氧化石墨烯對(duì)嗜熱四膜蟲(chóng)的毒性效應(yīng)

廖苑辰,常葉倩,徐晨珂,崔益斌,李 梅*

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氧化石墨烯對(duì)嗜熱四膜蟲(chóng)的毒性效應(yīng)

廖苑辰1,常葉倩1,徐晨珂1,崔益斌2,李 梅1*

(1.南京大學(xué)環(huán)境學(xué)院,污染控制與資源化研究國(guó)家重點(diǎn)實(shí)驗(yàn)室,江蘇 南京 210023；2.生態(tài)環(huán)境部南京環(huán)境科學(xué)研究所,江蘇 南京 210042)

以嗜熱四膜蟲(chóng)()作為受試生物,考察了納米材料氧化石墨烯(GO)對(duì)其細(xì)胞生長(zhǎng)率、乙酰膽堿酯酶(AchE)和氧化應(yīng)激酶活性、生物膜損傷及細(xì)胞凋亡的影響,以探究GO的毒性效應(yīng).結(jié)果表明,GO濃度高于32mg/L時(shí)顯著抑制嗜熱四膜蟲(chóng)的細(xì)胞增殖(<0.05),細(xì)胞存活率低于50%;在0~64mg/L實(shí)驗(yàn)范圍內(nèi),隨GO暴露濃度增加,細(xì)胞內(nèi)活性氧自由基(ROS)和超氧化物歧化酶(SOD)水平呈先升后降的趨勢(shì),AchE活性受抑;GO抑制位于線粒體內(nèi)膜的琥珀酸脫氫酶(SDH)活性,促進(jìn)細(xì)胞質(zhì)中乳酸脫氫酶(LDH)的釋放;64mg/L GO導(dǎo)致四膜蟲(chóng)細(xì)胞出現(xiàn)明顯凋亡現(xiàn)象.以上結(jié)果顯示,中低濃度GO(0~8mg/L)暴露下,氧化應(yīng)激機(jī)制對(duì)細(xì)胞毒性起主要貢獻(xiàn)作用;高濃度GO(32和64mg/L)作用下,四膜蟲(chóng)凋亡現(xiàn)象的產(chǎn)生可能是GO抑制其生長(zhǎng)作用導(dǎo)致的.

氧化石墨烯(GO)；嗜熱四膜蟲(chóng)；毒性效應(yīng)；氧化應(yīng)激

納米材料氧化石墨烯(GO)因其獨(dú)特的物理及化學(xué)性質(zhì),在生物、醫(yī)學(xué)和材料科學(xué)等領(lǐng)域具有廣闊的應(yīng)用前景,尤其作為藥物的靶向載體,被廣泛用于生物醫(yī)藥領(lǐng)域.然而,GO在生產(chǎn)、貯存、運(yùn)輸、消費(fèi)或廢棄過(guò)程中不可避免地進(jìn)入到環(huán)境中,造成暴露風(fēng)險(xiǎn),最終可能通過(guò)直接排放或地表徑流等方式遷移進(jìn)入水體,對(duì)水生生態(tài)系統(tǒng)產(chǎn)生危害.研究表明,GO可進(jìn)入溶酶體、線粒體、細(xì)胞核和內(nèi)質(zhì)網(wǎng),誘導(dǎo)氧化應(yīng)激和細(xì)胞凋亡[1],并通過(guò)改變細(xì)胞活力和形態(tài)、破壞膜完整性、誘導(dǎo)DNA損傷[2-4]等方式，對(duì)紅細(xì)胞、成纖維細(xì)胞和大鼠腎上腺嗜鉻細(xì)胞瘤細(xì)胞(PC12)等產(chǎn)生毒性.目前,有關(guān)GO對(duì)水生生物的毒性效應(yīng)研究主要圍繞藻類(lèi)[5-7]、魚(yú)類(lèi)[8-10]、雙殼類(lèi)動(dòng)物[11]和部分浮游動(dòng)物[12-13]展開(kāi),而對(duì)原生動(dòng)物的研究較少[3].

