久效磷农药对黄鳝染色体损伤的研究❋

冯永亮， 闫建国， 赵　飞， 汝少国

(中国海洋大学海洋生命学院，山东 青岛 266003)



久效磷农药对黄鳝染色体损伤的研究❋

冯永亮， 闫建国， 赵飞， 汝少国❋❋

(中国海洋大学海洋生命学院，山东 青岛 266003)

摘要：以0.25、0.50、1.00和2.00 mg/L浓度久效磷农药暴露黄鳝96 h，采用微核试验和染色体畸变试验方法研究了久效磷农药对外周血红细胞和肾细胞染色体的损伤作用。结果表明：0.50～2.00和0.25～2.00 mg/L的久效磷农药暴露显著升高了红细胞核异常细胞率及总核异常细胞率；1.00和2.00 mg/L暴露组肾细胞的染色体数目总异常率和染色体裂隙率显著升高，0.50～2.00 mg/L暴露组染色体结构总畸变率显著升高；2.00 mg/L暴露组肾脏组织RNA含量及RNA/DNA比值显著降低。结果表明久效磷农药能够引起黄鳝染色体损伤，导致遗传毒性效应。

关键词：久效磷农药；黄鳝；遗传毒性；染色体损伤

FENG Yong-Liang, YAN Jian-Guo, ZHAO Fei, et al. Chromosomal damage induced by monocrotophos pesticide on theMonopterusalbus[J]. Periodical of Ocean University of China, 2016, 46(2): 69-75.

1研究背景

久效磷农药是一种用于农林业害虫防治的高毒有机磷农药，目前在印度和巴基斯坦等发展中国家仍然被广泛使用[1-2]，蔬菜中的残留量在0.023～1.140 mg/kg之间[3]，水源地中浓度为0.165 μg/L[4]，工业废水中浓度达到(8.32±3.9) μg/L[5]。残留的久效磷农药通过雨水冲刷、地表径流和食物等方式进入水环境和生物体[3,5]，造成生物体遗传物质的损伤。采用Ames实验研究发现久效磷农药能够导致鼠伤寒沙门氏菌(Salmonellatyphimurium)和大肠杆菌(Escherichiacoli)基因突变[6-7]；采用彗星电泳实验研究证实久效磷农药能够造成鱼类外周血细胞DNA链断裂，还可导致小鼠外周血细胞、人淋巴细胞以及淡水硬骨鱼Channapunctatus鳃、肾、淋巴细胞[8-11]的DNA链断裂；但关于久效磷农药暴露是否会造成鱼类的染色体损伤研究还未见报道。在东亚地区广泛分布的黄鳝(Monopterusalbus)是一种具有较高经济价值的淡水鱼类，其染色体数目(2n=24)较少，染色体形态较大且为端着丝粒染色体，有利于观察统计染色体畸变类型。因此，本研究采用微核实验和染色体畸变实验研究了久效磷农药暴露对黄鳝外周血红细胞和肾细胞染色体的损伤作用，以期为全面评价久效磷农药的遗传毒性效应提供基础数据。

2材料与方法

2.1 试验材料与动物

久效磷农药(3-hydroxyl-N-methyl-cis-crotonamide dimethyl phosphate)购自青岛农药厂，为40%水溶性制剂。鱼精DNA和酵母RNA购自Sigma公司(St. Louis, MO, USA)，其它试剂均为分析纯，购自国药集团化学试剂有限公司(Beijing, P.R.China)。

试验用黄鳝(Monopterusalbus)购自青岛市南山市场，黄鳝体长(25.3±5.9)cm，体重(20.7±9.1)g。在实验室条件下驯养7 d后用于暴露实验。

