크롬 노출 근로자의 말초혈액 임파구내 소핵출현에 관한 연구

김해준,나중호,김영환,최재욱 大韓産業醫學會 1999 대한직업환경의학회지 Vol.11 No.3

Objectives: The goal of this study is to observe the associations between the chrome concentrations (blood and urine) and number of lymphocyte with micronuclei among workers exposed to chrome. Additionally to test appropriateness of micronuclei test for biologic monitoring markers such as urine or blood chrome. Methods: Twenty-seven pairs of subjects exposed to chromate environment and non-exposed were matched with age and smoking habit, and all subjects were composed of male workers only. Results: In control group, age-related increment in number of cultured lymphocytes with micronuclei was observed statistically significant. But, in exposed group, similar results were revealed without statistically significance. Number of lymphocytes with micronuclei was significantly increased with the increment of blood or urinary Cr concentration. The number of lymphocytes with micronuclei were highly correlated with age and smoking habit in control group. But, in chromate exposed group, age and logarithmic converted concentration of urinary Cr were significantly correlated with appearance of micronuclei. In both of control and exposed group, age, urinary concentration of Cr and logarithmic converted concentration of blood Cr were thought to be important variables. Conclusions: There was close relationship between the micronuclei assay for evaluation of mutagenesis and biological exposure markers such as blood or urinary Cr concentration of chromate workers. And obtained results suggested that further studies for establishing age-related reference frequencies of the micronuclei appearance for general people of Korean were needed to clarify physical increment with aging.

Frequency of Micronuclei in Lymphocytes Following Gamma and Fast-neutron Irradiations

김성호(Sung-Ho Kim),조철구 (Chul-Koo Cho),김태환 (Tae-Hwan Kim),정인용 (In-Yong Chung),류성렬 (Seong- Yul),고경환 (Kyoung-Hwan Koh),윤형근 (Hyong-Geun Yun). 대한방사선종양학회 1993 Radiation Oncology Journal Vol.11 No.1

원자력 시설 이용 증대에 따른 불의의 방사선 사고에 대비하여 방사선 직업종사자의 피폭시 진단을 위한 검사방법이 필요하다. 이 방법은 검사를 위한 검체의 체취가 용이하고, 짧은 시간내에 간편하게 많은 sample을 처리하여야 한다는 조건을 만족시켜야 한다. 인체의 다양한 조직 및 세포 중에서 위의 조건을 만족시킬 수 있는 말초 혈액의 림파구는 비교적 방사선에 대한 감수성이 높다고 알려져 있으며, 채집 또한 용이하여 생물학적 선량 측정의 도구로써 이용가치가 높아 방사선 작업 종사자나 피폭 가능성이 있는 사람의 screening test에 사용될 수 있다. 정상인에 있어서의 림파구내 미세핵 존재 여부와 방사선 피폭량에 따른 미세핵 발생빈도를 시험관내 실험을 통하여 표준화시켜 향후 방사선 피폭시 피폭선량을 역으로 산출해 낼 수 있는 방사선 장해의 평가 기술 개발의 기초자료를 마련하기 위하여 본 실험을 시행하였다. 정상인으로부터 혈액을 채취하여 림파구만을 Ficoll-Hypaque gradient 방법으로 추출하여 배양한 다음, 본 치료방사선과의 중성자 치료기 (MC-50, scanditronix)와 Co-60 teletherapy unit (Theratron-780, AECL)를 이용하여 방사선 조사를 시행하였다. Cytokinesis-block method를 이용하여 첫 번째 분열을 한 림파구에서 미세핵 (Micronucleus)을 현미경을 통하여 계수한 다음, 이의 선량-반응 관계식을 linear-quadratic model을 사용하여 구하고, 이를 근거로 하여 gamma-ray에 대한 중성자의 Relative biological effectiveness (RBE) 를 산출하였다. 방사선에 피폭되지 않은 림파구의 미세핵 발생빈도는 binucleated cell 한 개당 0.013±0.0002로써 사람에 따라 통계학적으로 큰 차이를 보이지 않았다. 그림 2와 3에서 보는 바와 같이 개개인으로부터 얻은 data는 감마선과 중성자선 모두에서 선량 -반응 곡선의 linear-qudratic equation에 잘 일치하였다. 감마선과 중성자선 모두에서 선량에 따른 미세핵의 발생빈도는 선량이 높을수록 비례하여 증가하였는데, 감마선의 경우에는 r²=1.000, x²=0.7074, p=0.95였으며, 중성자선인 경우에는 r²=0.996, x²=7.6834, p=0.11 였다. 이를 linear-quadratic model로 분석하면, 가장 적합한 선은 감마선인 경우에는 y=(0.31±0.049) D=(0.0022±0.0002) D²+(13.19±1.854) 였으며, 중성자선인 경우에는 y=(0.99±0.528) D=(0.0093±0.0047) D²+(13.31±7.309) 였었다. 감마선에 대한 중성자선의 상대적 생물학적 효과비 (RBE)는 y=aD+bD²+c를 다음과 같은 식으로 변형시켜 계산하였다. 미세핵 발생빈도가 세포당 0.05와 0.8 사이에서의 중성자선의 상대적 생물학적 효과비는 2.37±0.17이었다. 이상의 결과를 종합하여 볼 때 선량에 따른 미세핵 발생빈도는 기존의 방사선 감수성 test의 결과와 대동소이하여, 앞으로 q아사선 감수성을 측정하는 방법으로 이용할 수 있으며, 또한 실험방법이 비교적 간단하며 짧은 시간에 결과를 도출할 수 있어 생물학적 건량측정 도구로써 널리 이용될 수 있을 것으로 생각되어 진다. The dose response of the number of micronuclei in cytokinesis-blocked (CB) lymphocytes after in vitro irradiation with -rays and neutrons in the 5 dose ranges was studied for a heterogeneous population of 4 donors. One thousand binucleated cells were systematically scored for micronuclei. Measurements performed after irradiation showed a dose-dependent increase in micronuclei (MN) frequency in each of the donors studied. The dose-response curves were analyzed by a linear-quadratic model, frequencies per 1000 CB cells were (0.31±0.049) D+(0.0022±0.0002) D²+(13.19±1.854) (r²=0.996, x²=7.6834, p=0.11) following neutrons irradiation (D is irradiation dose in cGy). The relative biological effectiveness (RBE) of neutrons compared with -rays was estimated by best fitting linear-quadratic model. In the micronuclei freqyency between 0.05 and 0.8 per cell, the RBE of neutrons was 2.37±0.17. since the MN assay is simple and rapid, it may be a good tool for evaluating the -ray and neutron response.

