久效磷農藥對黃鱔染色體損傷的研究?

馮永亮， 閆建國， 趙　飛， 汝少國

(中國海洋大學海洋生命學院，山東 青島 266003)

?

久效磷農藥對黃鱔染色體損傷的研究?

馮永亮， 閆建國， 趙飛， 汝少國??

(中國海洋大學海洋生命學院，山東 青島 266003)

摘要：以0.25、0.50、1.00和2.00 mg/L濃度久效磷農藥暴露黃鱔96 h，采用微核試驗和染色體畸變試驗方法研究了久效磷農藥對外周血紅細胞和腎細胞染色體的損傷作用。結果表明：0.50～2.00和0.25～2.00 mg/L的久效磷農藥暴露顯著升高了紅細胞核異常細胞率及總核異常細胞率；1.00和2.00 mg/L暴露組腎細胞的染色體數目總異常率和染色體裂隙率顯著升高，0.50～2.00 mg/L暴露組染色體結構總畸變率顯著升高；2.00 mg/L暴露組腎臟組織RNA含量及RNA/DNA比值顯著降低。結果表明久效磷農藥能夠引起黃鱔染色體損傷，導致遺傳毒性效應。

關鍵詞：久效磷農藥；黃鱔；遺傳毒性；染色體損傷

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

久效磷農藥是一種用于農林業害蟲防治的高毒有機磷農藥，目前在印度和巴基斯坦等發展中國家仍然被廣泛使用[1-2]，蔬菜中的殘留量在0.023～1.140 mg/kg之間[3]，水源地中濃度為0.165 μg/L[4]，工業廢水中濃度達到(8.32±3.9) μg/L[5]。殘留的久效磷農藥通過雨水沖刷、地表徑流和食物等方式進入水環境和生物體[3,5]，造成生物體遺傳物質的損傷。采用Ames實驗研究發現久效磷農藥能夠導致鼠傷寒沙門氏菌(Salmonellatyphimurium)和大腸桿菌(Escherichiacoli)基因突變[6-7]；采用彗星電泳實驗研究證實久效磷農藥能夠造成魚類外周血細胞DNA鏈斷裂，還可導致小鼠外周血細胞、人淋巴細胞以及淡水硬骨魚Channapunctatus鰓、腎、淋巴細胞[8-11]的DNA鏈斷裂；但關于久效磷農藥暴露是否會造成魚類的染色體損傷研究還未見報道。在東亞地區廣泛分布的黃鱔(Monopterusalbus)是一種具有較高經濟價值的淡水魚類，其染色體數目(2n=24)較少，染色體形態較大且為端著絲粒染色體，有利于觀察統計染色體畸變類型。因此，本研究采用微核實驗和染色體畸變實驗研究了久效磷農藥暴露對黃鱔外周血紅細胞和腎細胞染色體的損傷作用，以期為全面評價久效磷農藥的遺傳毒性效應提供基礎數據。

2材料與方法

2.1 試驗材料與動物

久效磷農藥(3-hydroxyl-N-methyl-cis-crotonamide dimethyl phosphate)購自青島農藥廠，為40%水溶性制劑。魚精DNA和酵母RNA購自Sigma公司(St. Louis, MO, USA)，其它試劑均為分析純，購自國藥集團化學試劑有限公司(Beijing, P.R.China)。

試驗用黃鱔(Monopterusalbus)購自青島市南山市場，黃鱔體長(25.3±5.9)cm，體重(20.7±9.1)g。在實驗室條件下馴養7 d后用于暴露實驗。