2.2 久效磷农药暴露方法与样品制备

采用半静态暴露实验，容器为70 L玻璃水族箱，盛50 L连续曝气24 h的自来水，每组2个水族箱，每箱6条鱼，共12条鱼。根据急性毒性预试验，久效磷农药暴露黄鳝96 h的LC50为3.27 mg/L，设置久效磷农药暴露为0.25、0.50、1.00、2.00 mg/L，同时设对照组。为保持久效磷农药浓度每天换水50%，并补加农药至暴露浓度。试验期间不投饵，水温保持在(20±2) ℃，溶解氧(7.0±0.1) mg/L，pH=7.6±0.2，光暗比为14∶10。暴露90 h时，为获得足够数量的处于有丝分裂中期的细胞进行染色体核型分析，取每组6条鱼进行秋水仙素(0.05%，10 μL/g体重)肌肉注射，96 h时断尾充分放血，解剖取肾脏用于染色体畸变实验。暴露96 h，另外组取6条鱼采用75 mg/L间氨基苯甲酸乙酯甲磺酸盐(MS-222; Sigma, St. Louis, MO, USA)麻醉，用1%(W/V)肝素钠溶液润洗的注射器尾静脉取血，用于微核实验；同时解剖取黄鳝肾脏组织，液氮速冻，—80 ℃保存，用于DNA、RNA提取。

2.3 微核试验

微核试验参照Fenech[12]和Palus等[13]的方法进行。制备外周血血涂片并晾干，甲醇-冰醋酸(3∶1，V∶V)固定15 min后，用5%(V∶V)的Giemsa染色15 min后晾干，用Olympus CX31显微镜(油镜，1 000×)随机观察，每组随机观察6 500个具有完整细胞膜和核膜的有核红细胞，统计微核率、核异常细胞率及总核异常细胞率。微核细胞率=带有微核的细胞数/观察的细胞总数×1 000‰；核异常细胞率=具有核异常(除微核外)的细胞总数/观察细胞的总数×1 000‰；总核异常细胞率=微核细胞率+核异常细胞率。

2.4 染色体畸变试验

染色体畸变试验参照Ansari等[14]的方法进行。取肾脏组织剪碎，置于8 mL 0.075 mol/L的KCl溶液中匀浆，室温低渗30 min后，加入1.5 mL甲醇-冰醋酸(3∶1,V∶V)预固定，1 500 r/s离心10 min后弃上清液；8 mL甲醇-冰醋酸(3∶1,V∶V)固定20 min，1 500 r/s离心10 min后弃上清液，重复3次；取细胞液滴在浸泡于60%冰乙醇的载波片上，轻吹；常规空气干燥法制片，干燥后用3%(V∶V)的Giemsa染色10 min，在Olympus CX31显微镜(油镜，1 200×)下挑选图像清晰、染色体分散良好的分裂相进行观察。

2.5 DNA、RNA含量的测定

参照杨光彩等[15]的试验方法测定肾脏DNA、RNA的含量，分别采用鱼精DNA、酵母RNA作为标准，单位为mg DNA/g组织。

2.6 数据处理

微核试验和染色体畸变试验的结果进行卡方检验，当P<0.05认为差异显著。DNA、RNA含量及RNA/DNA比值的试验结果以平均值±标准差表示，采用单因素方差分析和Tukey多重检验分析显著性，当P<0.05认为差异显著。

3结果与分析

3.1 久效磷农药对黄鳝红细胞核异常的诱导作用

对照组黄鳝红细胞的细胞核为圆形或椭圆形，细胞膜完整，见图1a；久效磷农药暴露后红细胞出现的核异常主要包括核质外凸(见图1b)，核质内凹(见图1c)，核变形(见图1d)，核内空泡(见图1e)、双核(见图1f)和无丝分裂(见图1g)等，0.50～2.00 mg/L暴露组核异常细胞率及0.25～2.00 mg/L暴露组总核异常细胞率与对照相比显著升高(P<0.05)。暴露组还诱导红细胞产生了微核，位于胞质中，为圆形或椭圆形，直径约为主核直径1/5至1/20，微核的染色深度与主核一致或略浅于主核(见图1h)，但是不同浓度久效磷农药暴露组的微核率与对照组相比均无显著性变化(见表1)。

