirubin, calcium, albimin, blood urea nitrogen, glucose, sodium, potassium, chloride 을혈액생화학분석기 (FUJI DRI-CHEM 4000i, FUJI, JAPAN) 를이용하여분석하였다. 실험결과는평균 ±SE 로표시하였으며모든성적의분석은 Graph PAD In program (GPIP, Graph PAD software, USA) 을사용하였고 Student s t-test 를이용하여통계적분석을실시하였다. 유의성있는평가는 0.05 이상을기준으로삼았다. 160 JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012
Table 2. Absolute and Relative Organ Weight. Gy Male Female 0 0.02 0.2 2 0 0.02 0.2 2 Body weight (g) 26.18±.62 26.82±.8 26.86±.83 26.88±.95 19.6±1.7 19.54±1.1 19.48±1.5 19.46±1.1 Liver (g) 1.15±.09 1.45±.10 1.43±.04 1.43±.15 0.92±.10 1.04±.11 1.00±.13 1.00±.06 4.39±.31 5.39±.28 5.32±.16 5.32±.50 4.68±.18 5.31±.38 5.21±1.02 5.13±.41 Spleen (g) 0.104±.006 0.109±.008 0.107±.007 0.089±.005 0.104±.016 0.106±.010 0.097±.012 0.090±.008 0.396±.025 0.405±.020 0.398±.027 0.331±.013 0.531±.055 0.543±.045 0.501±.084 0.446±.087 Heart (g) 0.13±.010 0.139±.010 0.131±.011 0.132±.015 0.114±.008 0.103±.008 0.101±.004 0.100±.005 0.495±.032 0.518±.031 0.489±.043 0.491±.066 0.588±.036 0.528±.045 0.524±.055 0.528±.019 Kidney-L (g) 0.192±.030 0.218±.018 0.216±.015 0.207±.015 0.133±.027 0.147±.016 0.122±.021 0.125±.006 0.732±.104 0.810±.046 0.805±.068 0.770±.770 0.674±.097 0.743±.065 0.633±.144 0.664±.051 Kidney-R (g) 0.192±.040 0.219±.018 0.213±.017 0.211±.012 0.135±.016 0.136±.006 0.122±.020 0.125±.017 0.733±.145 0.815±.046 0.792±.065 0.786±.037 0.686±.038 0.692±.031 0.633±.141 0.661±.102 Lung (g) 0.177±.018 0.164±.017 0.158±.004 0.160±.020 0.141±.008 0.133±.007 0.126±.010 0.144±.025 0.675±.070 0.612±.049 0.588±.005 0.593±.061 0.72±.053 0.684±.059 0.65±.08 0.725±.097 Testis-L (g) 0.103±.005 0.096±.006 0.101±.006 0.063±.006* - - - - 0.393±.020 0.357±.023 0.374±.013 0.235±.027* - - - - Testis-R (g) 0.099±.006 0.103±.011 0.102±.002 0.066±.011* - - - - 0.379±.019 0.382±.035 0.380±.012 0.246±.048* - - - - Ovary-L (g) - - - - 0.009±.004 0.008±.004 0.003±.001* 0.003±.001* - - - - 0.044±.018 0.041±.022 0.015±.006* 0.017±.004* Ovary-R (g) - - - - 0.011±.003 0.008±.002 0.004±.001* 0.004±.001* - - - - 0.041±.013 0.032±.007 0.019±.007* 0.018±.002* Uterus (g) - - - - 0.077±.009 0.079±.005 0.087±.035 0.055±.012* - - - - 0.398±.069 0.404±.042 0.458±.22 0.285±.071 Brain (g) 0.452±.009 0.457±.023 0.456±.012 0.484±.046 0.449±.019 0.453±.011 0.442±.019 0.449±.009 1.726±.046 1.707±.131 1.700±.035 1.805±.202 2.303±.192 2.322±.080 2.285±.259 2.314±.152 treatment groups were placed on shelves in the same facility and were shielded from the radiation. Each value represents Mean±SE of five animals. *P<0.05 as compared to the each sex sham control. 