馮永亮， 閆建國， 趙　飛， 汝少國

(中國海洋大學(xué)海洋生命學(xué)院，山東 青島 266003)

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久效磷農(nóng)藥對(duì)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(xué)海洋生命學(xué)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(nóng)藥暴露黃鱔96 h，采用微核試驗(yàn)和染色體畸變?cè)囼?yàn)方法研究了久效磷農(nóng)藥對(duì)外周血紅細(xì)胞和腎細(xì)胞染色體的損傷作用。結(jié)果表明：0.50～2.00和0.25～2.00 mg/L的久效磷農(nóng)藥暴露顯著升高了紅細(xì)胞核異常細(xì)胞率及總核異常細(xì)胞率；1.00和2.00 mg/L暴露組腎細(xì)胞的染色體數(shù)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(jié)構(gòu)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(jié)果表明久效磷農(nóng)藥能夠引起黃鱔染色體損傷，導(dǎo)致遺傳毒性效應(yīng)。

關(guān)鍵詞：久效磷農(nóng)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(nóng)藥是一種用于農(nóng)林業(yè)害蟲防治的高毒有機(jī)磷農(nóng)藥，目前在印度和巴基斯坦等發(fā)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(yè)廢水中濃度達(dá)到(8.32±3.9) μg/L[5]。殘留的久效磷農(nóng)藥通過雨水沖刷、地表徑流和食物等方式進(jìn)入水環(huán)境和生物體[3,5]，造成生物體遺傳物質(zhì)的損傷。采用Ames實(shí)驗(yàn)研究發(fā)現(xiàn)久效磷農(nóng)藥能夠?qū)е率髠抽T氏菌(Salmonellatyphimurium)和大腸桿菌(Escherichiacoli)基因突變[6-7]；采用彗星電泳實(shí)驗(yàn)研究證實(shí)久效磷農(nóng)藥能夠造成魚類外周血細(xì)胞DNA鏈斷裂，還可導(dǎo)致小鼠外周血細(xì)胞、人淋巴細(xì)胞以及淡水硬骨魚Channapunctatus鰓、腎、淋巴細(xì)胞[8-11]的DNA鏈斷裂；但關(guān)于久效磷農(nóng)藥暴露是否會(huì)造成魚類的染色體損傷研究還未見報(bào)道。在東亞地區(qū)廣泛分布的黃鱔(Monopterusalbus)是一種具有較高經(jīng)濟(jì)價(jià)值的淡水魚類，其染色體數(shù)目(2n=24)較少，染色體形態(tài)較大且為端著絲粒染色體，有利于觀察統(tǒng)計(jì)染色體畸變類型。因此，本研究采用微核實(shí)驗(yàn)和染色體畸變實(shí)驗(yàn)研究了久效磷農(nóng)藥暴露對(duì)黃鱔外周血紅細(xì)胞和腎細(xì)胞染色體的損傷作用，以期為全面評(píng)價(jià)久效磷農(nóng)藥的遺傳毒性效應(yīng)提供基礎(chǔ)數(shù)據(jù)。

2材料與方法

2.1 試驗(yàn)材料與動(dòng)物

久效磷農(nóng)藥(3-hydroxyl-N-methyl-cis-crotonamide dimethyl phosphate)購自青島農(nóng)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(tuán)化學(xué)試劑有限公司(Beijing, P.R.China)。

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

2.2 久效磷農(nóng)藥暴露方法與樣品制備

采用半靜態(tài)暴露實(shí)驗(yàn)，容器為70 L玻璃水族箱，盛50 L連續(xù)曝氣24 h的自來水，每組2個(gè)水族箱，每箱6條魚，共12條魚。根據(jù)急性毒性預(yù)試驗(yàn)，久效磷農(nóng)藥暴露黃鱔96 h的LC50為3.27 mg/L，設(shè)置久效磷農(nóng)藥暴露為0.25、0.50、1.00、2.00 mg/L，同時(shí)設(shè)對(duì)照組。為保持久效磷農(nóng)藥濃度每天換水50%，并補(bǔ)加農(nóng)藥至暴露濃度。試驗(yàn)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(shí)，為獲得足夠數(shù)量的處于有絲分裂中期的細(xì)胞進(jìn)行染色體核型分析，取每組6條魚進(jìn)行秋水仙素(0.05%，10 μL/g體重)肌肉注射，96 h時(shí)斷尾充分放血，解剖取腎臟用于染色體畸變實(shí)驗(yàn)。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(rùn)洗的注射器尾靜脈取血，用于微核實(shí)驗(yàn)；同時(shí)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(yàn)