原生動(dòng)物是無(wú)細(xì)胞壁的單細(xì)胞真核生物,直接暴露在水環(huán)境中,對(duì)污染物更為敏感,在水環(huán)境污染物毒性研究中發(fā)揮重要作用.四膜蟲(chóng)()外觀呈橢圓形長(zhǎng)梨狀,體長(zhǎng)約50μm.作為一種模式生物,四膜蟲(chóng)在過(guò)去幾十年已成為一種有效的檢測(cè)工具,被廣泛應(yīng)用于外源化學(xué)物質(zhì)的潛在毒性研究[14].

本研究以嗜熱四膜蟲(chóng)作為研究對(duì)象,結(jié)合細(xì)胞數(shù)和乙酰膽堿酯酶活性、氧化應(yīng)激酶活性、生物膜損傷和凋亡形態(tài)學(xué)等指標(biāo),考察GO對(duì)四膜蟲(chóng)的細(xì)胞毒性、氧化應(yīng)激、生物膜系統(tǒng)損傷和細(xì)胞凋亡的影響,為評(píng)估GO對(duì)四膜蟲(chóng)的毒性效應(yīng)及其作用機(jī)制提供基礎(chǔ)數(shù)據(jù)和科學(xué)依據(jù).

1 材料與方法

1.1 試驗(yàn)材料

嗜熱四膜蟲(chóng)株系()由中國(guó)科學(xué)院水生生物研究所贈(zèng)予.采用SPP培養(yǎng)基:2%胰蛋白胨,0.1%酵母膏提取物,0.2%葡萄糖,0.003%西奎斯特林(13% Fe-EDTA)培養(yǎng),加入100μL 100′青鏈霉素合劑,121℃滅菌20min,冷卻后4℃保存.

GO購(gòu)自南京先豐納米材料科技有限公司.

1.2 試驗(yàn)方法

1.2.1 GO懸液配制及表征 GO懸液現(xiàn)配現(xiàn)用.將GO固體薄片置于滅菌超純水中,使懸液中GO濃度為1mg/mL,超聲處理15min(功率200W,10s脈沖,5s間隔),使體系充分分散.用超純水稀釋如下濃度:5, 20,80,320,640mg/L作為懸液母液,對(duì)照組為滅菌超純水.

GO形貌特征表征采用透射電鏡(TEM, Tecnai 12,荷蘭Philips公司)、拉曼光譜(HR-800,法國(guó)Horiba Jobin Yvon公司)和傅里葉變換紅外光譜儀(FTIR, Nicolet iS5,美國(guó)Thermo Fisher Scientific公司)進(jìn)行.

1.2.2 GO對(duì)嗜熱四膜蟲(chóng)細(xì)胞生長(zhǎng)率的影響 根據(jù)預(yù)試驗(yàn)結(jié)果,確定正式試驗(yàn)GO濃度分別為:0.5, 8,32,64mg/L.取處于對(duì)數(shù)培養(yǎng)期的四膜蟲(chóng)培養(yǎng)液9mL裝至50mL螺紋離心管中,加入1mL相應(yīng)濃度GO懸液母液進(jìn)行培養(yǎng),使GO終濃度為0.5,8,32, 64mg/L.每組設(shè)置3個(gè)平行,另設(shè)空白對(duì)照,置于光強(qiáng)3000lx左右,30℃的恒溫培養(yǎng)箱中培養(yǎng),分別于24h 和48h進(jìn)行細(xì)胞密度計(jì)數(shù).根據(jù)實(shí)驗(yàn)結(jié)果作時(shí)間-劑量-效應(yīng)圖,并利用T檢驗(yàn)確定是否具有顯著性.

1.2.3 酶活性指標(biāo)的測(cè)定 培養(yǎng)方法同前,72h后離心(1000r/min,3min,4℃),取上清液測(cè)定乳酸脫氫酶(LDH)活性.再取細(xì)胞沉淀,加入磷酸鹽緩沖液(PBS)后超聲破碎細(xì)胞(JY88-II型超聲細(xì)胞破碎儀,超聲20s,間隔10s,重復(fù)6次).將破碎后的細(xì)胞懸液離心(12000r/min,5min,4℃),取上清液測(cè)定活性氧自由基(ROS)、超氧化物歧化酶(SOD)、乙酰膽堿酯酶(AchE)和琥珀酸脫氫酶(SDH)活性.試驗(yàn)用試劑盒及方法均來(lái)自南京建成生物工程研究所.

