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沿海地区核设施的建设使得水生生态环境成为放射性污染物的主要接受者,核电正常运行时的液态流出物或事故后释出的大量放射性污染物都会对海洋生态环境产生危害。近年来,由于核电排污的增加,导致氚的排放量增加,并引起了社会各界的广泛关注。此外,氚可以迅速融入环境和生物系统,因此研究电离辐射在一定剂量范围内对水生生物的影响很有必要。氚(3H)是氢元素的放射性同位素之一,会随核反应装置中的冷却水排放入环境[1]。氚流动性极强,可通过食物链进入人体,对人类健康造成影响[2-3]。水生模式动物斑马鱼的基因与人类基因有着高达87%的相似度[4]。有研究结果表明,对非人类物种的研究结果可在一定程度上外推至人体[5]。针对斑马鱼早期发育阶段氚暴露的生物效应也有诸多研究,其结果显示氚暴露可诱发斑马鱼生长发育的改变[6-8]。此外,鱼类早期发育阶段被认为是其生命周期中最敏感的发育时期,可用作评估污染物毒性作用[9-10]。因此,本研究选择处于早期发育阶段的斑马鱼为研究对象,在前期斑马鱼辐射剂量评估的理论基础上[11],尝试将观察到的氚水对斑马鱼的辐射生物学效应与理论剂量计算相结合,以研究剂量-效应关系。
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野生型斑马鱼(AB品系)购于苏州木芮生物科技有限公司。实验经苏州大学伦理委员会同意,符合《实验动物护理和使用指南》的要求。
甲基纤维素(M0387-100G)由美国Sigma-Aldrich®公司提供;氚水(5 mCi,185 MBq)由美国PerkinElmer股份有限公司提供;闪烁液(UItima Gold LLT, PerkinElmer)由美国PerkinElmer股份有限公司提供。甲基纤维素溶液、E3溶液由本实验室自行制备。
根据购买氚水的出厂时间,计算配置溶液时氚水的活度,使用E3溶液分别稀释得到3.7×103、3.7×104 、3.7×105 Bq/ml实验用氚水。
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分别取培养皿中3.7×103、3.7×104、3.7×105 Bq/ml 3种浓度氚水各4.5 ml,与6.5 ml闪烁液于20 ml液闪瓶中混合均匀,避光8~12 h。使用检出限为5 Bq/L的液体闪烁计数器(Tri-Carb 2910TR,Quantulus 1220,美国PerkinElmer股份有限公司)测量。使用公式(1)对氚样品活度浓度进行计算:
$ \mathrm{A}=\frac{\mathrm{c}\mathrm{p}\mathrm{m}_{\mathrm{s}}-\mathrm{c}\mathrm{p}\mathrm{m}_{\mathrm{b}}}{\mathrm{V}\cdot\eta} $ 式中,A为氚样品的活度浓度,单位 Bq/ml;cpms为待测氚样品总计数率,单位计数每秒;cpmb为本底样品总计数率,单位计数每秒;V为测量所用溶液的体积,单位为ml;
$ \eta $ 为计数效率,无量纲。根据生物实验中所关注的发育阶段及斑马鱼发育形态的差异,选用斑马鱼受精后24 h(24 hours post fertilization,24 hpf)胚胎和96 hpf幼鱼为建模对象,使用前期建立的胚胎和幼鱼模型[11]。考虑到散射影响,本研究模拟斑马鱼在培养皿中的实际情况建立相应模型:培养皿直径为90 mm,高度为20 mm,内有30枚斑马鱼胚胎或10条斑马鱼幼鱼和25 ml溶液(根据模拟条件不同有所变化,具体如下文所述)。模拟内照射条件时,斑马鱼胚胎和幼鱼几何条件不变,培养皿中溶液为水,氚在胚胎、幼鱼头部、躯干及卵黄囊中均匀分布,源为各向同性。模拟外照射时,氚在溶液中均匀分布,源为培养皿中圆柱体体积源,直径与培养皿内径相同,源为各向同性。表1为斑马鱼材料组成,游囊内气体以空气替代。应用蒙特卡罗软件GATE (the geant4 application for tomographic emission)8.2版,结合实验条件对斑马鱼吸收剂量率进行计算[12]。
发育
阶段器官或
组织体积
(cm3)密度
(g/cm3)材料组成 96 hpf 幼鱼 头 0.0002 1.10 C、H、N、O、Na、P、S、Cl 身体 0.0005 1.10 C、H、N、O、Na、P、S、Cl 24 hpf 胚胎 游囊 0.0001 0.00129 N、O、Ar、C 受精卵 0.0022 1.00 H、O 卵黄囊 0.0004 1.04 C、H、N、O、Na、P、S、Cl、K 注:96 hpf为受精后96 h;24 hpf为受精后24 h 表 1 斑马鱼受精后24 h胚胎和96 h幼鱼模型的器官体 积、密度和组成
Table 1. Volume, density and composition of organs of zebrafish 24 hours post fertilization embryos and 96 hours post fertilization larvae models
建立斑马鱼早期发育阶段氚染毒剂量计算模型(图1)。
图 1 早期发育阶段斑马鱼受精后24 h胚胎和受精后96 h幼鱼氚染毒剂量计算模型
Figure 1. A model for the calculation of tritium contamination dose for 24 hous post fertilization embryos and 96 hours post fertilization larvae of zebrafish at early developmental stages