图1　久效磷农药暴露对黄鳝外周血

3.2 久效磷农药对黄鳝染色体畸变的诱导作用

对照组黄鳝肾细胞染色体核型可知2n=24(见图2a)，久效磷农药暴露96 h诱导了染色体的非整倍体和多倍体(见图2b)的形成，1.00和2.00 mg/L暴露组染色体数目总异常率显著升高(P<0.05，见表2)；除染色体数目异常外久效磷农药暴露还导致了染色体的断片(见图2c)、裂隙(见图2d)、着丝粒环(见图2e)、着丝粒融合(见图2f)等结构畸变，1.00～2.00 mg/L暴露组染色体裂隙率、0.50～2.00 mg/L暴露组染色体结构总畸变率与对照相比显著升高(P<0.05，见表3)。

表1　不同浓度久效磷农药对黄鳝外周

注:各暴露浓度组与相应对照组间的显著性差异，以* (P<0.05)表示。An asterisk (P<0.05) denotes the significant difference between the exposure groups and control group.

表2　不同浓度久效磷农药对黄鳝肾细胞

注:各暴露浓度组与相应对照组间的显著性差异，以* (P<0.05)表示。An asterisk (P<0.05) denotes the significant difference between the exposure groups and control group.

表3　不同浓度久效磷农药对黄鳝肾细胞

注:各暴露浓度组与相应对照组间的显著性差异，以*(P<0.05)表示。An asterisk (P<0.05) denotes the significant difference between the exposure groups and control group.

3.3 对肾脏DNA、RNA含量和RNA/DNA比值的影响

由图3可知，久效磷农药暴露96 h后各暴露组黄鳝肾脏DNA含量与对照相比无显著变化，只有2.00 mg/L最高浓度暴露组RNA含量及RNA/DNA比值与对照相比显著降低(P<0.05)。

4讨论

研究发现烷化剂能够诱导染色体结构和数目异常、姐妹染色单体交换、基因突变及细胞死亡[16]，久效磷农药等有机磷农药带有2～3个烷基，具有亲电子性，作为烷化剂可能与细胞内的亲核物质反应，导致DNA、蛋白等大分子损伤。目前鱼类的研究中发现久效磷农药能够诱导基因突变和DNA损伤，但是否会诱导鱼类细胞染色体的损伤尚不清楚，本研究的结果发现久效磷农药暴露后能够造成黄鳝红细胞形成异常核型、肾细胞出现染色体数目和结构畸变，表明久效磷农药同时能够诱导鱼类染色体损伤。

图2　久效磷农药暴露对黄鳝肾细胞染色体的影响(1 200×)

微核是无着丝粒的染色体片段或因纺锤体受损而丢失的整个染色体，在细胞分裂后期仍留在子细胞的胞质内而形成的结构[17]，很多研究报道出现微核的细胞中常同时出现其他类型的核异常[18-20]。Bolognesi等[21]和Ergene等[22]发现核质外凸和微核形成之间具有正相关关系；Shimizu等[23]认为，复制后的DNA选择性地定位于细胞核外周的特定位置，通过核质外凸最终形成微核与细胞核分离，因此遗传毒性物质可能通过诱导核异常最终导致微核形成。黄鳝正常体细胞的微核率在0.17‰～1.05‰之间，核异常细胞率在7.82‰～17.44‰，总核异常细胞率在7.98‰～18.09‰之间[24-26]，本研究统计发现对照组红细胞微核率为0.31‰、核异常细胞率为10.61‰、总核异常细胞率为10.92‰，久效磷农药暴露后核异常细胞率在0.50～2.00 mg/L暴露组显著上升，而总核异常细胞率在各暴露浓度组均显著上升。Fenech和Crott[27]认为叶酸缺失能够通过损伤DNA双链、干扰DNA复制导致人淋巴细胞核质外凸、核质桥形成等核异常，核异常与微核一样是遗传毒性物质作用于染色体和纺锤体产生的一种遗传毒性效应；本研究中0.25 mg/L的久效磷农药暴露即可引起黄鳝红细胞总核异常细胞率显著升高，表明久效磷农药具有致染色体断裂剂和纺锤体毒剂作用。本研究微核率在各暴露浓度条件下与对照相比均无显著变化，可能与鱼类外周血红细胞分裂指数较低[28]有关；试验结果表明当以微核试验检测久效磷农药的遗传毒性时，核异常细胞率及总核异常细胞率比微核率具有更高的敏感性。

(各暴露浓度组与相应对照组间的显著性差异，以* (P<0.05)表示。An asterisk(P<0.05) denotes the significant difference between the exposure group and control group.

