曲玉楹,張彩杰,沈秋岑,張 婧,于 紅

吐納麝香對(duì)東海原甲藻的毒性作用機(jī)制

曲玉楹,張彩杰,沈秋岑,張 婧*,于 紅

(中國海洋大學(xué)化學(xué)化工學(xué)院,山東 青島 266100)

為研究吐納麝香(AHTN)對(duì)海洋微藻的毒性作用機(jī)制,以東海原甲藻()為受試對(duì)象,測(cè)定5種濃度的AHTN(1,10,50,200和400μg/L)對(duì)微藻的生長、光合色素含量、細(xì)胞形態(tài)結(jié)構(gòu)、細(xì)胞膜通透性、抗氧化系統(tǒng)和光合系統(tǒng)的影響.結(jié)果表明,AHTN顯著抑制東海原甲藻的生長,對(duì)東海原甲藻具有高毒性(96h-EC50=48.21μg/L),其毒性作用機(jī)制為抑制光合色素產(chǎn)生,破壞其細(xì)胞結(jié)構(gòu)和細(xì)胞膜完整性,誘發(fā)抗氧化系統(tǒng)響應(yīng),影響光合作用性能.其中,1μg/L AHTN處理對(duì)東海原甲藻生長有促進(jìn)作用,1和10μg/L AHTN處理促進(jìn)光合色素合成,并使超氧化物歧化酶活性和部分光合參數(shù)升高.低濃度AHTN(1μg/L)可能會(huì)誘導(dǎo)東海原甲藻赤潮爆發(fā),高濃度AHTN(10～400μg/L)則會(huì)抑制該藻種群生長,從而對(duì)海洋生態(tài)系統(tǒng)產(chǎn)生潛在威脅.

吐納麝香；東海原甲藻；毒性機(jī)制；抗氧化系統(tǒng)；光合性能

合成麝香(SMs)廣泛使用于香水、洗發(fā)水、乳液、洗衣粉和除臭劑等[1],通過廢水進(jìn)入到各種環(huán)境,存在于地表水、海水、水體顆粒相、水體沉積物、大氣樣品及生物體中[1-2].在我國水環(huán)境中各類麝香被頻繁檢出,如廣州的化妝品污水中含有高達(dá)595.48μg/L的SMs[3];Zhang等[4]在新加坡的海水樣品中檢測(cè)到8種合成麝香化合物,其中佳樂麝香和吐納麝香的總濃度(溶解+顆粒)范圍分別為1730～ 23300和270～1950pg/L,麝香酮濃度在170～1260pg/ L之間;王杰等[5]在膠州灣北岸潮間帶沉積物中檢出佳樂麝香、吐納麝香和酮麝香3種麝香污染物,含量分別為1.84～4.35,n.d.～10.9和6.90～10.9ng/g[5].麝香具有較強(qiáng)的親脂性和持久性,這些物質(zhì)往往會(huì)從環(huán)境中進(jìn)入水生生物體中積累,并隨著食物鏈傳遞和放大[6].

在各類合成麝香中,多環(huán)麝香占市場(chǎng)的主導(dǎo)地位,其中吐納麝香(AHTN)在環(huán)境和生物群中的濃度相對(duì)較高,經(jīng)常在環(huán)境中被檢測(cè)到[3,7].前期研究發(fā)現(xiàn),AHTN具有雌激素效應(yīng),并且對(duì)水生動(dòng)物有毒性[8];Parolini等[9]發(fā)現(xiàn)高濃度AHTN暴露會(huì)引起斑馬貽貝脂質(zhì)過氧化、蛋白質(zhì)羰基化和DNA鏈斷裂; Wollenberger等[10]的研究表明4種合成麝香對(duì)海洋橈足類幼蟲的5d-EC50范圍為0.03～0.16mg/L,能強(qiáng)烈抑制幼蟲的發(fā)育.這些研究結(jié)果表明, AHTN對(duì)水生生物具有毒性效應(yīng),對(duì)水生生態(tài)系統(tǒng)存在潛在風(fēng)險(xiǎn).