1.2.4 四膜蟲(chóng)細(xì)胞凋亡檢測(cè) 培養(yǎng)方法同前,72h后離心(1000r/min,3min,4℃),棄上清液.用細(xì)滴管將余下的0.5mL細(xì)胞懸液逐滴加入4℃預(yù)冷的1.5mL無(wú)水乙醇(99%)中,邊加邊震蕩,4℃冰箱過(guò)夜,固定18h.細(xì)胞固定后2周內(nèi)進(jìn)行實(shí)驗(yàn).通過(guò)加入PBS調(diào)整懸液濃度為1′106個(gè)細(xì)胞/mL,取1mL上述細(xì)胞液,離心后(1000r/min,3min,4℃)用PBS洗滌,重復(fù)3次,去除殘留乙醇,棄上清,留細(xì)胞沉淀.加入RNA酶A(100μg/mL),37℃水浴中孵育30min.孵育結(jié)束再加入0.5mg/mL碘化丙啶(PI)染液50μL進(jìn)行DNA染色,4℃再孵育60min.上機(jī)前用200目尼龍濾網(wǎng)過(guò)濾,流式細(xì)胞儀于488nm激發(fā)條件下對(duì)細(xì)胞凋亡情況進(jìn)行分析.每個(gè)樣品檢測(cè)微粒數(shù)為2′104個(gè).

1.3 數(shù)據(jù)分析

利用SPSS 20.0軟件處理實(shí)驗(yàn)所得數(shù)據(jù),進(jìn)行正態(tài)分布檢驗(yàn),并用T檢驗(yàn)和方差分析(ANOVA)進(jìn)行統(tǒng)計(jì)處理.以<0.05表示具有顯著性差異.

2 結(jié)果與討論

2.1 GO表征

通過(guò)TEM、拉曼光譜和FTIR對(duì)GO進(jìn)行相關(guān)表征,結(jié)果如圖1所示.本實(shí)驗(yàn)所用GO主要以單片層形式存在,其片層厚度相對(duì)均一,約1~2nm,在納米尺度范圍內(nèi).拉曼譜圖驗(yàn)證了GO在1330cm-1(D帶)和1576cm-1(G帶)附近有兩個(gè)特征峰.紅外譜圖顯示GO具有大量親水性官能團(tuán),如3363cm-1附近出現(xiàn)明顯吸收峰,代表O—H伸縮振動(dòng);1732cm-1處特征峰代表GO石墨片層邊緣C=O伸縮振動(dòng)[15];1371cm-1附近的峰歸屬O—H的彎曲振動(dòng);1074cm-1處為C—O的伸縮振動(dòng).該納米材料在水中的分散情況良好.

2.2 GO對(duì)四膜蟲(chóng)的毒性效應(yīng)

2.2.1 GO對(duì)四膜蟲(chóng)細(xì)胞生長(zhǎng)率的影響 分別于培養(yǎng)24和48h后計(jì)數(shù),得到四膜蟲(chóng)在不同時(shí)間及不同濃度GO下的存活數(shù)量(圖2).結(jié)果顯示在培養(yǎng)細(xì)胞中加入一定量GO后,相同暴露時(shí)間內(nèi),隨GO濃度的增加,四膜蟲(chóng)細(xì)胞數(shù)量減少.高濃度GO(32和64mg/L)明顯抑制細(xì)胞的增殖(<0.05).經(jīng)驗(yàn)證,GO對(duì)四膜蟲(chóng)活性影響在48h內(nèi)與時(shí)間關(guān)系不顯著.Zhao等[16]研究了GO對(duì)小球藻()的毒性效應(yīng),結(jié)果顯示,GO可通過(guò)遮蔽效應(yīng)抑制約16%藻類(lèi)的生長(zhǎng).石柳等[12]研究發(fā)現(xiàn),GO對(duì)大型溞()的毒性表現(xiàn)出一定劑量-效應(yīng)關(guān)系,在65~ 143mg/L范圍內(nèi),隨GO濃度的升高,大型溞死亡率從10%逐漸增加到100%.另有研究表明,GO對(duì)小眼蟲(chóng)()的96h EC50為(3.76±0.74)mg/L,當(dāng)GO濃度超過(guò)2.5mg/L時(shí)產(chǎn)生明顯不良反應(yīng)(<0.01),抑制生物生長(zhǎng)[3].上述研究結(jié)果與本實(shí)驗(yàn)結(jié)果存在一定差異,因?yàn)椴煌镏gGO毒性大小有所區(qū)別,且培養(yǎng)基營(yíng)養(yǎng)成分的不同也可能對(duì)GO的毒性造成影響[3].