图3不同浓度久效磷农药对黄鳝肾脏DNA、RNA含量和RNA/DNA比值的影响

Fig.3Impacts of different doses of monocrotophos pesticide on the RNA and DNA contents and

RNA/DNA ratio in the kidney tissues ofMonopterusalbus

本研究发现对照组中也存在一定比例的染色体数目和结构畸变细胞，这与陈刚等[24]的结果一致；与对照组相比，0.25～2.00 mg/L久效磷暴露96 h后，黄鳝肾细胞染色体数目总异常率达到16.36%～27.78%，染色体数目的畸变主要表现为染色体非整倍体率升高，表明在细胞有丝分裂过程中久效磷农药能够作用于纺锤体微管，导致微管断裂使部分染色体在分裂过程中丢失，最终引起子细胞染色体非整倍体率升高，久效磷农药具有非整倍体诱导剂作用。采用哺乳动物作为实验动物，Bhunya和Behera[29]发现1.25～5 mg/kg的久效磷农药暴露能够导致小鼠骨髓细胞染色体结构畸变；Wang等[30]报道8.0～1 000.0 μg/mL的久效磷农药(纯度为98%)暴露能够诱导中国仓鼠卵巢(CHO)细胞姐妹染色单体交换，1 000.0 μg/mL还能诱导染色体畸变的发生；本研究中各个暴露浓度组染色体结构总畸变率达到14.55%～31.38%，久效磷农药还可直接作用于黄鳝肾细胞染色体而产生染色体断片、裂隙、着丝粒环、着丝粒融合等结构畸变，具有染色体断裂剂作用。与哺乳动物体外实验相比，本研究中0.50mg/L的久效磷农药即可诱导黄鳝肾细胞染色体结构总畸变率显著升高，表明鱼类体内暴露实验对于久效磷农药染色体损伤效应的检测更为敏感。

生物体每个细胞中的DNA含量是稳定的，而RNA含量随着蛋白合成速率的不同而不同，因而组织中RNA/DNA比值能够对代谢过程和蛋白合成过程进行表征，RNA/DNA比值能够反映污染物胁迫对体细胞生长的延迟作用[31-32]。本研究中0.25 mg/L久效磷农药暴露96 h后黄鳝肾细胞中RNA含量略有上升，可能是因为低浓度久效磷农药的胁迫上调了机体某些基因的表达、促进了蛋白的翻译和合成，从而产生某种能够抵抗外源有毒物质的应激蛋白；但当久效磷农药浓度进一步升高时，主要发挥毒性作用，最终导致暴露组中RNA的含量逐渐降低。Rath和Misra[33]的研究发现暴露于亚致死浓度的敌敌畏后，莫桑比克罗非鱼(Tilapiamossambica)肝脏DNA、RNA和蛋白质含量均下降，且RNA/DNA比值也降低，这与本文的研究结果类似；久效磷暴露96 h后肾脏DNA含量无显著性变化，这可能与生物体内的DNA含量相对比较稳定以及久效磷农药暴露时间较短有关。

5结语

本研究采用微核试验和染色体畸变试验，证实久效磷农药暴露能够损伤鱼类细胞染色体，产生遗传毒性效应。暴露后黄鳝外周血红细胞核异常细胞率和总核异常细胞率显著升高，肾细胞的染色体数目总异常率、染色体裂隙率及染色体结构总畸变率均显著性升高，久效磷农药具有染色体断裂剂、纺锤体毒剂、非整倍体诱导剂和染色体断裂剂作用。此外，2.00 mg/L久效磷农药暴露还能显著降低肾脏组织RNA含量及RNA/DNA比值。