3. 결과 3.1 사망률및임상증상시험전기간동안대조군을포함한모든실험군에서암 수모두사망동물은관찰되지않았다. 임상증상을관찰한결과역시대조군및방사선조사군모두에서방사선조사에의한외형의변화및이상행동등은관찰되지않았다 (Table 1). 3.2 사료및음수섭취량시험기간중사료섭취량과음수섭취량에서도체중의 증가에따른자연스러운사료및음수의섭취량증가를보였을뿐, 대조군을포함한모든실험군간에통계적으로의미있는사료및음수섭취량의차이는보이지않았다 (data not shown). 3.3 체중변화군분리 (randomization) 를위한시험개시전체중측정후부검전까지매주체중을측정한결과모든군에서고른체중증가가관찰되었으며대조군을포함한모든실험군간의유의한체중증가의차이는암 수모두보이지않았다 (data not shown). JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012 161
Table. 3. Urinalysis Values. Sex Male Female Parameter(Gy) 0 0.02 0.2 2 0 0.02 0.2 2 Specific Gravity Bilirubin (mg/dl) Nitrite leukocyte (WBC/ul) ph Ketone (mg/dl) Urobilinogen(mg/dl) Blood (RBC/ul) Protein (mg/dl) Glucose (mg/dl) 1.005 4 0 0 0 0 0 0 0 1.010 1 4 1 0 0 0 0 0 1.015 0 1 3 4 0 3 2 0 1.020 0 0 1 1 1 2 2 3 1.025 0 0 0 0 3 0 1 2 1.03 0 0 0 0 1 0 0 0 -/- neg 5 5 5 5 5 5 5 5 +/ - 0 0 0 0 0 0 0 0 +/ + 0.5 0 0 0 0 0 0 0 0 -/- 5 5 5 5 5 5 5 5 +/ - 0 0 0 0 0 0 0 0 +/ + 0 0 0 0 0 0 0 0 -/- neg 1 5 5 5 3 5 5 4 +/ - 10 2 0 0 0 1 0 0 1 +/ + 25 2 0 0 0 1 0 0 0 6 0 0 2 1 1 0 0 0 7 2 0 0 0 2 2 4 3 8 3 5 3 4 2 3 1 2 9 0 0 0 0 0 0 0 0 -/- neg 1 1 1 0 3 2 2 1 +/ - 5 3 2 3 5 2 2 3 3 +/ + 10 1 2 1 0 0 1 0 1 normal 0.1 3 5 5 5 3 4 5 5 + 10 2 0 0 1 2 1 0 0 -/- neg 5 4 4 4 3 3 5 4 + 10 0 1 1 1 2 0 0 0 ++ 50 0 0 0 0 0 2 0 1 +++ 250 0 0 0 0 0 0 0 0 -/- neg 0 0 2 0 0 0 0 0 +/ - 10 1 0 0 0 1 0 0 0 + 30 1 2 1 3 3 4 2 2 ++ 100 2 3 2 2 1 1 3 3 +++ 300 0 0 0 0 0 0 0 0 -/- neg 3 5 3 1 3 2 1 1 +/ - 100 2 0 2 4 2 2 1 2 + 250 0 0 0 0 0 1 3 2 ++ 500 0 0 0 0 0 0 0 0 treatment groups were placed on shelves in the same facility and were shielded from the radiation. After radiation exposure, urines from individual mice were collected for 24h before autopsy by using metabolic cage. Each value represents incidence of five animals. 3.4. 부검및장기무게실험종료후시료채취를위한부검시각장기의육안적소견관찰및절대적 / 상대적무게를측정하였다. 간, 비장, 뇌, 심장, 폐, 신장의경우대조군을포함한모든실험군에서암 수모두유의한절대적 / 상대적장기무게의변화는관찰되지않았다. 다만수컷의경우 2Gy 조사군에서통계적으로유의한정소의절대적 / 상대적장기무게의감소를보였으며, 암컷의경우 2Gy 조사군에서통계적으로유의한난소및자궁의절대적 / 상대적장기중량감소가관찰되었다 (Table 2). 3.5 혈액학적검사혈액학적검사결과암컷과수컷모두적혈구, 백혈구수치 (k/ul) 에서실험군간의의미있는차이는관찰되지않았다 (Table 3). 기타 hemoglobin, hematocrit, MCV, MCH, MCHC 및 RDW 수치도대조군을포함한모든실험군들에서유의한차이를보이지않았다. 백혈구의감별진단결과, 암컷과수컷에서 2Gy 조사군에서대조군과비교하여유의한호중구수치증가 (p<0.05% of control value) 가관찰되었다. 162 JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012