目前關(guān)于AHTN對(duì)海洋生物的毒性研究較少,缺乏毒性數(shù)據(jù)和水生生物標(biāo)準(zhǔn),因而對(duì)AHTN進(jìn)行環(huán)境風(fēng)險(xiǎn)評(píng)估存在困難[8].海洋微藻是海洋生態(tài)系統(tǒng)的初級(jí)生產(chǎn)者,其對(duì)水質(zhì)具有敏感性,經(jīng)常被用作生態(tài)毒理實(shí)驗(yàn)的目標(biāo)微生物.因此本文以長江口海域的優(yōu)勢(shì)赤潮藻東海原甲藻為研究對(duì)象,結(jié)合微藻生長情況、光合活性、氧化應(yīng)激、細(xì)胞膜完整性及形態(tài)結(jié)構(gòu)等參數(shù)探究AHTN暴露對(duì)海洋微藻的毒性及作用機(jī)制,旨在為AHTN在長江口等近岸海域生態(tài)系統(tǒng)中的風(fēng)險(xiǎn)評(píng)價(jià)提供毒性數(shù)據(jù).

1 材料與方法

1.1 實(shí)驗(yàn)材料

東海原甲藻()購自上海光語公司.藻種培養(yǎng)采用添加f/2培養(yǎng)基的滅菌天然海水,使用錐形瓶置于光照培養(yǎng)箱(GXZ-380B,寧波江南儀器公司,中國)中搖床震蕩培養(yǎng),培養(yǎng)溫度(21±1)℃,光照強(qiáng)度3000lux,光暗比為12h:12h.吐納麝香(AHTN)為標(biāo)準(zhǔn)品(分子式C18H26O),購自美國霍尼韋爾公司;使用二甲亞砜(DMSO)為助溶劑配制母液,購自上海滬試公司,為分析純.

1.2 毒性生長實(shí)驗(yàn)

將東海原甲藻培養(yǎng)至對(duì)數(shù)生長期進(jìn)行實(shí)驗(yàn).將藻液分裝于250mL錐形瓶,每瓶100mL,初始密度為4.5×104cells/mL.根據(jù)預(yù)實(shí)驗(yàn)設(shè)計(jì)AHTN濃度梯度(對(duì)藻類生長產(chǎn)生0～100%抑制效應(yīng)),共設(shè)置1個(gè)對(duì)照組和5個(gè)處理組(1,10,50,200和400μg/L),對(duì)照組添加0.1%的DMSO以消除溶劑影響(經(jīng)預(yù)實(shí)驗(yàn),該濃度的DMSO對(duì)東海原甲藻無生長抑制),每組3個(gè)平行.實(shí)驗(yàn)條件與培養(yǎng)條件一致,共培養(yǎng)96h,每24h取樣,使用流式細(xì)胞儀(Accuri C6Plus,BD,美國)計(jì)數(shù)并繪制生長曲線,比生長率()使用藻細(xì)胞密度均值計(jì)算,公式如下:

式中:N和0分別代表時(shí)刻和0時(shí)刻(初始接種)的藻密度.抑制率計(jì)算如下:

式中:C和μ分別代表對(duì)照組和實(shí)驗(yàn)組的比生長率平均值.使用概率單位-濃度對(duì)數(shù)法計(jì)算96h的半最大效應(yīng)濃度(96h-EC50).

1.3 生理生化參數(shù)測(cè)定

1.3.1 光合色素含量測(cè)定 采用叢海兵等[11]的方法測(cè)定不同濃度AHTN脅迫對(duì)東海原甲藻細(xì)胞內(nèi)葉綠素a(chl-a)和葉綠素c(chl-c)含量的影響.在生長實(shí)驗(yàn)96h后,取20mL藻液經(jīng)0.45 μm濾膜過濾,將濾膜浸泡到5mL 90%乙醇中,4 ℃條件下黑暗萃取24h.以90%乙醇作為對(duì)照,使用分光光度計(jì)測(cè)定萃取液在630,647,664和750nm波長處的吸光值,用下式計(jì)算chl-a和chl-c含量(mg/L):

1.3.2 細(xì)胞形貌及內(nèi)部結(jié)構(gòu)觀察 收集正常東海原甲藻和400μg/L AHTN處理96h后的藻細(xì)胞,使用2.5%的戊二醛溶液固定,磷酸鹽緩沖溶液(PBS)清洗3次后,加入1%的鋨酸溶液固定,再使用PBS清洗3次,使用乙醇梯度(30%、50%、70%、80%、90%和95%濃度)脫水處理細(xì)胞,包埋后切片染色,使用透射電子顯微鏡(TEM,H-7650,日立,日本)觀察細(xì)胞內(nèi)部結(jié)構(gòu);同上述步驟完成梯度洗脫后,先后使用乙醇與醋酸異戊酯的混合液和純醋酸異戊酯處理樣品,臨界點(diǎn)干燥,鍍膜,使用掃描電子顯微鏡(SEM,SU8010,日立,日本)觀察細(xì)胞形貌.