2.2 久效磷農藥暴露方法與樣品制備

采用半靜態暴露實驗，容器為70 L玻璃水族箱，盛50 L連續曝氣24 h的自來水，每組2個水族箱，每箱6條魚，共12條魚。根據急性毒性預試驗，久效磷農藥暴露黃鱔96 h的LC50為3.27 mg/L，設置久效磷農藥暴露為0.25、0.50、1.00、2.00 mg/L，同時設對照組。為保持久效磷農藥濃度每天換水50%，并補加農藥至暴露濃度。試驗期間不投餌，水溫保持在(20±2) ℃，溶解氧(7.0±0.1) mg/L，pH=7.6±0.2，光暗比為14∶10。暴露90 h時，為獲得足夠數量的處于有絲分裂中期的細胞進行染色體核型分析，取每組6條魚進行秋水仙素(0.05%，10 μL/g體重)肌肉注射，96 h時斷尾充分放血，解剖取腎臟用于染色體畸變實驗。暴露96 h，另外組取6條魚采用75 mg/L間氨基苯甲酸乙酯甲磺酸鹽(MS-222; Sigma, St. Louis, MO, USA)麻醉，用1%(W/V)肝素鈉溶液潤洗的注射器尾靜脈取血，用于微核實驗；同時解剖取黃鱔腎臟組織，液氮速凍，—80 ℃保存，用于DNA、RNA提取。

2.3 微核試驗

微核試驗參照Fenech[12]和Palus等[13]的方法進行。制備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顯微鏡(油鏡，1 000×)隨機觀察，每組隨機觀察6 500個具有完整細胞膜和核膜的有核紅細胞，統計微核率、核異常細胞率及總核異常細胞率。微核細胞率=帶有微核的細胞數/觀察的細胞總數×1 000‰；核異常細胞率=具有核異常(除微核外)的細胞總數/觀察細胞的總數×1 000‰；總核異常細胞率=微核細胞率+核異常細胞率。

2.4 染色體畸變試驗

染色體畸變試驗參照Ansari等[14]的方法進行。取腎臟組織剪碎，置于8 mL 0.075 mol/L的KCl溶液中勻漿，室溫低滲30 min后，加入1.5 mL甲醇-冰醋酸(3∶1,V∶V)預固定，1 500 r/s離心10 min后棄上清液；8 mL甲醇-冰醋酸(3∶1,V∶V)固定20 min，1 500 r/s離心10 min后棄上清液，重復3次；取細胞液滴在浸泡于60%冰乙醇的載波片上，輕吹；常規空氣干燥法制片，干燥后用3%(V∶V)的Giemsa染色10 min，在Olympus CX31顯微鏡(油鏡，1 200×)下挑選圖像清晰、染色體分散良好的分裂相進行觀察。

2.5 DNA、RNA含量的測定

參照楊光彩等[15]的試驗方法測定腎臟DNA、RNA的含量，分別采用魚精DNA、酵母RNA作為標準，單位為mg DNA/g組織。

2.6 數據處理

微核試驗和染色體畸變試驗的結果進行卡方檢驗，當P<0.05認為差異顯著。DNA、RNA含量及RNA/DNA比值的試驗結果以平均值±標準差表示，采用單因素方差分析和Tukey多重檢驗分析顯著性，當P<0.05認為差異顯著。

3結果與分析

3.1 久效磷農藥對黃鱔紅細胞核異常的誘導作用

對照組黃鱔紅細胞的細胞核為圓形或橢圓形，細胞膜完整，見圖1a；久效磷農藥暴露后紅細胞出現的核異常主要包括核質外凸(見圖1b)，核質內凹(見圖1c)，核變形(見圖1d)，核內空泡(見圖1e)、雙核(見圖1f)和無絲分裂(見圖1g)等，0.50～2.00 mg/L暴露組核異常細胞率及0.25～2.00 mg/L暴露組總核異常細胞率與對照相比顯著升高(P<0.05)。暴露組還誘導紅細胞產生了微核，位于胞質中，為圓形或橢圓形，直徑約為主核直徑1/5至1/20，微核的染色深度與主核一致或略淺于主核(見圖1h)，但是不同濃度久效磷農藥暴露組的微核率與對照組相比均無顯著性變化(見表1)。

圖1　久效磷農藥暴露對黃鱔外周血

3.2 久效磷農藥對黃鱔染色體畸變的誘導作用

對照組黃鱔腎細胞染色體核型可知2n=24(見圖2a)，久效磷農藥暴露96 h誘導了染色體的非整倍體和多倍體(見圖2b)的形成，1.00和2.00 mg/L暴露組染色體數目總異常率顯著升高(P<0.05，見表2)；除染色體數目異常外久效磷農藥暴露還導致了染色體的斷片(見圖2c)、裂隙(見圖2d)、著絲粒環(見圖2e)、著絲粒融合(見圖2f)等結構畸變，1.00～2.00 mg/L暴露組染色體裂隙率、0.50～2.00 mg/L暴露組染色體結構總畸變率與對照相比顯著升高(P<0.05，見表3)。

表1　不同濃度久效磷農藥對黃鱔外周

注:各暴露濃度組與相應對照組間的顯著性差異，以* (P<0.05)表示。An asterisk (P<0.05) denotes the significant difference between the exposure groups and control group.