根据公式(2)计算斑马鱼的吸收剂量率:
$ \mathrm{D}=\left(\mathrm{D}\mathrm{C}_{\mathrm{e}\mathrm{x}\mathrm{t}}\cdot\mathrm{A}_{\mathrm{w}}+\mathrm{D}\mathrm{C}_{\mathrm{i}\mathrm{n}\mathrm{t}}\cdot\mathrm{A}_{\mathrm{o}}\right)\times10^3 $ 式中,D为剂量率,单位μGy/h;DCext为外照射剂量系数,单位μGy·L·Bq−1·h−1;DCint为内照射剂量系数,单位μGy·kg·Bq−1·h−1;Aw为水中氚的活度浓度,单位Bq/L;Ao为生物体中氚的活度浓度,单位Bq/kg。
实际实验中,生物体内氚活度由于条件限制难以测量,因此采用浓度比的方法进行计算。本研究中采用Arcanjoa等[13]文献中的浓度比公式进行计算。
$ \mathrm{C}\mathrm{F}=\frac{{\mathrm{A}}_{\mathrm{o}}}{{\mathrm{A}}_{\mathrm{w}}} $ 式中,CF为浓度比,也称为浓集因子,无量纲。定义为生物体内放射性核素的活度浓度与环境介质中放射性核素的活度浓度的比值。Ao为生物体中氚的活度浓度,单位Bq/kg;Aw为水中氚的活度浓度,单位Bq/L。
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取10对成年斑马鱼,雌雄1∶1配比放入生殖缸,待受精后收集胚胎,28℃恒温保存在E3培养液中。对照组为E3溶液,处理组氚水的3种浓度分别为3.7×103、3.7×104 、3.7×105 Bq/ml。将3 hpf胚胎分别暴露于E3溶液和 3种不同浓度的氚水中,每组50枚,并重复3次实验。
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受精后24 h,在上述3种不同浓度的氚水中用简单随机抽样方法各选取30枚斑马鱼胚胎,体式显微镜(中国麦克奥迪实业集团有限公司,Motic SMZ-168)测定胚胎1 min翻转次数。
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受精后96 h,在上述3种不同浓度的氚水中用简单随机抽样方法各选取10条斑马鱼幼鱼,将其放入甲基纤维素中进行固定,测定20 s心率。各阶段结果取平均值。
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应用SPSS 20.0软件对数据进行统计学分析。符合正态分布的数据以
$\bar x \pm s $ 表示。多组间比较采用单因素方差分析,对照组与处理组之间的比较采用LSD-t检验(方差齐)。P<0.05为差异有统计学意义。 -
由表2可知,氚活度浓度的测定结果与理论浓度值的误差在5%以内, 24 hpf斑马鱼胚胎和96 hpf幼鱼的内照射剂量系数一致,外照射剂量系数均高于内照射剂量系数,这可能是因为实验条件无法满足“均匀各项同性模型”的假设条件,培养皿的材料中产生了一定的散射线所致;外照射剂量系数96 hpf幼鱼要略高于24 hpf胚胎,这可能与外形和材料组成的不同有关。因此,虽然斑马鱼幼鱼氚的实际浓度比较低,但是最终氚的吸收剂量率幼鱼略高于胚胎。
发育阶段 理论浓度( Bq/ml) 测量浓度Aw( , Bq/ml)$\bar x \pm s $ DCe×t(μGy·L·Bq−1·h−1) DCint(μGy·kg·Bq−1·h−1) CF 吸收剂量率(μGy/h) 24 hpf 胚胎 3.7×103 3.15×103±1.27×102 4.67×10−6 3.27×10−6 0.66 2.15×10 3.7×104 3.23×104±6.33×102 2.21×102 3.7×105 3.74×105±3.21×103 2.55×103 96 hpf 幼鱼 3.7×103 3.05×103±1.86×102 7.68×10−6 3.27×10−6 0.61 2.95×10 3.7×104 3.14×104±9.48×102 3.03×102 3.7×105 3.59×105±4.84×103 3.47×103 注:24 hpf为受精后24 h;96 hpf为受精后96 h; 为氚活度浓度;$ {\mathrm{A}}_{\mathrm{w}} $ 为外照射剂量系数;$ {\mathrm{D}\mathrm{C}}_{\mathrm{e}\mathrm{x}\mathrm{t}} $ 为内照射剂量系数;CF为浓度比$ {\mathrm{D}\mathrm{C}}_{\mathrm{i}\mathrm{n}\mathrm{t}} $ 表 2 受精后24 h斑马鱼胚胎与96 h幼鱼在不同浓度氚水中的测量浓度、浓度比、内外照射剂量系数和吸收剂量率
Table 2. Measured concentration, concentration ratio, internal and external radiation dose coefficient and absorbed dose rate after exposure of 24 hours post fertilization embryos and 96 hours post fertilization larvae of zebrafish in different concentrations of tritium water