1.3.3 細(xì)胞膜通透性測(cè)定 使用碘化丙啶(PI)染色測(cè)定生長實(shí)驗(yàn)后48和96h后的藻細(xì)胞膜通透性.參照王執(zhí)偉等[12]的方法,使用PBS配制1mmol/L的PI儲(chǔ)備液,4℃保存.取1mL藻液,加入PI染液的濃度為50μmol/L,25℃避光孵育20min.使用流式細(xì)胞儀檢測(cè),在FL2通道處收集PI熒光信號(hào),結(jié)果表示為強(qiáng)熒光區(qū)細(xì)胞數(shù)量占檢測(cè)細(xì)胞的百分比.

1.3.4 抗氧化系統(tǒng)活性測(cè)定 取生長實(shí)驗(yàn)96h后的藻液40mL,于4℃下6000r/min離心10min.去除上清液,細(xì)胞沉淀使用PBS清洗2次后,再加入2mL PBS重懸,使用超聲細(xì)胞破碎儀在冰浴下破碎至鏡檢無完整細(xì)胞.將破碎后的藻液離心(7500r/min, 15min,4℃),上清液即為粗酶液.使用試劑盒(南京建成生物工程研究所,中國)檢測(cè)總可溶性蛋白(TSP)含量、超氧化物歧化酶(SOD)活性、還原性谷胱甘肽含量(GSH)和丙二醛(MDA)含量.

1.3.5 葉綠素?zé)晒鈪?shù)測(cè)定 取生長實(shí)驗(yàn)0,24,48, 72,96h后的藻液2mL,使用浮游植物分類熒光儀(PHYTO-PAM,WALZ,德國)直接測(cè)定微藻實(shí)際量子產(chǎn)率(φPSⅡ);藻液避光暗處理15min,用于檢測(cè)PSⅡ最大量子產(chǎn)率(Fv/Fm)、實(shí)際量子產(chǎn)率(φPSⅡ)、光限制斜率()、最大相對(duì)電子傳遞速率(rETRmax).

1.4 數(shù)據(jù)分析

采用SPSS 20.0軟件,使用單因素方差分析和雙因素方差分析實(shí)驗(yàn)結(jié)果,以多重比較(Duncan)檢驗(yàn)組間差異,<0.05即表示存在顯著性差異.

2 結(jié)果與分析

2.1 AHTN對(duì)東海原甲藻生長的影響

如圖1所示,在實(shí)驗(yàn)的48h內(nèi),所有的AHTN處理組表現(xiàn)為對(duì)東海原甲藻的抑制效應(yīng),藻細(xì)胞密度相對(duì)于對(duì)照組下降了2.95%～63.23%,10～400μg/L處理組效應(yīng)顯著(<0.05).72h后,1μg/L AHTN濃度處理下的藻細(xì)胞密度超過對(duì)照組,表現(xiàn)為促進(jìn)東海原甲藻生長;96h時(shí)在50～400μg/L濃度處理下細(xì)胞密度隨濃度增大降低了59.57%～78.72%,表現(xiàn)為抑制作用,與對(duì)照組差異顯著(<0.05).10μg/L AHTN處理組在前72h內(nèi)對(duì)東海原甲藻表現(xiàn)為生長抑制,在第96h細(xì)胞密度恢復(fù)至與對(duì)照組無明顯差異(> 0.05).

圖1 東海原甲藻生長對(duì)AHTN處理的毒性響應(yīng)

2.2 AHTN對(duì)東海原甲藻生理的影響

2.2.1 AHTN對(duì)東海原甲藻光合色素的影響 如圖2所示,不同濃度AHTN處理96h的東海原甲藻chl-a、chl-c含量與生長結(jié)果類似,1和10μg/L AHTN處理組的chl-a含量與對(duì)照組相比分別增加了7.18%、6.93%,chl-c含量分別增加了29.77%、8.05%,但與對(duì)照組比較無顯著性差異.50～400μg/L處理組顯著抑制光合色素產(chǎn)生,chl-a含量相對(duì)于對(duì)照組減少了54.96%～87.58%,chl-c含量減少了50.45%～79.23%.