Effect of Spirulina maxima and Its Protein Extract on Micronuclei Induction by Hydroxyurea in Pregnant Mice and Their Fetuses

Isela A ´ lvarez-Gonza´lez,Jorge Va´zquez-Sa´nchez,Germa´n Chamorro-Cevallos,Eduardo Madrigal-Bujaidar 한국식품영양과학회 2013 Journal of medicinal food Vol.16 No.11

The purpose of the present report was to determine the inhibitory effect of Spirulina maxima (Sm) and its protein extract (PE), mainly consisting of C-phycocyanin, on the increase in micronuclei and bone marrow cytotoxicity induced by hydroxyurea (HU) in pregnant mice and their fetuses. The two tested antimutagenic agents were administered daily from day 10 to day 18 of pregnancy, and HU (300 mg/kg) was administered once on day 16 of the assay. The experimental design also included mice that were administered only Sm or PE (1000 and 400 mg/kg, respectively), two control groups that were administered with vehicles (water and 0.5% Tween 80), and one additional group that was treated solely with HU. Blood samples from the pregnant mice and their fetuses were examined at day 19 of pregnancy. Significant increases in the number of micronucleated polychromatic erythrocytes and in the total number of micronucleated erythrocytes were observed in all HU-treated animals. In contrast, similarly low numbers of micronuclei were observed in the two control groups and in the groups treated with Sm and PE alone. The administration of Sm (100, 500, and 1000 mg/kg) and PE (100, 200, and 400 mg/kg) to HU-treated animals conferred moderate genotoxic protection (*30%) and some protection against the cytotoxicity induced by HU in mice. The obtained results provide new information regarding the capacity of the tested agents to confer protection to adult mice and transplacentally, as well as on a specific subclass of micronuclei.