表2　不同濃度久效磷農藥對黃鱔腎細胞

注:各暴露濃度組與相應對照組間的顯著性差異，以* (P<0.05)表示。An asterisk (P<0.05) denotes the significant difference between the exposure groups and control group.

表3　不同濃度久效磷農藥對黃鱔腎細胞

注:各暴露濃度組與相應對照組間的顯著性差異，以*(P<0.05)表示。An asterisk (P<0.05) denotes the significant difference between the exposure groups and control group.

3.3 對腎臟DNA、RNA含量和RNA/DNA比值的影響

由圖3可知，久效磷農藥暴露96 h后各暴露組黃鱔腎臟DNA含量與對照相比無顯著變化，只有2.00 mg/L最高濃度暴露組RNA含量及RNA/DNA比值與對照相比顯著降低(P<0.05)。

4討論

研究發現烷化劑能夠誘導染色體結構和數目異常、姐妹染色單體交換、基因突變及細胞死亡[16]，久效磷農藥等有機磷農藥帶有2～3個烷基，具有親電子性，作為烷化劑可能與細胞內的親核物質反應，導致DNA、蛋白等大分子損傷。目前魚類的研究中發現久效磷農藥能夠誘導基因突變和DNA損傷，但是否會誘導魚類細胞染色體的損傷尚不清楚，本研究的結果發現久效磷農藥暴露后能夠造成黃鱔紅細胞形成異常核型、腎細胞出現染色體數目和結構畸變，表明久效磷農藥同時能夠誘導魚類染色體損傷。

圖2　久效磷農藥暴露對黃鱔腎細胞染色體的影響(1 200×)

微核是無著絲粒的染色體片段或因紡錘體受損而丟失的整個染色體，在細胞分裂后期仍留在子細胞的胞質內而形成的結構[17]，很多研究報道出現微核的細胞中常同時出現其他類型的核異常[18-20]。Bolognesi等[21]和Ergene等[22]發現核質外凸和微核形成之間具有正相關關系；Shimizu等[23]認為，復制后的DNA選擇性地定位于細胞核外周的特定位置，通過核質外凸最終形成微核與細胞核分離，因此遺傳毒性物質可能通過誘導核異常最終導致微核形成。黃鱔正常體細胞的微核率在0.17‰～1.05‰之間，核異常細胞率在7.82‰～17.44‰，總核異常細胞率在7.98‰～18.09‰之間[24-26]，本研究統計發現對照組紅細胞微核率為0.31‰、核異常細胞率為10.61‰、總核異常細胞率為10.92‰，久效磷農藥暴露后核異常細胞率在0.50～2.00 mg/L暴露組顯著上升，而總核異常細胞率在各暴露濃度組均顯著上升。Fenech和Crott[27]認為葉酸缺失能夠通過損傷DNA雙鏈、干擾DNA復制導致人淋巴細胞核質外凸、核質橋形成等核異常，核異常與微核一樣是遺傳毒性物質作用于染色體和紡錘體產生的一種遺傳毒性效應；本研究中0.25 mg/L的久效磷農藥暴露即可引起黃鱔紅細胞總核異常細胞率顯著升高，表明久效磷農藥具有致染色體斷裂劑和紡錘體毒劑作用。本研究微核率在各暴露濃度條件下與對照相比均無顯著變化，可能與魚類外周血紅細胞分裂指數較低[28]有關；試驗結果表明當以微核試驗檢測久效磷農藥的遺傳毒性時，核異常細胞率及總核異常細胞率比微核率具有更高的敏感性。

(各暴露濃度組與相應對照組間的顯著性差異，以* (P<0.05)表示。An asterisk(P<0.05) denotes the significant difference between the exposure group and control group.