圖2 不同濃度AHTN處理96h后東海原甲藻的光合色素含量

不同字母表示實(shí)驗(yàn)組間存在顯著差異,<0.05

2.2.2 AHTN對(duì)東海原甲藻細(xì)胞形貌和內(nèi)部結(jié)構(gòu)的影響 如圖3(a)所示,未經(jīng)AHTN處理的東海原甲藻,細(xì)胞成橢圓體,飽滿,細(xì)胞壁完整,表面長有尖刺并布有零星殼面小孔.圖3(b,c)為處理組藻細(xì)胞,明顯觀察到細(xì)胞嚴(yán)重變形并縮小.由圖3(d)可見,正常東海原甲藻細(xì)胞結(jié)構(gòu)清晰,各細(xì)胞器完整,細(xì)胞壁與細(xì)胞膜貼合緊密,細(xì)胞內(nèi)部具有一個(gè)大液泡. AHTN處理后的許多藻細(xì)胞出現(xiàn)質(zhì)壁分離的現(xiàn)象,細(xì)胞器被破壞,細(xì)胞質(zhì)溶解且空泡化(圖3e,f);圖3(f)顯示細(xì)胞不同程度的損傷,細(xì)胞內(nèi)物質(zhì)泄露.

2.2.3 AHTN對(duì)東海原甲藻細(xì)胞膜通透性的影響 PI是一種熒光染料,可以插入雙鏈核酸在藍(lán)光激發(fā)下產(chǎn)生紅色熒光.由于PI不能穿過完整的細(xì)胞膜,當(dāng)細(xì)胞膜受損時(shí)PI才能夠進(jìn)入細(xì)胞并染色,因此強(qiáng)PI熒光細(xì)胞的比例可以反映細(xì)胞膜通透性[13].如圖4所示,低濃度(1和10μg/L)處理48和96h時(shí),強(qiáng)熒光細(xì)胞百分比與對(duì)照組無顯著性差異,表明細(xì)胞膜通透性幾乎不受影響;且隨著暴露時(shí)間的延長,強(qiáng)熒光細(xì)胞百分比略微減少.第48h時(shí),50,200和400μg/L處理組強(qiáng)熒光細(xì)胞百分比分別為1.0%、1.2%和2.4%,與對(duì)照組相比有顯著性差異,細(xì)胞膜受損程度隨濃度呈增加趨勢(shì).第96h與48h相比,50,200和400μg/L處理組強(qiáng)熒光細(xì)胞百分比分別增加到17.9%、13.6%和4.4%,隨濃度增大呈減小趨勢(shì).

圖4 東海原甲藻在AHTN處理48,96h后的PI強(qiáng)熒光細(xì)胞百分比

不同字母表示同一時(shí)間實(shí)驗(yàn)組間存在顯著差異,<0.05

2.2.4 AHTN對(duì)東海原甲藻抗氧化系統(tǒng)活性的影響 如圖5(a)所示,在1,10μg/L AHTN處理下,TSP含量高于對(duì)照組,10μg/L處理組與對(duì)照組存在顯著性差異;50～400μg/L處理組TSP含量顯著低于對(duì)照組,且隨暴露濃度增大,藻細(xì)胞TSP含量降低.如圖5(b),在較低濃度AHTN(1,10μg/L)處理下,SOD活性與對(duì)照組無顯著性差異;在50μg/L AHTN處理下的SOD活性達(dá)到最大值,為對(duì)照組的2倍;高濃度AHTN(200,400μg/L)處理下,SOD活性相比50μg/L處理組逐漸降低,在實(shí)驗(yàn)濃度范圍內(nèi)呈先升高后下降趨勢(shì).如圖5(c),與SOD活性結(jié)果相似, GSH含量出現(xiàn)先升高后降低的現(xiàn)象,50μg/L AHTN處理下的GSH含量達(dá)最大值,為對(duì)照組的2倍.如圖5(d), AHTN處理組誘導(dǎo)東海原甲藻MDA含量增大,且隨AHTN濃度增大MDA含量顯著增多,與對(duì)照組相比增大0.8～4倍.

