생식독성 번역(한국어 원본)2. Materials and Methods
2.1. Grass shrimp embryos
Grass shrimp (Palaemonetes pugio) 배아는 미국 조지아주 Savannah 지역의 Skidaway River 하구(30º95‘N-81º43’W)에서 2009년 6월에 채집하였다. 수정된 grass shrimp 배아는 난낭에 둘러싸여 암컷의 외복부에 부착하여 발달하며, 발달과정은 형태학적으로 뚜렷한 10단계로 구분된다. 채집된 암컷의 외복부에 부착한 배아를 분리하고 현미경 하에서 발달단계를 확인한 후 배아를 낱개로 분리하여 노출실험에 사용하였다.
2.2. 부화율 측정
노출실험에 사용된 BDE-47,-99, -209은 Accustandard 사에서 구입하여 사용하였다. PBDEs 노출에 따른 부화율 실험의 경우 발달단계 중 4단계와 8단계의 배아를 분류하여 24 well plate에 mL당 1개의 배아가 포함되도록 하여 5, 50 100 ㎍/L로 노출하였다. UV를 조사한 PBDEs 노출에 따른 부화율 실험의 경우 PBDEs를 solar simulator(Sunset CPS, Heraeus Instruments)를 이용하여 270 w/m2 로 25℃에서 1시간 동안 UV를 조사하였으며, 부화율 실험에서와 동일한 배아 밀도로 배아발달 7단계에서 5, 50 ㎍/L로 노출하여 부화율을 측정하였다. 모든 실험에 대조구(0.1% dimethylsulfoxide, DMSO)를 포함하여 실시하였으며, 노출 온도는 28℃를 유지하였다.
2.3. Single-cell gel electrophoresis (comet) assay
DNA 손상차이를 측정하기 위해 간단하고 빠르며, 높은 감도로 측정할 수 있는 방법으로 알려진(McKelvey-Martin et al., 1993), 단일세포 전기영동법(single cell gel electrophoresis, 일명 comet assay)을 이용하여 DNA 손상차이를 측정하였으며, 실험방법은 다음과 같다.
발달 7단계의 배아를 수집하여 페트리디쉬에 10 embryos/4 mL 밀도로 PBDEs 5, 50 ㎍/L로 24시간 노출한 뒤 comet assay를 실시하였다. 부화율 실험에서와 동일하게 UV를 조사한 PBDEs에 대해 동일한 노출농도에서 반복 실험하였다. 실험에 사용한 모든 시약은 Sigma 또는 Fisher Scientific에서 구매하여 사용하였으며, comet assay는 Singh et al. (1988)의 실험방법을 수정하여 실시하였다. 실험을 실시하기 전 모든 슬라이드는 TAE 용액(0.04M trisacetate, 1mM EDTA)에 1% normal melting point agarose로 1차 코팅하여 사용하였다. 노출시킨 배아를 조직분쇄기로 분쇄하고 세포를 추출하여 원심분리용 튜브에 옮겨 담았다. 원심분리 후 상등액을 제거하고, 분리되어진 세포들은 Kenny’s salt solution (0.4 M NaCl, 9 mM KCl, 0.7 mM K2HPO4, 2 mM NaHCO3) 에 용해된 0.65% low-melting agarose gel(LMA) 30㎕로 재부유 시킨 후, 1차 코팅된 슬라이드에 놓고 커버글라스를 덮었다 (2차 코팅). 슬라이드를 얼음 위에서 3분간 응고시킨 후 다시한번 0.65% LMA로 3차 코팅하여 다시 슬라이드를 얼음 위에서 3분간 응고시켰다. 응고시킨 슬라이드는 커버글라스를 제거한 후 lysis buffer solution(2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris–HCl, 10% dimethyl sulfoxide, 1% Triton X-100)에 넣고 2시간 이상 냉장 보관하였다. 그 후, 4℃ 증류수에 슬라이드를 2분 간격으로 3번 옮기면서 염을 제거한 뒤, DNA unwinding buffer solution(0.2 N NaOH, 1 mM EDTA, >pH 13)이 담긴 coplin jar에 슬라이드를 넣어 15분간 냉장 보관하였다. 그 다음 슬라이드를 동일한 DNA unwinding buffer solution으로 채워진 전기영동기에 넣고 25분간 25V, 300 mA에서 전기영동을 실시하였다. 전기 영동한 슬라이드를 0.4 M Tris 용액(pH 7.5)에 2분씩 3회 반복 세척하였다. 최종적으로 슬라이드를 찬 에탄올에 5분간 넣어둔 후 실온에서 건조시켰다. 건조된 슬라이드는 ethidium bromide 용액(20㎍/mL)으로 염색시켜 형광현미경(Nikon Eclipse E200)으로 200배 배율에서 관찰하였다. 염색되어진 DNA의 영상을 CCD 카메라를 통해 컴퓨터로 불러들인 후, 이미지 자동분석 소프트웨어(Komet version 5, Kinet Imaging Ltd)를 이용하여 DNA tail moment(부서진 DNA 꼬리의 길이(㎛)×꼬리부분에 있는 DNA(%))를 계산하였다.