에틸렌옥사이드(Ethylene oxide)에 노출된 병원 근로자들의 소핵 빈도와 유전적 감수성 지표와의 연관성

이선영,김양지,최영주,이중원,이영현,신미연,김원,윤충식,김성균,정해원,Lee, Sun-Yeong,Kim, Yang-Jee,Choi, Young-Joo,Lee, Joong-Won,Lee, Young-Hyun,Shin, Mi-Yeon,Kim, Won,Yoon, Chung-Sik,Kim, Sung-Kyoon,Chung, Hai-Won 한국환경보건학회 2011 한국환경보건학회지 Vol.37 No.6

Objectives: Ethylene oxide (EtO) is classified as a human carcinogen, but EtO is still widely used to sterilize heat-sensitive materials in hospitals. Employees working around sterilizers are exposed to EtO after sterilization. The aim of the present study was to assess the exposure of EtO level, coupled with occupationally induced micronuclei from hospital workers. The influence of genetic polymorphisms of detoxifying genes (GSTT1 and GSTM1) and DNA repair genes (XRCC1 and XRCC3) on the frequencies of micronuclei in relation to exposure of EtO was also investigated. Methods: The study population was composed of 35 occupationally exposed workers to EtO, 18 student controls and 44 unexposed hospital controls in Korea. Exposure to EtO is measured by passive personal samplers. We analyzed the frequencies of micronuclei by performing cytokinesis-block micronucleus assay (CBMN assay) and GSTM1, GSTT1, XRCC1, and XRCC3 were also genotyped by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The frequencies of micronuclei in EtO exposure group, student controls and hospital controls were $18.00{\pm}7.73$, $10.47{\pm}7.96$ and $13.86{\pm}6.35$ respectively and their differences were statistically significant, but no significant differences according to the level of EtO were observed. There was a dose-response relationship between the frequencies of micronuclei and cumulative dose of EtO, but no significantly differences were observed. We also investigated the influence of genetic polymorphisms (GSTM1, GSTT1, XRCC1, and XRCC3) on the frequencies of micronuclei, but there were no differences in the frequencies of micronuclei by genetic polymorphisms. Conclusions: The frequencies of micronuclei in EtO exposure group was significantly higher than control groups. A dose-response relationship was found between the level of EtO exposure and the frequencies of micronuclei, but no statistically differences were observed. We also found that the frequencies of micronuclei were increased according to cumulative EtO level. There was no association of the genetic GSTM1, GSTT1, XRCC1, and XRCC3 state with the frequency of micronuclei induced by EtO exposure.

저 선량 X-선 조사에 의한 마우스의 혈구세포 및 염색체 DNA 에 미치는 영향 : 2. 백혈구의 Pyknosis 및 Micronuclei 생성 2. Occurrence of Pyknosis and Micronuclei in Lenkocytes

이덕규,강호조 한국수의공중보건학회 2002 예방수의학회지 Vol.26 No.2

The present study was carried out to investigate the effect of low dose X-irradiation (0.1∼1.5 Gy) on the frequency of pyknosis and micronuclei in leukocytes in mice. For observation of pyknosis and micronuclei in leukocytes, blood smears were performed, then stained with Hematoxylin-Eosine. The frequencies of pyknosis and micronuclei cells were observed under microscopic field. The frequency of pyknotic leukocytes with degeneration, disintegration and dark-stained nuclei was signifantly high in groups of 0.5, 1.0 and 1.5 Gy irradiation (P<0.05, P<0.001 and P<0.0001, respectively), compared with the control. The occurrence of micronuclei of leukocytes was also significantly high in groups of 1.0 Gy (P<0.05) and 1.5 Gy irradiation (P<0.05) compared with the control. These results suggest that the irradiation with 0.5∼1.5 Gy damaged to leucocytes in a dose-dependent manner.

Relationship between Chromosome Aberrations and Micronuclei in Circulating Lymphocytes after Fractionated Irradiation in vitro

Joksic´, Gordana 한국유전학회 2004 Genes & Genomics Vol.26 No.3

The aim of this study is to establish relationship between incidence of chromosomal aberrations and micronuclei in circulating human lymphocytes after fractionated irradiation in vitro. Blood samples (of males of mean age 32, range 30-34) were irradiated using X-rays (300 kVp, 10 mA, 2.7 ㎜ CuHVT) employing radiation doses of 1-4 Gy (dose-rate 1 Gy/min). Each dose was given in two equal fractions with four different time intervals between two irradiations: 30 minutes, 1 hour, 2 and 3 hours. Acute irradiated samples serve as positive controls. For each experimental point parallel cultures were set up: for chromosome aberration and micronuclei analysis. The results obtained in present study have shown that the best correlation was found between dicentrics and total chromosome aberrations versus micronuclei in all employed doses. In contrast to samples acutely irradiated where chromatid aberrations were no found, they are observed in all samples where radiation dose was delivered with the longest pause between two fractions (3 hours). Incidence of chromosomal aberrations decline faster than micronuclei at all given doses if the time interval between two irradiations is longer than 2 hours. The study highlighted that micronuclei is reliable parameter of the DNA damage in conditions of fractionated irradiation, that could be particularly important for estimation of radiosensitivity and recovery time of lymphocytes for patients undergoing radiotherapy.