圖5 AHTN對(duì)東海原甲藻TSP含量、SOD活性、GSH含量和MDA含量的影響

不同字母表示實(shí)驗(yàn)組間存在顯著差異,<0.05

圖6 不同濃度AHTN和處理時(shí)間對(duì)東海原甲藻葉綠素?zé)晒鈪?shù)的影響

2.2.5 AHTN對(duì)東海原甲藻光合活性的影響 如表1所示,AHTN濃度、處理時(shí)間和二者交互作用(濃度*時(shí)間)對(duì)Fv/Fm、φPSⅡ、、rETRmax4項(xiàng)葉綠素?zé)晒鈪?shù)均有顯著影響,其中對(duì)φPSⅡ的影響最為顯著.由圖6和多重比較可見,AHTN高濃度(350μg/L)處理組的4項(xiàng)葉綠素?zé)晒鈪?shù)顯著低于低濃度處理組(1,10μg/L)和對(duì)照組,且隨著處理濃度的增大,AHTN對(duì)4項(xiàng)參數(shù)抑制作用增強(qiáng).1μg/L處理組對(duì)東海原甲藻的Fv/Fm、、rETRmax有刺激作用,10μg/L處理組對(duì)Fv/Fm和有刺激作用,但作用不顯著(>0.05).根據(jù)多重比較結(jié)果,Fv/Fm、φPSⅡ隨處理時(shí)間增長而減小.

表1 AHTN濃度和處理時(shí)間對(duì)東海原甲藻光合活性參數(shù)的雙因素方差分析和多重比較(Duncan)結(jié)果

注:不同字母表示實(shí)驗(yàn)組間存在顯著差異,按升序排列,即a

3 討論

3.1 AHTN對(duì)東海原甲藻生長有低促高抑的作用

本文中,AHTN對(duì)東海原甲藻生長的作用表現(xiàn)為低濃度時(shí)促進(jìn)、高濃度時(shí)抑制.1μg/L實(shí)驗(yàn)組為探究接近環(huán)境濃度的低濃度AHTN對(duì)微藻的影響,結(jié)果表明,在1μg/L AHTN處理下,東海原甲藻被刺激生長,產(chǎn)生毒物興奮效應(yīng)(hormesis)[14],這種效應(yīng)反映的是一種生物的過度補(bǔ)償[15],因此AHTN可能會(huì)在實(shí)際環(huán)境中的低濃度下引發(fā)赤潮危害.10μg/L AHTN處理組對(duì)東海原甲藻的生長抑制在第96h恢復(fù),可能由于AHTN降解為低毒產(chǎn)物,或者藻細(xì)胞在低濃度脅迫下自身解毒機(jī)制被激活[16].AHTN對(duì)東海原甲藻的生長抑制表現(xiàn)出明顯的劑量-效應(yīng)關(guān)系,由抑制率計(jì)算得到AHTN對(duì)東海原甲藻的96h- EC50為48.21μg/L,根據(jù)《新化學(xué)危害評(píng)估導(dǎo)則》[17]中的分級(jí)標(biāo)準(zhǔn),AHTN對(duì)東海原甲藻為高毒性(EC50<1mg/L),可能因?yàn)锳HTN有較高的logK而易在生物體內(nèi)積蓄,和含有致毒的苯環(huán)結(jié)構(gòu).但由于AHTN有揮發(fā)性,實(shí)驗(yàn)得到的生物毒性可能被低估.由此可見,AHTN能夠抑制海洋微藻種群的生長,而微藻作為初級(jí)生產(chǎn)者,AHTH的毒害作用將會(huì)沿著食物鏈放大累積,進(jìn)而對(duì)整個(gè)海洋生態(tài)系統(tǒng)造成潛在威脅.

當(dāng)受到污染脅迫時(shí),光合色素含量通常是表征藻類生長的一個(gè)指標(biāo)[18].本實(shí)驗(yàn)中chl-a與chl-c的合成也有低促高抑的現(xiàn)象:低AHTN濃度(1和10μg/L)促進(jìn)光合色素的合成,這可能是藻細(xì)胞一種自我保護(hù)機(jī)制,用于清除葉綠體中因外界脅迫積累的活性氧(ROS)[19];高濃度AHTN(350μg/L)抑制光合色素的生成,且隨濃度增大抑制作用增強(qiáng),光合色素的減少也是植物和微藻的常見脅迫反應(yīng),可能是類囊體脂質(zhì)的過氧化和光合系統(tǒng)PSII復(fù)合物的降解所導(dǎo)致的[20].當(dāng)葉綠素含量下降時(shí),可能會(huì)阻礙植物正常的光吸收和散射[21],從而影響光合系統(tǒng)活性.