微核試驗(yàn)參照Fenech[12]和Palus等[13]的方法進(jìn)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(jī)觀察，每組隨機(jī)觀察6 500個(gè)具有完整細(xì)胞膜和核膜的有核紅細(xì)胞，統(tǒng)計(jì)微核率、核異常細(xì)胞率及總核異常細(xì)胞率。微核細(xì)胞率=帶有微核的細(xì)胞數(shù)/觀察的細(xì)胞總數(shù)×1 000‰；核異常細(xì)胞率=具有核異常(除微核外)的細(xì)胞總數(shù)/觀察細(xì)胞的總數(shù)×1 000‰；總核異常細(xì)胞率=微核細(xì)胞率+核異常細(xì)胞率。

2.4 染色體畸變?cè)囼?yàn)

染色體畸變?cè)囼?yàn)參照Ansari等[14]的方法進(jìn)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(yù)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(fù)3次；取細(xì)胞液滴在浸泡于60%冰乙醇的載波片上，輕吹；常規(guī)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(jìn)行觀察。

2.5 DNA、RNA含量的測(cè)定

參照楊光彩等[15]的試驗(yàn)方法測(cè)定腎臟DNA、RNA的含量，分別采用魚精DNA、酵母RNA作為標(biāo)準(zhǔn)，單位為mg DNA/g組織。

2.6 數(shù)據(jù)處理

微核試驗(yàn)和染色體畸變?cè)囼?yàn)的結(jié)果進(jìn)行卡方檢驗(yàn)，當(dāng)P<0.05認(rèn)為差異顯著。DNA、RNA含量及RNA/DNA比值的試驗(yàn)結(jié)果以平均值±標(biāo)準(zhǔn)差表示，采用單因素方差分析和Tukey多重檢驗(yàn)分析顯著性，當(dāng)P<0.05認(rèn)為差異顯著。

3結(jié)果與分析

3.1 久效磷農(nóng)藥對(duì)黃鱔紅細(xì)胞核異常的誘導(dǎo)作用

對(duì)照組黃鱔紅細(xì)胞的細(xì)胞核為圓形或橢圓形，細(xì)胞膜完整，見圖1a；久效磷農(nóng)藥暴露后紅細(xì)胞出現(xiàn)的核異常主要包括核質(zhì)外凸(見圖1b)，核質(zhì)內(nèi)凹(見圖1c)，核變形(見圖1d)，核內(nèi)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(xì)胞率及0.25～2.00 mg/L暴露組總核異常細(xì)胞率與對(duì)照相比顯著升高(P<0.05)。暴露組還誘導(dǎo)紅細(xì)胞產(chǎn)生了微核，位于胞質(zhì)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(nóng)藥暴露組的微核率與對(duì)照組相比均無顯著性變化(見表1)。

圖1　久效磷農(nóng)藥暴露對(duì)黃鱔外周血

3.2 久效磷農(nóng)藥對(duì)黃鱔染色體畸變的誘導(dǎo)作用

對(duì)照組黃鱔腎細(xì)胞染色體核型可知2n=24(見圖2a)，久效磷農(nóng)藥暴露96 h誘導(dǎo)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(shù)目總異常率顯著升高(P<0.05，見表2)；除染色體數(shù)目異常外久效磷農(nóng)藥暴露還導(dǎo)致了染色體的斷片(見圖2c)、裂隙(見圖2d)、著絲粒環(huán)(見圖2e)、著絲粒融合(見圖2f)等結(jié)構(gòu)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(jié)構(gòu)總畸變率與對(duì)照相比顯著升高(P<0.05，見表3)。

表1　不同濃度久效磷農(nóng)藥對(duì)黃鱔外周

注:各暴露濃度組與相應(yīng)對(duì)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(nóng)藥對(duì)黃鱔腎細(xì)胞