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由表2可知,24 hpf斑马鱼胚胎在3.7×103、3.7×104、3.7×105 Bq/ml 3种不同浓度的氚水中对应的吸收剂量率分别为2.15×10、2.21×102、2.55×103 μGy/h。氚水暴露后,斑马鱼胚胎翻转频率和斑马鱼幼鱼心率结果如图2所示。各组间斑马鱼胚胎翻转频率的差异有统计学意义(F=7.64,P<0.001),与对照组相比,3.7×103 Bq/ml氚水处理组斑马鱼胚胎翻转频率明显减少(t=3.94,P<0.001);3.7×104 Bq/ml氚水处理组斑马鱼胚胎翻转频率虽有升高趋势,但差异无统计学意义(t=−0.06,P=0.95);3.7×105 Bq/ml氚水处理组斑马鱼胚胎翻转频率无明显改变(t=0.17,P=0.87)。
图 2 氚水对受精后24 h斑马鱼胚胎翻转频率和受精后96 h斑马鱼幼鱼心率的影响
Figure 2. Effects of tritiated water on the frequency of zebrafish embryo flipping at 24 hours post fertilization and on the heart rate of zebrafish larvae at 96 hours post fertilization
96 hpf斑马鱼幼鱼在3种不同浓度的氚水暴露下,相对应的剂量率分别为2.95×10、3.03×102、3.47×103 μGy/h,3组间斑马鱼幼鱼心率的比较,差异有统计学意义(F=93.85,P<0.001)。由图2可见,与对照组相比,3.7×103 Bq/ml氚水处理组斑马鱼幼鱼心率明显下降(t=2.86,P=0.01),而3.7×105 Bq/ml氚水处理组斑马鱼幼鱼心率上升(t=−12.12,P<0.001),3.7×104 Bq/ml氚水处理组斑马鱼幼鱼的心率无明显变化(t=1.27,P=0.21)。
氚水对斑马鱼早期发育阶段的剂量-效应关系研究
Study on the dose-effect relationship of tritium water in the early growth and development of zebrafish
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摘要:
目的 研究氚水对早期发育阶段的斑马鱼造成的辐射剂量,并与其造成的生物效应相结合,初步观察剂量-效应关系。 方法 模拟真实实验条件,建立物理模型,计算斑马鱼受精后24 h(24 hpf )胚胎和 96 hpf 幼鱼在 3.7×103、3.7×104、 3.7×105 Bq/ml 3种不同浓度氚水中的吸收剂量率。观察经3种不同浓度氚水染毒后24 hpf 胚胎和 96 hpf 幼鱼的翻转频率和心率,并与对照组(E3溶液)进行比较。多组间比较采用单因素方差分析,对照组与处理组之间的比较采用 LSD-t 检验。 结果 随着斑马鱼发育时间的延长,其吸收剂量有所提高。24 hpf 斑马鱼胚胎在3种不同浓度的氚水中对应的吸收剂量率分别为2.15×10、2.21×102、2.55×103 μGy/h;96 hpf 斑马鱼幼鱼对应的吸收剂量率分别为 2.95×10、3.03×102、 3.47×103 μGy/h。24 hpf 斑马鱼胚胎翻转实验结果显示,与对照组比较,3.7×103 Bq/ml 氚水处理组的翻转频率明显减少(t=3.94,P<0.001)。96 hpf 斑马鱼幼鱼心率随着不同氚水浓度的变化明显,与对照组比较,3.7×103 Bq/ml 氚水处理组心率明显下降(t=2.86,P=0.01),而 3.7×105 Bq/mL 氚水处理组心率上升(t=−12.12,P<0.001)。 结论 随着吸收剂量率的变化,24 hpf 斑马鱼胚胎翻转频率和 96 hpf斑马鱼幼鱼心率均出现显著改变 。 Abstract:Objective To study the effect of tritium water radiation dose on zebrafish at early developmental stages and integration with biological effects for the preliminary observation of dose–effect relationships. Methods The real experimental conditions were simulated, the physical conditions were modeled, and the absorbed dose rates were calculated for 24 hours post fertilization (hpf)embryos and 96 hpf larvae in three different concentrations of tritium water: 3.7×103, 3.7×104, and 3.7×105 Bq/ml. The embryos or larvae turnover frequency and heart rate of 24 and 96 hpf were observed and compared with those of the control group. Comparisons between multiple groups were analyzed by one-way ANOVA, and differences between the control and treatment groups were compared for significance using the LSD-t test. Results With the extension of development time, the absorbed dose of zebrafish increased in three different concentrations of tritiated water. 