3.2 AHTN損傷東海原甲藻的形貌結(jié)構(gòu)及細(xì)胞膜完整性

細(xì)胞結(jié)構(gòu)的完整性是細(xì)胞增殖和發(fā)育的重要因素[22].由兩種電鏡結(jié)果可以看出,400μg/L AHTN對(duì)藻細(xì)胞的形態(tài)結(jié)構(gòu)造成了一定的破壞.根據(jù)SEM圖片和陸斗定等[23]的報(bào)道分析,東海原甲藻表面均勻分布的球形突起可以增大毒性物質(zhì)與藻細(xì)胞的接觸面積,零星的殼面小孔有利于毒性物質(zhì)進(jìn)入藻體,這可能是東海原甲藻易受AHTN損傷的關(guān)鍵.TEM結(jié)果顯示,AHTN可能破壞細(xì)胞膜并誘導(dǎo)細(xì)胞內(nèi)物質(zhì)泄漏,導(dǎo)致藻細(xì)胞死亡;部分藻細(xì)胞觀察到葉綠體結(jié)構(gòu)被破壞,這將會(huì)導(dǎo)致葉綠素合成受阻,這可能也是上節(jié)討論中高濃度AHTN處理組光合色素含量減少的原因之一.

細(xì)胞膜是細(xì)胞選擇性的動(dòng)態(tài)屏障,在調(diào)節(jié)生理生化過程中發(fā)揮重要作用[24].MDA是膜脂質(zhì)過氧化的產(chǎn)物,可衡量藻細(xì)胞受逆境脅迫后膜結(jié)構(gòu)損傷的程度[25].AHTN對(duì)東海原甲藻細(xì)胞膜的影響可通過膜的通透性和MDA含量變化來反映.根據(jù)實(shí)驗(yàn)結(jié)果分析,低濃度AHTN(1和10μg/L)下的膜受損細(xì)胞的比例隨著處理時(shí)間增長略微減小,可能是由于隨著東海原甲藻的生長,AHTN對(duì)總?cè)后w的毒性作用逐漸減弱,或由于藻細(xì)胞自身修復(fù).高濃度AHTN (350μg/L)處理組隨暴露時(shí)間的增長對(duì)細(xì)胞膜的破壞增強(qiáng),在96h時(shí)隨濃度升高強(qiáng)熒光細(xì)胞百分比逐漸減小,可能由于部分細(xì)胞在高濃度AHTN脅迫下破壞程度較大,胞內(nèi)核酸物質(zhì)泄露(圖3f),導(dǎo)致流式細(xì)胞儀檢測(cè)到的熒光細(xì)胞數(shù)量減少.細(xì)胞膜通透性的改變可能導(dǎo)致細(xì)胞體積控制過程中的滲透壓調(diào)節(jié)功能被破壞[26],這可以用來解釋SEM觀察到的細(xì)胞皺縮的現(xiàn)象(圖3b,c).MDA含量呈濃度依賴性變化,表明AHTN處理濃度增大使膜脂質(zhì)過氧化加劇,進(jìn)而導(dǎo)致細(xì)胞膜完整性的損害.這些結(jié)果表明, AHTN可以破壞藻細(xì)胞膜完整性進(jìn)入細(xì)胞體內(nèi),從而影響藻類生長.