圖3不同濃度久效磷農藥對黃鱔腎臟DNA、RNA含量和RNA/DNA比值的影響

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

RNA/DNA ratio in the kidney tissues ofMonopterusalbus

本研究發現對照組中也存在一定比例的染色體數目和結構畸變細胞，這與陳剛等[24]的結果一致；與對照組相比，0.25～2.00 mg/L久效磷暴露96 h后，黃鱔腎細胞染色體數目總異常率達到16.36%～27.78%，染色體數目的畸變主要表現為染色體非整倍體率升高，表明在細胞有絲分裂過程中久效磷農藥能夠作用于紡錘體微管，導致微管斷裂使部分染色體在分裂過程中丟失，最終引起子細胞染色體非整倍體率升高，久效磷農藥具有非整倍體誘導劑作用。采用哺乳動物作為實驗動物，Bhunya和Behera[29]發現1.25～5 mg/kg的久效磷農藥暴露能夠導致小鼠骨髓細胞染色體結構畸變；Wang等[30]報道8.0～1 000.0 μg/mL的久效磷農藥(純度為98%)暴露能夠誘導中國倉鼠卵巢(CHO)細胞姐妹染色單體交換，1 000.0 μg/mL還能誘導染色體畸變的發生；本研究中各個暴露濃度組染色體結構總畸變率達到14.55%～31.38%，久效磷農藥還可直接作用于黃鱔腎細胞染色體而產生染色體斷片、裂隙、著絲粒環、著絲粒融合等結構畸變，具有染色體斷裂劑作用。與哺乳動物體外實驗相比，本研究中0.50mg/L的久效磷農藥即可誘導黃鱔腎細胞染色體結構總畸變率顯著升高，表明魚類體內暴露實驗對于久效磷農藥染色體損傷效應的檢測更為敏感。

生物體每個細胞中的DNA含量是穩定的，而RNA含量隨著蛋白合成速率的不同而不同，因而組織中RNA/DNA比值能夠對代謝過程和蛋白合成過程進行表征，RNA/DNA比值能夠反映污染物脅迫對體細胞生長的延遲作用[31-32]。本研究中0.25 mg/L久效磷農藥暴露96 h后黃鱔腎細胞中RNA含量略有上升，可能是因為低濃度久效磷農藥的脅迫上調了機體某些基因的表達、促進了蛋白的翻譯和合成，從而產生某種能夠抵抗外源有毒物質的應激蛋白；但當久效磷農藥濃度進一步升高時，主要發揮毒性作用，最終導致暴露組中RNA的含量逐漸降低。Rath和Misra[33]的研究發現暴露于亞致死濃度的敵敵畏后，莫桑比克羅非魚(Tilapiamossambica)肝臟DNA、RNA和蛋白質含量均下降，且RNA/DNA比值也降低，這與本文的研究結果類似；久效磷暴露96 h后腎臟DNA含量無顯著性變化，這可能與生物體內的DNA含量相對比較穩定以及久效磷農藥暴露時間較短有關。

5結語

本研究采用微核試驗和染色體畸變試驗，證實久效磷農藥暴露能夠損傷魚類細胞染色體，產生遺傳毒性效應。暴露后黃鱔外周血紅細胞核異常細胞率和總核異常細胞率顯著升高，腎細胞的染色體數目總異常率、染色體裂隙率及染色體結構總畸變率均顯著性升高，久效磷農藥具有染色體斷裂劑、紡錘體毒劑、非整倍體誘導劑和染色體斷裂劑作用。此外，2.00 mg/L久效磷農藥暴露還能顯著降低腎臟組織RNA含量及RNA/DNA比值。

參考文獻：

[1]Tariq M I, Afzal S, Hussain I. Pesticides in shallow groundwater of bahawalnagar, Muzafargarh, DG Khan and Rajan Pur districts of Punjab, Pakistan [J]. Environment International, 2004, 30(4): 471-479.

[2]Krause k H, Van thriel C, De sousa p A, et al. Monocrotophos in Gandaman village: India school lunch deaths and need for improved toxicity testing [J]. Archives of Toxicology, 2013, 87(10): 1877-1881.

[3]Swarnam T P, Velmurugan A. Pesticide residues in vegetable samples from the Andaman Islands, India [J]. Environmental Monitoring and Assessment, 2013, 185(7): 6119-6127.