Table. 4. Hematological Values. Indices Male Female Gy 0 0.02 0.2 2 0 0.02 0.2 2 WBC (k/ul) 4.54±.63 5.36±.90 5.14±.12 4.33±.91 4.19±.60 5.91±.00 5.28±1.84 4.47±.55 NEU (k/ul) 0.60±.27 0.62±.13 0.81±.61 0.91±.18* 0.54±.19 0.7±.32 0.71±.59 0.93±.19* LYM (k/ul) 3.61±.77 4.33±.71 3.91±.41 3.16±.75 3.44±.63 4.75±.99 3.91±.60 3.10±.36 MO (k/ul) 0.25±.07 0.32±.05 0.31±.09 0.16±.05 0.27±.12 0.25±.10 0.33±.11 0.13±.02 EO (k/ul) 0.06±.05 0.06±.02 0.08±.11 0.04±.02 0.04±.02 0.06±.03 0.05±.03 0.03±.02 BASO(k/ul) 0.02±.02 0.03±.02 0.02±.03 0.01±.01 0.01±.01 0.02±.01 0.01±.01 0.01±.01 RBC (M/ul) 9.00±.55 9.57±.05 8.90±.39 9.19±.76 9.04±.39 9.46±.76 9.70±.53 9.19±.49 Hb (g/dl) 12.44±.49 12.48±.57 11.40±.74 11.88±.61 12.66±.79 12.48±.76 12.66±.68 12.02±.44 HCT (%) 53.64±.62 59.68±.56 53.76±.55 57.62±1.6 54.18±.61 56.95±.84 60.20±.02 56.14±.36 MCV (fl) 59.42±.90 61.26±.92 60.80±.21 62.58±.86 61.32±.13 60.23±.83 60.72±.63 61.20±.93 MCH (pg) 13.80±.00 12.66±.98 12.82±.42 12.86±.70 14.56±.13 13.23±.43 12.86±.90 13.12±.39 MCHC(g/dl) 23.32±.31 20.60±.42 21.24±.16 20.58±.89 23.66±.16 21.98±.85 21.16±.97 21.46±.19 RDW (%) 20.18±.95 20.30±.40 20.04±.54 21.00±.88 20.62±.9 20.60±.30 20.1±.19 20.91±.58 PLT (k/ul) 834.8±75.2 966.6±93.4 731.6±22.4 873.6±15.6 628.4±55.8 774.3±40.4 743.2±55.5 573.0±28.6 treatment groups were placed on shelves in the same facility and were shielded from the radiation. WBC: white blood cell, NEU: neutrophil, LYM: lymphocyte, MO: monocyte, EO: eosinophil, BASO: basophil, RBC: red blood cell, Hb: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean cell hemoglobin concentration, RDW: red cell distribution width. Each value represents Mean±SE of five animals. *P<0.05 as compared to the each sex sham control. Table. 5. Serum Biochemical Values Indices Male Female Gy 0 0.02 0.2 2 0 0.02 0.2 2 TP 4.2±.1 4.2±.2 4.2±.2 4.0±.1 3.9±.1 3.3±.7 3.7±.1 3.6±.4 TCHO 100.0±.6 104.7±1.0 102.7±.4 87.7±.9 58.7±.4 53.5±16 62.0±.4 59.7±.1 CRE 0.2±.0 0.2±.1 0.2±.1 0.2±.1 0.2±.1 0.2±.1 0.5±.3 0.2±.1 IP 6.8±.5 8.5±.0 7.6±.5 8.8±.5 7.2±.0 6.3±.9 9.1±.1 8.8±.7 TBIL 0.6±.1 0.5±.1 0.5±.1 0.5±.0 0.6±.1 0.5±.0 1.0±.6 0.5±.1 Ca 8.9±.5 8.6±.3 8.7±.3 9.0±.6 8.9±.3 7.6±.1 9.0±.3 8.4±.4 ALB 2.2±.1 2.3±.1 2.3±.1 2.1±.1 2.1±.1 1.8±.2 2.1±.5 1.9±.3 BUN 19.5±.9 23.8±.4 21.9±.8 21.6±.8 18.9±2.1 21.0±.5 23.0±4.5 24.0±.8 GLU 178.2±13.2 161.9±68.1 93.2±82.8 164.0±5.2 207.5±6.1 150.3±43.5 163.1±29.1 275.5±83. Na 144±.7 146±.5 146.3±.2 145±.2 142.3±.5 151.0±.1 136.3±8.2 146.5±.2 K 3.8±.1 4.4±.4 4.3±.3 4.0±.1 4.3±.2 3.9±.2 4.2±.5 5.1±.04 Cl 106.7±.5 108.3±.2 109.3±.6 108.