3.3 AHTN使東海原甲藻的生理生化功能產(chǎn)生氧化應(yīng)激

在藻細(xì)胞正常生理代謝過程中,葉綠體和線粒體等細(xì)胞器均可能產(chǎn)生ROS[12],低濃度的ROS可以調(diào)節(jié)細(xì)胞周期、免疫和基因組完整性[27].當(dāng)藻細(xì)胞受到外界脅迫時(shí),可能會(huì)誘導(dǎo)ROS快速產(chǎn)生并累積,造成細(xì)胞氧化損傷.ROS主要包括超氧陰離子自由基(O2?)、過氧化氫(H2O2)和羥基自由基(·OH)等,藻細(xì)胞可通過體內(nèi)的抗氧化酶類和抗氧化物質(zhì)清除ROS和還原其為毒性較小的物質(zhì)來減少氧化損傷[28].大多數(shù)氧化損傷始于O2?的產(chǎn)生,SOD可以將O2?轉(zhuǎn)化為H2O2,因此是抵抗ROS的第一道防線[29].在50～400μg/L AHTN處理下SOD活性升高,這是因?yàn)樵寮?xì)胞通過增加SOD活力來對(duì)抗AHTN引起的氧化脅迫;當(dāng)濃度繼續(xù)增大時(shí),SOD活性降低,說明此時(shí)的氧化脅迫已經(jīng)超出細(xì)胞的防御能力,藻細(xì)胞受到損傷.當(dāng)酶清除系統(tǒng)不堪重負(fù)時(shí),會(huì)使H2O2累積并分解產(chǎn)生·OH,·OH可以抑制PSII的電子傳遞并減少光合作用相關(guān)的基因(B,D1)的數(shù)量,從而阻礙光合作用進(jìn)程[30],這可能是本研究中高濃度AHTN脅迫下光合活性顯著降低的原因.TSP主要參與各種代謝酶,用作藻類細(xì)胞對(duì)污染物抗性的指標(biāo)[31].TSP在高濃度AHTN(350μg/L)下顯著降低,可能與微藻生物量的減少有關(guān).MDA含量增加說明膜系統(tǒng)受到了氧化損傷,蛋白質(zhì)也會(huì)因膜系統(tǒng)受到干擾而加速降解[32].另外,蛋白質(zhì)是植物光合作用的部分產(chǎn)物,chl-a的減少也影響了蛋白質(zhì)的生產(chǎn)[33].GSH是藻細(xì)胞內(nèi)重要的抗氧化劑,同時(shí)也是谷胱甘肽過氧化物酶和谷胱甘肽轉(zhuǎn)移酶的底物[34].GSH含量在暴露濃度范圍內(nèi)出現(xiàn)先升高后降低的現(xiàn)象表明, GSH首先被誘導(dǎo)產(chǎn)生,但隨著酶的清除或細(xì)胞破損使GSH含量減少或流失,但處理組GSH含量基本顯著高于對(duì)照組,說明即使在SOD等抗氧化酶受到抑制時(shí),GSH仍能發(fā)揮其抗氧化作用,這與王執(zhí)偉[35]的研究結(jié)果一致.

葉綠素?zé)晒饧夹g(shù)常用于監(jiān)測(cè)植物和藻類的光合性能,因此可以通過葉綠素?zé)晒鈪?shù)來評(píng)估在逆境脅迫下植物的光合作用能力的改變[36].Fv/Fm可以反映藻細(xì)胞的最大光合作用能力,在高濃度AHTN(350μg/L)處理下Fv/Fm下降表明,AHTN能顯著抑制東海原甲藻的光合性能,并且脅迫程度隨著暴露時(shí)間增長而增加;低濃度(1和10 μg/L)處理下則能夠增強(qiáng)微藻的光合能力.φPSⅡ反映了PSⅡ反應(yīng)中心實(shí)際光化學(xué)效率,φPSⅡ下降表明,AHTN使細(xì)胞同化功能受損,影響了碳的固定和同化[36].α反映了微藻對(duì)光能的利用效率[37],這種效能也表現(xiàn)出在低濃度AHTN(1和10μg/L)下增強(qiáng),在高濃度AHTN(350μg/L)下減弱的現(xiàn)象.rETRmax反映了藻類的潛在最大光合速率,常用于指示微藻的“生長潛能”[38],10μg/L濃度下微藻的生長潛能在72h逐漸恢復(fù),與細(xì)胞密度恢復(fù)的結(jié)果相一致;高濃度AHTN (350μg/L)組rETRmax減小表明AHTN阻礙了東海原甲藻PSⅡ中心的電子傳遞過程.藻細(xì)胞吸收的光能如果無法通過光合作用耗散,這部分過剩能量可能會(huì)以ROS的形式聚集,從而對(duì)細(xì)胞造成損傷[39].因此,AHTN可以通過影響東海原甲藻的光合系統(tǒng)活性來抑制細(xì)胞生長.