[4]康躍惠, 張干, 盛國英, 等. 固相萃取法測定水源水中的有機磷農藥[J]. 中國環境科學, 2000, 20(1): 1-4. Kang Y H, Zhang G, Sheng G Y, Fu J M. Analysis of organophosphorous pesticides from source water using solid-phase extraction technique [J]. China Environmental Science, 2000, 20(1): 1-4.

[5]Anjum r, Malik a. Evaluation of mutagenicity of wastewater in the vicinity of pesticide industry [J]. Environmental Toxicology and Pharmacology, 2013, 35(2): 284-291.

[6]Moriya m, Ohta T, Watanabe K, et al. Further mutagenicity studies on pesticides in bacterial reversion assay systems [J]. Mutation Research, 1983, 116(3-4): 185-216.

[7]Klopman G, Contreras R, Rosenkranz H S, et al. Structure-genotoxic activity relationships of pesticides: comparison of the results from several short-term assays [J]. Mutation Research, 1985, 147(6): 343-356.

[8]Saleha banu B, Danadevi K, Rahman M F, et al. Genotoxic effect of monocrotophos to sentinel species using comet assay [J]. Food and Chemical Toxicology, 2001, 39(4): 361-366.

[9]Mahboob M, Rahman M F, Danadevi k, et al. Detection of DNA damage in mouse peripheral blood leukocytes by the comet assay after oral administration of monocrotophos [J]. Drug and Chemical Toxicology, 2002, 25(1): 65-74.

[10]Jamil K, Shaik A P, Mahboob M, et al. Effect of organophosphorus and organochlorine pesticides (monochrotophos, chlorpyriphos, dimethoate, and endosulfan) on human lymphocytes in-vitro [J]. Drug and Chemical Toxicology, 2004, 27(2): 133-144.

[11]Ali D, Kumar S. Long-term genotoxic effect of monocrotophos in different tissues of freshwater fishChannapunctatus(Bloch) using alkaline single cell gel electrophoresis [J]. Science of the Total Environment, 2008, 405(1-3): 345-350.

[12]Fenech M. Cytokinesis-block micronucleus cytome assay [J]. Nature Protocols, 2007, 2: 1084-1104.

[13]Palus J, Rydzynski K, Dziubaltowska E, et al. Genotoxic effects of occupational exposure to lead and cadmium [J]. Mutation Research, 2003, 540(1): 19-28.

[14]Ansari R A, Rahman S, Kaur M, et al. In vivo cytogenetic and oxidative stress-inducing effects of cypermethrin in freshwater fish,ChannapunctataBloch [J]. Ecotoxicology and Environmental Safety, 2011, 74(1): 150-156.

[15]楊光彩. 生物化學實驗指導[M]. 廣州: 華南理工大學出版社, 2001: 136-139.

Yang G C. Biochemical Experiment Instruction [M]. Guang Zhou: South China University of Technology Press, 2001: 136-139.

[16]Wilhelm D, Bender K, Knebel A, et al. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents [J]. Molecular and Cellular Biology, 1997, 17(8): 4792-800.

[17]Heddle J A, Cimino M C, Hayashi M, et al. Micronuclei as an index of cytogenetic damage: Past, present, and future [J]. Environmental and Molecular Mutagenesis, 1991, 18(4): 277-291.

[18]Furnus G N, Caffetti J D, Garcia E M, et al. Baseline micronuclei and nuclear abnormalities frequencies in native fishes from the Paraná River (Argentina) [J]. Brazilian Journal of Biology, 2014, 74(1): 217-221.

[19]Lajmanovich r C, Cabagna-zenklusen M C, Attademo A M, et al. Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinellaarenarum) treated with the herbicides Liberty?and glufosinate-ammonium [J]. Mutation Research, 2014, 769: 7-12.

[20]Sisman T. Genotoxic effects of water pollution on two fish species living in Karasu River, Erzurum, Turkey [J]. Environmental Monitoring and Assessment, 2014, 186(11): 8007-8016.

[21]Bolognesi C, Perrone E, Roggieri P, et al. Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions [J]. Aquatic Toxicology, 2006, 78 (Suppl 1): 93-98.