注:各暴露濃度組與相應(yīng)對(duì)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(nóng)藥對(duì)黃鱔腎細(xì)胞

注:各暴露濃度組與相應(yīng)對(duì)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(duì)腎臟DNA、RNA含量和RNA/DNA比值的影響

由圖3可知，久效磷農(nóng)藥暴露96 h后各暴露組黃鱔腎臟DNA含量與對(duì)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(duì)照相比顯著降低(P<0.05)。

4討論

研究發(fā)現(xiàn)烷化劑能夠誘導(dǎo)染色體結(jié)構(gòu)和數(shù)目異常、姐妹染色單體交換、基因突變及細(xì)胞死亡[16]，久效磷農(nóng)藥等有機(jī)磷農(nóng)藥帶有2～3個(gè)烷基，具有親電子性，作為烷化劑可能與細(xì)胞內(nèi)的親核物質(zhì)反應(yīng)，導(dǎo)致DNA、蛋白等大分子損傷。目前魚類的研究中發(fā)現(xiàn)久效磷農(nóng)藥能夠誘導(dǎo)基因突變和DNA損傷，但是否會(huì)誘導(dǎo)魚類細(xì)胞染色體的損傷尚不清楚，本研究的結(jié)果發(fā)現(xiàn)久效磷農(nóng)藥暴露后能夠造成黃鱔紅細(xì)胞形成異常核型、腎細(xì)胞出現(xiàn)染色體數(shù)目和結(jié)構(gòu)畸變，表明久效磷農(nóng)藥同時(shí)能夠誘導(dǎo)魚類染色體損傷。

圖2　久效磷農(nóng)藥暴露對(duì)黃鱔腎細(xì)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(gè)染色體，在細(xì)胞分裂后期仍留在子細(xì)胞的胞質(zhì)內(nèi)而形成的結(jié)構(gòu)[17]，很多研究報(bào)道出現(xiàn)微核的細(xì)胞中常同時(shí)出現(xiàn)其他類型的核異常[18-20]。Bolognesi等[21]和Ergene等[22]發(fā)現(xiàn)核質(zhì)外凸和微核形成之間具有正相關(guān)關(guān)系；Shimizu等[23]認(rèn)為，復(fù)制后的DNA選擇性地定位于細(xì)胞核外周的特定位置，通過核質(zhì)外凸最終形成微核與細(xì)胞核分離，因此遺傳毒性物質(zhì)可能通過誘導(dǎo)核異常最終導(dǎo)致微核形成。黃鱔正常體細(xì)胞的微核率在0.17‰～1.05‰之間，核異常細(xì)胞率在7.82‰～17.44‰，總核異常細(xì)胞率在7.98‰～18.09‰之間[24-26]，本研究統(tǒng)計(jì)發(fā)現(xiàn)對(duì)照組紅細(xì)胞微核率為0.31‰、核異常細(xì)胞率為10.61‰、總核異常細(xì)胞率為10.92‰，久效磷農(nóng)藥暴露后核異常細(xì)胞率在0.50～2.00 mg/L暴露組顯著上升，而總核異常細(xì)胞率在各暴露濃度組均顯著上升。Fenech和Crott[27]認(rèn)為葉酸缺失能夠通過損傷DNA雙鏈、干擾DNA復(fù)制導(dǎo)致人淋巴細(xì)胞核質(zhì)外凸、核質(zhì)橋形成等核異常，核異常與微核一樣是遺傳毒性物質(zhì)作用于染色體和紡錘體產(chǎn)生的一種遺傳毒性效應(yīng)；本研究中0.25 mg/L的久效磷農(nóng)藥暴露即可引起黃鱔紅細(xì)胞總核異常細(xì)胞率顯著升高，表明久效磷農(nóng)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(duì)照相比均無顯著變化，可能與魚類外周血紅細(xì)胞分裂指數(shù)較低[28]有關(guān)；試驗(yàn)結(jié)果表明當(dāng)以微核試驗(yàn)檢測(cè)久效磷農(nóng)藥的遺傳毒性時(shí)，核異常細(xì)胞率及總核異常細(xì)胞率比微核率具有更高的敏感性。