±.04 107.7±.2 115.5±6.4 105.5±2.9 117±.03 treatment groups were placed on shelves in the same facility and were shielded from the radiation. TP: total protein, TCHO: total cholesterol, CRE: creatinine, IP: inorganic phosphorus, TBIL: total bilirubin, Ca: calcium, ALB: albumin, BUN: blood urea nitrogen, GLU: glucose, Na: sodium, K: potassium, Cl: chloride. Each value represents Mean±SE of five animals. 3.6 혈액화학학적검사본실험결과측정된혈액화학적항목모두에서암컷과수컷공히방사선조사와관련하여어떠한의미있는변화도관찰되지않았다 (Table 4). 3.7 뇨검사소견수컷의경우방사선조사선량에따라뇨비중이증가하는경향을보인다. 기타케톤, 단백질, 잠혈, 백혈구, ph 등은각군간의유의한차이를보이지않았다. 빌리루빈의경우도모든실험군에서음성을나타내었다. 암컷의경우는전항목에걸쳐방사선조사선량과관련한실험 군간의의미있는차이는보이지않았다 (Table 5). 4. 논의 본연구는동남권원자력의학원에구축된저선량율방사선조사시설을이용, SPF 환경에서저선량율 (3.49 mgy h -1 ) 방사선조사가생체에미치는영향을평가하기위해방사선에민감한실험동물종으로널리알려진 BALB/c mouse 를이용하여저선량율방사선조사 (0, 0.02, 0.2, 2Gy) 에따른임상증상, 체중및식이 음수섭 JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012 163
취량의변화, 육안적부검소견및장기무게의변화, 혈액학적분석, 혈액화학적분석, 뇨분석등을실시하였다. 시험전기간동안대조군을포함한암수모두저선량율방사선조사군에서사망동물은관찰되지않았으며, 유의할만한행동변화및이상증상은관찰되지않았다 (Table 1). 체중의변화에있어서도총 21 일간의방사선조사기간중유의성있는차이는발견되지않았다. 이는 0.2 및 2 Gy 조사후대조군을포함한각실험군간의체중증가의변화를보이지않은 Kang 등의연구결과와도일치한다 [9]. 물론일부보고에서 0.07Gy 의방사선이조사된후체중및장기의무게가감소하는것으로보고된바있으나 [9], 이러한차이는조사된방사선의선량율차이및실험에사용된마우스종 (CBAxC57BL/6 F1 mice) 에따른차이로판단된다. 사료섭취량및음수량에있어서도시험기간중유의성있는차이는발견되지않았다. 부검및장기중량측정결과에서는대조군을포함한모든저선량율방사선조사군에서조사후간, 뇌, 심장, 폐, 신장의무게변화는관찰되지않았다. 이는이전연구에서보고되었던 2 Gy 조사마우스에서간및신장의무게증가결과와는차이가있었다 [10]. 다른뇌와심장, 폐에서는무게감소가관찰되지않았으며이러한결과는이전연구결과와일치하였다 [9]. 이번실험결과저선량율방사선조사군가운데 2 Gy 조사군에서수컷마우스는고환의무게감소, 암컷마우스에서는난소와자궁의무게감소가관찰되었다. 이전저선량율방사선조사에의한장기무게변화연구에서는고환, 자궁, 난소와같은생식장기의무게측정은이루어지지않았다 [9]. 고환은방사선에가장민감한장기가운데하나이며저선량방사선조사에도고환의기능및형태에변화를주게된다. 방사선의조사는고환세포의염색체이상이나세포자멸사및고환세포수감소등의손상을유발하게된다 [11,12]. 방사선조사에의해고환세포는방사선선량과시간에따라다양한반응을나타낸다. 방사선치료환자에서 0.1 Gy 의낮은선량에서도정조세포는형태학적인변화와숫자의감소가보고되었으며, 2 Gy 의선량에서는정모세포의숫자의감소가보고되었다 [12]. 대부분많은논문에서저선량율방사선의조사는고선량율방사선조사와는달리유의성있는생물학적영향이없거나오히려다양한긍정적인영향을미치는것으로보고된바있다 [2,3,4]. 하지만 Moreno 등에보고에따르면마우스태아에서의고환 gonocyte 및 Sertoli cell 의경우고선량율의방사선조사보다저선량율의방사선조사에서더심각한고환독성을유도하는것으로보고되었다. 특히태아시기의고환의 germ cell 은고선량율방사선보다저선량율방사선에민감하게독성을보이는것으로보고된바있다 [5]. 