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责任编辑高蓓

Chromosomal Damage Induced by Monocrotophos Pesticide on theMonopterusalbus

FENG Yong-Liang, YAN Jian-Guo, ZHAO Fei, RU Shao-Guo

(College of Marine Life Science, Ocean University of China, Qingdao 266003, China)

Abstract:Monocrotophos is a high-toxic organophosphorus pesticide used for pest control in agriculture and forestry, and is still widely used in developing countries including India and Pakistan. Its residues in aquatic environment, with concentrations of 0.165～8.32 μg/L, can be absorbed by organisms via the food chain, and thereby induce genotoxicity. Researches have confirmed the gene mutation and DNA strand breaks caused by monocrotophos, the chromosomal damage produced by this pesticide, however, has not been reported in fish. Therefore, using Monopterus albus as the model animal, chromosomal damage caused by monocrotophos were investigated by performing the micronucleus test in the peripheral erythrocytes and the chromosome aberration test in the kidney cells. In the micronucleus test, nuclear abnormalities including blebbed, notched, deformed, and vacuolated nuclei, and binucleated cells and amitosis were observed after monocrotophos exposure. Results showed that the frequencies of erythrocytic nuclear abnormalities and total nuclear abnormalities, in the 0.50～2.00 mg/L and 0.25～2.00 mg/L treatment groups respectively, increased significantly compared with the control. The frequencies of micronuclei, however, exhibited no significant changes in treatment groups. Genotoxic substances may lead to the formation of micronuclei by inducing nuclear abnormalities, the presence of which could also be considered as an indicator of genotoxic effects. Therefore, our data indicated monocrotophos’ potential as a clastogen and a spindle toxin. In the chromosomal aberration test, the frequencies of the total numerical chromosome aberration were significantly increased by exposure of 1.00 and 2.00 mg/L monocrotophos. Since more cells exhibited an aneuploid chromosomal pattern compared with the multiploid pattern, the results implied that monocrotophos might act on the spindle microtubules in the progress of mitosis and lead to the loss of chromosomes, and consequently caused aneuploidy in cells. In addition, structural chromosomal aberrations including chromosomal fragmentation and gap, centric ring, and centric fusion were also induced by monocrotophos exposure. The frequencies of the chromosomal gap in the kidney cells and those of the total structural chromosomal aberration were also significantly elevated by exposure of 0.50～2.00 mg/L pesticide. Compared with a previous study conducting in Chinese Hamster Ovary cells, the effective concentration of monocrotophos to induce chromosomal aberration were much lower in our study, suggesting a higher sensitivity of fish cells to monocrotophos exposure. Impact of monocrotophos exposure on the somatic growth of fish cell was also investigated in this study, and results found a significant decrease in both RNA content and RNA/DNA ratio in the kidney tissue, revealing the pesticide’s adverse effect on fish growth. In conclusion, this study reported the chromosomal damage caused by monocrotophos in fish for the first time, and results of the present study further confirmed the genotoxicity of this pesticide.

Key words:monocrotophos pesticide； Monopterus albus； genotoxicity； chromosomal damage

DOI：10.16441/j.cnki.hdxb.20150116

中图法分类号：X174

文献标志码：A

文章编号：1672-5174(2016)02-069-07

作者简介：冯永亮(1987-)，男，博士，主要研究方向为生态学。E-mail：yongliangfeng0511@126.com❋❋通讯作者：E-mail：rusg@ouc.edu.cn

收稿日期：2015-04-01；

修订日期：2015-08-31

基金项目：❋国家自然科学基金项目(31202001)资助

引用格式：冯永亮， 闫建国， 赵飞， 等. 久效磷农药对黄鳝染色体损伤的研究[J]. 中国海洋大学学报(自然科学版), 2016, 46(2): 69-75.

Supported by National Natural Science Foundation of China (31202001)