2.4. Data analysis
각 슬라이드 당 50개의 핵을 무작위로 선정하여 분석하였으며, 이들로부터 얻어진 자료의 평균과 표준오차를 구하였다. 대조구와 처리구와의 차이는 student t-test를 통하여 산출하였으며, 유의수준(p)은 0.05를 기준으로 하였다. |
생식독성 번역(영어 번역본)2. Materials and Methods
2.1. Grass Shrimp Embryos
Grass shrimp (Palaemonetes pugio) embryos were collected in June 2009 at the mouth of Skidaway River(30º95‘N-81º43’W) in Savannah, Georgia. A fertilized grass shrimp embryo covered in an egg sac attaches to the female’s outer abdomen and develops, and the developmental process is morphologically divided into 10 distinct phases. The embryos were separated from female outer abdomens, had their developmental phases identified with a microscope, and finally divided in single embryos to be used in the exposure experiment.
2.2. Hatching Rate Measurement
BDE-47,-99, -209 used in the exposure experiment were purchased from AccuStandard. For the hatching rate experiment based on PBDEs exposure, Phase 4 and Phase 8 embryos were respectively placed in 24 well plates with 1 embryo/mL density and were exposed to 5, 50 100 ㎍/L concentrations. For the hatching rate experiment based on UV-radiated PBDEs exposure, PBDEs were UV-radiated for an hour using a solar simulator(Sunset CPS, Heraeus Instruments) on 270 w/m2 at 25℃, and Phase 7 embryos were exposed to 5, 50 ㎍/L concentrations using the same embryo density as the hatching rate experiment. A control group (0.1% dimethylsulfoxide, DMSO) was included in all experiments, and the exposure temperature was maintained at 28℃.
2.3. Single-cell Gel Electrophoresis (Comet Assay)
To measure difference in DNA damage, single-cell gel electrophoresis (comet assay), a simple, fast method that can measure with high sensitivity (McKelvey-Martin et al., 1993), was used, and the experiment method is as follows.
Phase 7 embryos were collected and placed in petri dishes with 10 embryos/4 mL density, and a comet assay was conducted after exposing them to PBDEs with 5, 50 ㎍/L for 24 hours. Like the hatching rate experiment, the experiment was repeated in the same exposure concentration to UV-radiated PBDEs. All reagents used in the experiment were purchased from Sigma or Fisher Scientific, and the comet assay was conducted based on a modification of the test method from Singh et al. (1988). Before conducting the experiment, all slides were initially coated with 1% normal melting point agarose in TAE solution (0.04M trisacetate, 1mM EDTA). The exposed embryos were grinded with a tissue grinder, and the cells were extracted and put in a centrifuge tube. After centrifugation, the supernatant was removed and the separated cells were re-suspended with 30㎕ of 0.65% low-melting agarose gel(LMA) dissolved in Kenny’s salt solution (0.4 M NaCl, 9 mM KCl, 0.7 mM K2HPO4, 2 mM NaHCO3). The cells were put on the initially coated slides and had cover glasses placed on top of them (2nd coating). After solidifying the slides on ice for 3 minutes, they were coated again with 0.65% LMA (3rd coating) and were solidified on ice for another 3 minutes. After the solidified slides had their cover glasses removed, they were put in a lysis buffer solution(2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris–HCl, 10% dimethyl sulfoxide, 1% Triton X-100) and refrigerated for more than 2 hours. Afterwards, salt was removed from the slides by moving them around in 4℃ distilled water 3 times with 2 minute intervals, and then the slides were put in a coplin jar that contains DNA unwinding buffer solution(0.2 N NaOH, 1 mM EDTA, >pH 13) and refrigerated for 15 minutes. The slides were put in an electrophoresis machine filled with the same DNA unwinding buffer solution and were electrophoresed for 25 minutes at 25V, 300 mA. The electrophoresed slides were repeatedly washed 3 times for 2 minutes each in 0.4 M Tris solution (pH 7.5). Finally, the slides were put in cold ethanol for 5 minutes and then dried in room temperature. The dried slides were dyed with ethidium bromide solution (20㎍/mL) and observed with a fluorescent microscope (Nikon Eclipse E200) at 200 times magnification. After uploading the image of the dyed DNA onto a computer with a CCD camera, the DNA tail moment (damaged DNA tail length (㎛)×fraction of total DNA(%)) was computed using an image auto-analysis software (Komet version 5, Kinet Imaging Ltd).
2.4. Data Analysis
50 nuclei from each slide were randomly selected and analyzed, and the average and standard deviation of the obtained data were computed. The difference between the control and treatment groups was computed using the Student’s t-test, with significance level (p) at 0.05. |