난소의 germ cell 역시방사선에매우민감하다 [13,14]. 방사선노출은난포내과립세포의세포자멸사를유발하며, 난자의염색체에손상을일으켜난소의기능을저하시킨다 [15]. 이번연구결과다른장기에서의특별한독성이나변화가관찰되지않는것과비교하여암수생식기에서는장기의무게감소를비롯한생식독성이관찰됨에따 라고환과난소를비롯한생식기에대한저선량율방사선에관한심도있는연구가필요할것으로보인다. 혈액학적검사결과혈소판수치의경우암컷과수컷의 2 Gy 방사선조사군에서혈소판수치가감소하는모습을보이나통계적인유의성은없었으며다른방사선조사군들과의방사선량 (dose)- 상관성등을고려했을때방사선조사와는관계가없는우발적인소견으로사료된다. 기타적혈구수나, hemoglobin, hematocrit, MCV, MCH 등의빈혈과관련된지표들은별다른이상소견을보이지않았고부검시육안적이상소견이없는바, 비장, 골수, 간의조혈기능이저선량율방사선조사와관련된이상은발견되지않았다. 마지막으로백혈구감별진단결과 2Gy 조사한암컷과수컷방사선조사군에서호중구수치의유의한증가 (p<0.05% of control value) 가관찰되었다. 비록이러한증가는 Wolford 등이제시한실험동물의혈액학적기초자료에서제시한측정치와비교하였을때, 측정치가모두정상범위내에속하는범주였다 [16]. 하지만기존에알려진바와같이방사선노출은골수에심각한손상을유도하는것으로알려져있으며, 0.5 Gy 선량의방사선노출에도조사후 1 일부터림프구와호중구를포함한백혈구감소를유도한다고보고된바있다 [17]. 하지만이번실험결과저선량율방사선 (3.49 mgy h -1 ) 의노출은호중구를증가시켰으며, 기존보고된실험결과와는차이가있었다 [17]. Li 등의보고에따르면 75 mgy min -1 의선량율로 75 mgy 정도의방사선에노출되었을때골수의 hematopoietic progenitor cell 에증식이유도되어지고말초혈액에혈구이동이유도되는것으로보고된바있다 [18]. 이러한실험결과는본실험에서보이는낮은선량율의방사선에노출시관찰되어지는방사선량의존적인말초호중구수의증가와일치하는결과로사료된다. 특히고선량율방사선노출시에가장민감하게감소하는림프구의변화가 [19], 이번저선량율방사선노출에서는관찰되지않았으며, 이러한변화는기존방사선연구에서사용되었던고선량율방사선과저선량율방사선이생물학적영향이서로다름을확인하는결과이다. 본실험결과측정된혈액화학적항목모두에서암컷과수컷공히방사선조사와관련하여어떠한의미있는변화도관찰되지않았다. 칼슘과 BUN 은운동실조와관련된항목이며, 빌리루빈은헤모글로빈의대사산물로서빌리루빈의증가는빈혈의소견으로앞서혈액학적인검사에서이상이없음을확인한소견과도일치한다. 기타항목들에서도방사선조사와관련된의미있는소견은보이지않았다. 뇨분석은비뇨기계의손상이나체내염증반응을파악하기위한가장간단한방법이면서도필수적인요소이다. 본실험결과수컷에서는방사선조사에따라뇨비중이증가하는경향을보이기도하나, 뇨검사의다른항목 ( 케톤, 단백질, 잠혈, 백혈구수 ) 과비교하여보았을때방사선조사선량상관성이결여된우발적인결과로판단된다. 이상의결과를통해저선량율방사선은조사선량에따라조사 24 시간후암컷과수컷의생식장기의절대적 / 상대적무게를감소시키며, 정상범위내에서호중구 164 JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012
수치를증가시킨다. 이는저선량율방사선조사에의한표적장기로생식장기가활용될수있음을확인하였다. 본연구는저선량율방사선을이용한연구의중요한참고자료로활용될수있으며, 차후실험을통해저선량율방사선조사후시간에따른생식장기의반응및그기전을규명하는연구가추가적으로필요하다고판단된다. 5. 결론 저선량율방사선에의한 BALB/c 마우스에서 3.49 mgy h -1 의낮은선량율의방사선에노출되었을때급성독성을평가하였다. SPF 시설에갖추어진방사선조사시설을이용하여정상대조군, 0.02, 0.2, 2 Gy 로노출시키고일반증상및음수량, 체중변화및뇨검사를평가하였고, 조사를마치고 1 일마우스를희생하여장기중량및부검소견, 혈구검사및혈청검사를통해독성유무를평가하였다. 실험결과일반증상및음수량, 체중변화에서는특이한차이점을관찰하지못하였다. 혈액검사결과 2Gy 조사군에서호중구의증가가관찰되었으나정상범위내의수치였고, 다른지표에서는특징적인변화가관찰되지않았다. 뇨검사및혈청생화학검사에서도독성은관찰되지않았다. 부검결과수컷에서는고환의크기가, 암컷에서는난소와자궁의크기가감소되었으며, 무게역시방사선량에의존적으로감소하였고최고선량 (2 Gy) 군에서는유의성있는감소가관찰되었다. 생식장기이외의다른장기무게변화는관찰되지않았다. 이는저선량율방사선조사에의한표적장기로생식장기가활용될수있음을확인한결과였다. 본연구는저선량율방사선을이용한연구의참고자료로활용될수있으며, 차후실험을통해저선량율방사선조사후시간에따른생식장기의반응및그기전을규명하는연구가추가적으로필요하다고판단된다. 감사의글이연구는교육과학기술부의재원으로동남권원자력의학원의지원을받아수행된연구입니다 (50496-2012). 