(各暴露濃度組與相應(yīng)對(duì)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(nóng)藥對(duì)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(fā)現(xiàn)對(duì)照組中也存在一定比例的染色體數(shù)目和結(jié)構(gòu)畸變細(xì)胞，這與陳剛等[24]的結(jié)果一致；與對(duì)照組相比，0.25～2.00 mg/L久效磷暴露96 h后，黃鱔腎細(xì)胞染色體數(shù)目總異常率達(dá)到16.36%～27.78%，染色體數(shù)目的畸變主要表現(xiàn)為染色體非整倍體率升高，表明在細(xì)胞有絲分裂過程中久效磷農(nóng)藥能夠作用于紡錘體微管，導(dǎo)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(xì)胞染色體非整倍體率升高，久效磷農(nóng)藥具有非整倍體誘導(dǎo)劑作用。采用哺乳動(dòng)物作為實(shí)驗(yàn)動(dòng)物，Bhunya和Behera[29]發(fā)現(xiàn)1.25～5 mg/kg的久效磷農(nóng)藥暴露能夠?qū)е滦∈蠊撬杓?xì)胞染色體結(jié)構(gòu)畸變；Wang等[30]報(bào)道8.0～1 000.0 μg/mL的久效磷農(nóng)藥(純度為98%)暴露能夠誘導(dǎo)中國倉鼠卵巢(CHO)細(xì)胞姐妹染色單體交換，1 000.0 μg/mL還能誘導(dǎo)染色體畸變的發(fā)生；本研究中各個(gè)暴露濃度組染色體結(jié)構(gòu)總畸變率達(dá)到14.55%～31.38%，久效磷農(nóng)藥還可直接作用于黃鱔腎細(xì)胞染色體而產(chǎn)生染色體斷片、裂隙、著絲粒環(huán)、著絲粒融合等結(jié)構(gòu)畸變，具有染色體斷裂劑作用。與哺乳動(dòng)物體外實(shí)驗(yàn)相比，本研究中0.50mg/L的久效磷農(nóng)藥即可誘導(dǎo)黃鱔腎細(xì)胞染色體結(jié)構(gòu)總畸變率顯著升高，表明魚類體內(nèi)暴露實(shí)驗(yàn)對(duì)于久效磷農(nóng)藥染色體損傷效應(yīng)的檢測(cè)更為敏感。

生物體每個(gè)細(xì)胞中的DNA含量是穩(wěn)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?qū)Υx過程和蛋白合成過程進(jìn)行表征，RNA/DNA比值能夠反映污染物脅迫對(duì)體細(xì)胞生長(zhǎng)的延遲作用[31-32]。本研究中0.25 mg/L久效磷農(nóng)藥暴露96 h后黃鱔腎細(xì)胞中RNA含量略有上升，可能是因?yàn)榈蜐舛染眯Я邹r(nóng)藥的脅迫上調(diào)了機(jī)體某些基因的表達(dá)、促進(jìn)了蛋白的翻譯和合成，從而產(chǎn)生某種能夠抵抗外源有毒物質(zhì)的應(yīng)激蛋白；但當(dāng)久效磷農(nóng)藥濃度進(jìn)一步升高時(shí)，主要發(fā)揮毒性作用，最終導(dǎo)致暴露組中RNA的含量逐漸降低。Rath和Misra[33]的研究發(fā)現(xiàn)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(zhì)含量均下降，且RNA/DNA比值也降低，這與本文的研究結(jié)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(nèi)的DNA含量相對(duì)比較穩(wěn)定以及久效磷農(nóng)藥暴露時(shí)間較短有關(guān)。

5結(jié)語

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

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責(zé)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(hào)：X174

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1672-5174(2016)02-069-07

作者簡(jiǎn)介：馮永亮(1987-)，男，博士，主要研究方向?yàn)樯鷳B(tài)學(xué)。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(xiàng)目：?國家自然科學(xué)基金項(xiàng)目(31202001)資助

引用格式：馮永亮， 閆建國， 趙飛， 等. 久效磷農(nóng)藥對(duì)黃鱔染色體損傷的研究[J]. 中國海洋大學(xué)學(xué)報(bào)(自然科學(xué)版), 2016, 46(2): 69-75.

Supported by National Natural Science Foundation of China (31202001)