스타이렌과 산화스타이렌에 의한 소핵 유도

이세영,이세훈 가톨릭대학산업의학센타 산업의학연구소 1999 韓國의 産業醫學 Vol.38 No.1

Styrene is a commercially important chemical used mainly in the production of polymers and reinforced plastics. Human erythrocytes, lymphocytes can oxidize styrene to styrene-7,8-oxide in the absence of the co-factors required for metabolic reactions catalyzed by the microsomal cytochrome P-450 system. Styrene has been reported to produce chromosomal aberrations, micronuclei and sister chromatid exchanges in human whole-blood lymphocyte cultures. This study was designed to investigate whether styrene or styrene-7,8-oxide induce micronuclei(MN) in phytohaemagglutinin-stimulated human whole-blood lymphocytes culture. Fresh heparinized peripheral blood samples from a healthy donor (male, smoker) was cultured in the phytohaemagglutinin stimulated RPMI media. Styrene and styrene-7,8-oxide were injected into the cultures. Cytochalasin-B was added into the cultures for cytokinesis-block. Micronuclei were analyzed in 1000 binucleated lymphocytes from each duplicated cultures on coded acridine orange stained slides. The results were as follows: 1. Average frequency of micronuclei in binucleated lymphocytes treated with styrene was not significantly increased at any concentration applied in this study. But, styrene-7,8-oxide showed a significant induction of micronuclei in 0.125, 0.500mM concentration. 2. Styrene and styrene-7,8-oxide significantly decresed replication index(RI). The results of this study demonstrated that styrene-7,8-oxide could contribrte to the genotoxicity of styrene in human whole-blood lymphocyte.

Baseline Micronuclei in Fetal Lymphocytes-a Higher Frequency in Females Relative to Males

Joksic´, Gordana,Guc´-Sˇc´ekic´, Marija,Stankovic´, Miroslava 한국유전학회 2003 Genes & Genomics Vol.25 No.4

This study provides information concerning base-line micronuclei (MN) and sister-chromatid exchanges (SCEs) in human-fetal lymphocytes. Fetal cord blood (75 samples) were used for purposes of prenatal diagnosis; small aliquots of each were set up into cultures for micronuclei and sister-chromatid exchage analysis. Obtained mean values of base-line MN in fetal lymphocytes are 21.48 ± 3.52 for males, 24.54 ± 5.31 for females, respectively. Although small number of fetuses carrying abnormal karyotype was found (6 out of 75), no statistical difference in base-line level of micronuclei between normal versus abnormal karyotype was found. The mean value of SCE is 4.06 ± 0.77 in female group, 4.03 ± 0.66 in male group, respectively. As in the case of base-line micronuclei in fetal lymphocytes carrying abnormal karyotype SCEs also are not affected by chromosomal imbalances. There was a lack of correlation between MN and SCE incidence (r = 0.12).

말초혈액을 이용한 초고속 유전독성평가법 개발 연구

안일영 ( Il Young Ahn ),김주환 ( Ju Hwan Kim ),임대현 ( Dae Hyun Lim ),양준영 ( Jun Young Yang ),김소영 ( So Young Kim ),이정선 ( Jung Sun Yi ),염영나 ( Young Na Yum ),임채형 ( Chae Hyung Lim ),이진하 ( Jin Ha Lee ),최기환 ( Ki Hw 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1

To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.

연구논문 : 말초혈액을 이용한 초고속 유전독성평가법 개발 연구

안일영 ( Il Young Ahn ),김주환 ( Ju Hwan Kim ),임대현 ( Dae Hyun Lim ),양준영 ( Jun Young Yang ),김소영 ( So Young Kim ),이정선 ( Jung Sun Yi ),염영나 ( Young Na Yum ),임채형 ( Chae Hyung Lim ),이진하 ( Jin Ha Lee ),최기환 ( Ki Hw 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1

To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.