참고문헌 01. Hall E. Radiobiology for the radiologist, 2nd. Harper & Row, Publishers, Inc. 1978:63-78. 02. De Saint-Georges L. Low-dose ionizing radiation exposure: understanding the risk for cellular transformation. J. Biol. Regul. Homeost. Agents 2004;18(2):96-100. 03.Nakamura H, Yasui Y, Saito N, Tachibana A, Komatsu K, Ishizaki K. DNA repair defect in AT cells and their hypersensitivity to low-dose-rate radiation. Radiat. Res. 2006;165(3):277-282. 04. Ina Y, Sakai K. Activation of immunological network by chronic low-dose-rate irradiation in wild-type mouse strains: analysis of immune cell populations and surface molecules. Int. J. Radiat. Biol. 2005;81(10):721-729. 05. Ina Y, Tanooka H, Yamada T, Sakai K. Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice. Radiat. Res. 2005;163(2):153-158. 06. Moreno SG, Dutrillaux B, Coffigny H. High sensitivity of rat foetal germ cells to low dose-rate irradiation. Int. J. Radiat. Biol. 2001;77(4):529-538. 07. UNSCER. Volume I and II. United Nations sales publication E.00.IX.3 and E.00.IX.4 United Nations, New York. 2000. 08. OECD (Organisation of Economic Co-operation and Development). Guidelines for the Testing of Chemicals. No. 401:Acute Oral Toxicity, Paris; 1987. 09. Kang YM, Shin SC, Jin YW, Kim HS. Changes in body and organ weights, hematological parameters, and frequency of micronuclei in the peripheral blood erythrocytes of ICR mice exposure to low dose rate Υ-radiation. Journal of Radiation Protection. 2009;34(3):102-106. 10. Tsiperson VP, Soloviev MY. The impact of chronic radioactive stress on the immuno-physiological condition of small mammals. Sci. Total Environ. 1997;203(2):105-113. 11. Jang JS, Kim JS, Kim JC, Kim SH. Evaluation of Radiation-induced Apoptosis in Seminiferous Tubule of ICR Mouse after Gamma Irradiation. Journal of Life Science 2009;19(6): 799-803. 12. Liu G, Gong P, Zhao H, Wang Z, Gong S, Cai L. Effect of low-level radiation on the death of male germ cells. Radiat. Res. 2006;165(4):379-389. 13. Lee YK, Chang HH, Kim WR, Kim JK, Yoon YD. Effects of gamma-radiation on ovarian follicles. Arh. Hig. Rada. Toksikol. 1998;49(2):147-153. 14. Dobson RL, Felton JS. Female germ cell loss from radiation and chemical exposures. Am. J. Ind. Med. 1983;4:175-190. 15. Chapman RM. Effect of cytotoxic therapy on sexuality and gonadal function. Semin. Oncol.1982; 9(1):84-94. 