[22]Ergene S, Cavas T, Celik A, et al. Evaluation of river water genotoxicity using the piscine micronucleus test [J]. Environmental and Molecular Mutagenesis, 2007, 48(6): 421-429.

[23]Shimizu N, Itoh N, Utiyama H, et al. Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase [J]. Journal of Cell Biology, 1998, 140(6): 1307-1320.

[24]陳剛, 耿德貴, 朱必才, 等. 除草劑精禾草克對黃鱔細胞遺傳毒性的研究[J]. 動物學雜志， 2000， 35(5): 15-19.

Chen G, Geng D G, Zhu B C, et al. Genetic toxicity to the cells of Monopterus albus by the herbicide NC-302D(+) [J]. Chinese Journal of Zoology, 2000， 35(5): 15-19.

[25]劉賢德, 程偉. 除草劑精克草能對黃鱔的遺傳毒性及三種物質的抑制作用[J]. 徐州師范大學學報, 2000, 18(2): 56-58.

Liu X D, Cheng W. Genetic toxicity in cells ofMonopterusalbusby the herbicide Jing Ke Cao Neng and inhibition of three substances [J]. J of Xuzhou Normal Uni (Natural Sciences), 2000, 18(2): 56-58.

[26]耿德貴, 屈艾, 劉曉峰, 等. 5種物質對除草劑精克草星誘發黃鱔外周血紅細胞微核和核異常的抑制作用[J]. 癌變畸變突變， 1999， 1(3)： 134-139.

Geng D G, Qu A, Liu X F, et al. Inhibition of five substances on micronuclei and nuclear anomalies induced by the herbicide Jing Ke Cao Xing in the peripheral blood erythrocytes of monopterus albus [J]. Carcino Genesis, Terato Genesis & Muta Genesis, 1999， 1(3)： 134-139.

[27]FenecH M, Crott J W. Micronuclei, nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakage-fusion-bridge cycles in the cytokinesis-block micronucleus assay [J]. Mutation Research, 2002, 504(1-2): 131-136.

[28]賀維順, 王蕊芳. 蝌蚪(Bufobufoandrewsi)血紅細胞微核和核異常監測水質污染的研究[J]. 動物學研究, 1990, 11(1): 1-6.

He W S, Wang R F. Detection of water pollution by micronuclei and other nuclear anomalies of erythrocytes of tadpoies (Bufobufoandrewsi). Zoological Research, 1990, 11(1): 1-6.

[29]Bhunya S P, Behera B C. Mutagenicity assay of an organophosphorate pesticide, monocrotophos in mammalian in vivo test system [J]. Cytologia, 1988, 53(4): 801-807.

[30]Wang T C, Lin C M, LO L W. Genotoxicity of methoxyphosphinyl insecticide in mammalian cells [J]. Zoological Studies, 2003, 42(3): 462-469.

[31]Barron M G, Adelman I R. Nucleic acid, protein content, and growth of larval fish sublethally exposed to various toxicants [J]. Canadian Journal of Fisheries and Aquatic Sciences, 1984, 41(1): 141-150.

[32]Varo I, Navarro J C, Numes B, et al. Effects of dichlorovos aquaculture treatments on selected biomarkers of gilthead sea bream (SparusaurataL.) fingerlings [J]. Aquaculture, 2007, 266(1-4): 87-92.

[33]Rath S, Misra B N. Changes in nucleic acids and protein content ofTilapiamossambicaexposed to dichlorvos [J]. Indian Journal of Fisheries, 1980, 27: 76-81.

責任編輯高蓓

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

中圖法分類號：X174

文獻標志碼：A

文章編號：1672-5174(2016)02-069-07

作者簡介：馮永亮(1987-)，男，博士，主要研究方向為生態學。E-mail：yongliangfeng0511@126.com??通訊作者：E-mail：rusg@ouc.edu.cn

收稿日期：2015-04-01；

修訂日期：2015-08-31

基金項目：?國家自然科學基金項目(31202001)資助

引用格式：馮永亮， 閆建國， 趙飛， 等. 久效磷農藥對黃鱔染色體損傷的研究[J]. 中國海洋大學學報(自然科學版), 2016, 46(2): 69-75.

Supported by National Natural Science Foundation of China (31202001)