24 hpf embryos corresponding to dose rates of 2.15×10, 2.21×102, 2.55×103 μGy/h; 96 hpf larvae corresponding to dose rates of 2.95×10, 3.03×102, 3.47×103 μGy/h. Comparison with control group, the results of 24 hpf embryo flipping showed that the significantly fewer embryos turned over in the tritium water-stained group whith 3.7×103 Bq/ml( t=3.94, P<0.001); and 96 hpf larvae heart rate changes significantly with tritium water concentration. Comparison with control group, the 3.7×103 Bq/ml tritium water-treated group had a significant decrease in heart rate(t=2.86, P=0.01), while the 3.7×105 Bq/ml tritium water-treated group had an increase in heart rate (t=−12.12, P<0.001). Conclusion Significant changes in embryonic turnover at 24 hpf and heart rate of larvae at 96 hpf were observed with changes in absorbed dose rate. -
Key words:
- Zebrafish /
- Tritium /
- Dose-response relationship, radiation
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表 1 斑马鱼受精后24 h胚胎和96 h幼鱼模型的器官体 积、密度和组成
Table 1. Volume, density and composition of organs of zebrafish 24 hours post fertilization embryos and 96 hours post fertilization larvae models
发育
阶段器官或
组织体积
(cm3)密度
(g/cm3)材料组成 96 hpf 幼鱼 头 0.0002 1.10 C、H、N、O、Na、P、S、Cl 身体 0.0005 1.10 C、H、N、O、Na、P、S、Cl 24 hpf 胚胎 游囊 0.0001 0.00129 N、O、Ar、C 受精卵 0.0022 1.00 H、O 卵黄囊 0.0004 1.04 C、H、N、O、Na、P、S、Cl、K 注:96 hpf为受精后96 h;24 hpf为受精后24 h 表 2 受精后24 h斑马鱼胚胎与96 h幼鱼在不同浓度氚水中的测量浓度、浓度比、内外照射剂量系数和吸收剂量率
Table 2. Measured concentration, concentration ratio, internal and external radiation dose coefficient and absorbed dose rate after exposure of 24 hours post fertilization embryos and 96 hours post fertilization larvae of zebrafish in different concentrations of tritium water
发育阶段 理论浓度( Bq/ml) 测量浓度Aw( , Bq/ml)$\bar x \pm s $ DCe×t(μGy·L·Bq−1·h−1) DCint(μGy·kg·Bq−1·h−1) CF 吸收剂量率(μGy/h) 24 hpf 胚胎 3.7×103 3.15×103±1.27×102 4.67×10−6 3.27×10−6 0.66 2.15×10 3.7×104 3.23×104±6.33×102 2.21×102 3.7×105 3.74×105±3.21×103 2.55×103 96 hpf 幼鱼 3.7×103 3.05×103±1.86×102 7.68×10−6 3.27×10−6 0.61 2.95×10 3.7×104 3.14×104±9.48×102 3.03×102 3.7×105 3.59×105±4.84×103 3.47×103 注:24 hpf为受精后24 h;96 hpf为受精后96 h; 为氚活度浓度;$ {\mathrm{A}}_{\mathrm{w}} $ 为外照射剂量系数;$ {\mathrm{D}\mathrm{C}}_{\mathrm{e}\mathrm{x}\mathrm{t}} $ 为内照射剂量系数;CF为浓度比$ {\mathrm{D}\mathrm{C}}_{\mathrm{i}\mathrm{n}\mathrm{t}} $ -
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