16. Wolford ST, Schroer RA, Gohs FX, Gallo PP, Brodeck M, Falk HB, Ruhren R. Reference range data base for serum chemistry and hematology values in laboratory animals. J. Toxicol. Environ. Health 1986;18(2):161-188. 17. Lee DK, Kang HJ. Effects on Heamopoietic Cells and Chromosomal DNA of Leukocytes in Mice Irradiated with Low Dose of X-ray I. Changes of Leukocyte, Lymphocyte, Thrombocyte and JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012 165
Erythrocyte Number. Kor. J. Vet. Publ. Hlth. 2002; 26:143-152. 18. Li W, Wang G, Cui J, Xue L, Cai L. Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation. Exp. Hematol. 2004;32(11):1088-1096. 19. Kucerova M, Anderson AJ, Buckton KE, Evans HJ. X-ray-induced chromosome aberrations in human peripheral blood leucocytes: the response to low levels of exposure in vitro. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1972;21(4):389-396. Bioassay in BALB/c mice exposed to low dose rate radiation Sung Dae Kim *, Eun Ji Gong *, Minji Bae *, Kwangmo Yang *,, and Joong Sun Kim * * Dongnam Institute of Radiological & Medical Sciences, Korea Institute of Radiological & Medical Sciences Abstract - The present study was performed to investigate the toxicity of low-dose-rate irradiation in BALB/c mice. Twenty mice of each sex were randomly assigned to four groups of five mice each and were exposed to 0 (sham), 0.02, 0.2, or 2 Gy, equivalents to low-dose-rate irradiation to 3.49 mgy h -1. Urine, blood, and blood biochemistry were analyzed, and organ weight was measured. The low-dose-rate irradiation did not induce any toxicologically significant changes in mortality, clinical signs, body weight, food and water consumption, urinalysis, and serum biochemistry. However, the weights of reproductive organs including the testis, ovary, and uterus decreased in a dose-dependent manner. Irradiation at 2 Gy significantly decreased the testis, ovary, and uterus weights, but did not change the weights of other organs. There were no adverse effects on hematology in any irradiated group and only the number of neutrophils increased dose dependently. The low-dose-rate irradiation exposure did not cause adverse effects in mice at dose levels of 2 Gy or less, but the reproductive systems of male and female mice showed toxic effects. Keywords : Low-dose-rate irradiation, Toxicity, Mice, Testis, Ovary 166 JOURNAL OF RADIATION PROTECTION, VOL.37 NO.3 SEPTEMBER 2012