根據(jù)以上結(jié)果可見,東海原甲藻的抗氧化系統(tǒng)和光合系統(tǒng)等生理生化功能系統(tǒng)在AHTN脅迫下受到顯著影響.在低濃度AHTN脅迫下藻細(xì)胞可以通過自身調(diào)節(jié)作用維持正常生理功能,但隨著處理濃度的升高,抗氧化系統(tǒng)活性和光合作用性能的降低可能會(huì)導(dǎo)致細(xì)胞中ROS的累積,從而導(dǎo)致蛋白質(zhì)變性、脂質(zhì)過氧化和葉綠素漂白[40],最終影響藻類生長.張薛薇等[41]也報(bào)道了4種香料通過抑制藻細(xì)胞抗氧化酶活性和葉綠素含量導(dǎo)致藻類生長異常.另外,東海原甲藻的生理生化功能也在低AHTN濃度脅迫下出現(xiàn)毒性興奮效應(yīng),這與Liu等[29]、Wang等[42]和劉偉杰等[43]的研究結(jié)果相似.據(jù)報(bào)道,植物的毒性興奮效應(yīng)會(huì)影響其細(xì)胞和分子機(jī)制,包括光合作用、希爾反應(yīng)、葉綠素含量、信號(hào)傳導(dǎo)途徑和抗氧化酶,從而反映到作物產(chǎn)量[14],這解釋了東海原甲藻的生長在1μg/L AHTN處理下被促進(jìn)和在10μg/L AHTN處理下恢復(fù)生長的現(xiàn)象.

4 結(jié)論

4.1 低濃度AHTN(1μg/L)會(huì)促進(jìn)東海原甲藻生長,1,10μg/L AHTN能夠促進(jìn)其光合色素的合成,對(duì)細(xì)胞膜完整性無顯著影響,能夠激發(fā)抗氧化系統(tǒng)活性,促進(jìn)光合作用性能增強(qiáng),因而具有導(dǎo)致以東海原甲藻為優(yōu)勢(shì)種的赤潮爆發(fā)的潛在風(fēng)險(xiǎn).

4.2 較高濃度AHTN(10～400μg/L)對(duì)東海原甲藻的生長有抑制作用,50～400μg/L AHTN能夠抑制東海原甲藻光合色素產(chǎn)生,破壞藻細(xì)胞膜和內(nèi)部結(jié)構(gòu)的完整性,在誘導(dǎo)氧化應(yīng)激后破壞細(xì)胞抗氧化能力,造成光合系統(tǒng)損傷,證明AHTN對(duì)海洋生態(tài)環(huán)境存在潛在的威脅.

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Toxic mechanism of tonalide on.

QU Yu-ying, ZHANG Cai-jie, SHEN Qiu-cen, ZHANG Jing*, YU Hong

(College of Chemistry Engineering, Ocean University of China, Qingdao 266100, China)., 2022,42(3)：1401～1409

was selected to study the toxic mechanism of tonalide (AHTN) on marine microalgae. Five concentrations (1, 10, 50, 200 and 400μg/L) of AHTN were tested to evaluate its influence on the growth, photosynthetic pigment content, cell morphology and structure, cell membrane permeability and the antioxidant and photosynthetic systems. The results showed that AHTN significantly inhibited the growth ofwith high toxicity (96h-EC50= 48.21μg/L). The mechanism of realization of the algal toxicity of AHTN is the inhibition of photosynthetic pigment production, the destruction of cell structure and cell membrane integrity, the induction of antioxidant system response, and the effect of photosynthe performance. However, treatment with 1μg/L AHTN treatment promoted the growth of. Meanwhile, treatment with 1μg/L and 10μg/L AHTN promoted the synthesis of photosynthetic pigments and increased the activity of superoxide dismutase as well as some photosynthetic parameters. In conclusion, low concentrations of AHTN (1μg/L) may induce red tides of, while high concentrations of AHTN (10～400μg/L) could inhibit the population growth of this algae and cause a potential threat to marine ecosystems.

tonalide；；toxic mechanism；antioxidant system；photosynthetic characteristics

X55

A

1000-6923(2022)03-1401-09

曲玉楹(1997-),女,山東青島人,中國海洋大學(xué)化學(xué)化工學(xué)院碩士研究生,主要從事新興污染物的生態(tài)毒理學(xué)研究.

2021-08-17

國家自然科學(xué)基金青年科學(xué)基金資助項(xiàng)目(41806093)

*責(zé)任作者, 教授, zhangjouc@ouc.edu.cn