圖2 GO對(duì)嗜熱四膜蟲(chóng)細(xì)胞生長(zhǎng)率的影響Fig.2 Effects of GO on cell growth rate of T.thermophila與CK組相比有顯著差異,*P<0.05

用單個(gè)樣本K-S檢驗(yàn)、Q-Q圖分別對(duì)24和48h四膜蟲(chóng)細(xì)胞數(shù)的正態(tài)分布進(jìn)行檢驗(yàn)(圖3).在SPSS中執(zhí)行“分析-非參數(shù)檢驗(yàn)-單個(gè)樣本K-S檢驗(yàn)”,24h的值為0.745,值0.636>0.05;48h的值為0.606,值=0.856>0.05;Q-Q圖中,各點(diǎn)基本圍繞圖中對(duì)角線.正態(tài)檢驗(yàn)結(jié)果說(shuō)明本實(shí)驗(yàn)所得GO毒性數(shù)據(jù)呈近似正態(tài)分布.

2.2.2 GO對(duì)四膜蟲(chóng)的氧化應(yīng)激作用 氧化應(yīng)激被認(rèn)為是納米材料重要的毒性機(jī)制[16].細(xì)胞內(nèi)的ROS水平能直接反映細(xì)胞自由基的產(chǎn)生情況.SOD是機(jī)體抗氧化防御體系中的關(guān)鍵酶,可通過(guò)其在機(jī)體內(nèi)含量和活性的變化間接反映機(jī)體的氧化損傷程度,保護(hù)細(xì)胞免受ROS的不利影響[17].

結(jié)果表明GO處理下ROS和SOD水平均隨其濃度增大呈現(xiàn)先升后降的趨勢(shì)(圖4).在0~8mg/L范圍內(nèi),隨著GO暴露濃度增加,細(xì)胞內(nèi)ROS含量升高,說(shuō)明中低濃度GO可誘導(dǎo)四膜蟲(chóng)細(xì)胞產(chǎn)生氧化應(yīng)激,此時(shí)細(xì)胞中的SOD活性增強(qiáng)以清除ROS,減輕GO造成的氧化脅迫.高濃度GO(32和64mg/L)條件下,ROS和SOD均呈大幅下降,結(jié)合GO濃度高于32mg/L暴露下,四膜蟲(chóng)存活率不足50%的情況,推測(cè)此時(shí)嗜熱四膜蟲(chóng)難以生存,細(xì)胞中的抗氧化酶被破壞,導(dǎo)致酶活性降低.已有研究證實(shí),GO能誘導(dǎo)藻類(lèi)細(xì)胞和斑馬魚(yú)產(chǎn)生氧化應(yīng)激,引起ROS的產(chǎn)生,破壞細(xì)胞完整性[18]. Nogueira等[19]將綠藻暴露于0~ 100μg/L GO,發(fā)現(xiàn)當(dāng)其濃度為20μg/mL時(shí),綠藻的生長(zhǎng)抑制率為50%,且在96h孵育時(shí)間內(nèi),GO濃度與ROS產(chǎn)生呈顯著正相關(guān)(<0.05);Lu等[20]將斑馬魚(yú)暴露于10mg/L GO培養(yǎng)14d,發(fā)現(xiàn)GO誘導(dǎo)斑馬魚(yú)胚胎產(chǎn)生過(guò)量ROS,造成斑馬魚(yú)明顯的組織損傷.上述結(jié)果表明GO的毒性作用可能與其誘導(dǎo)細(xì)胞內(nèi)ROS含量增加有關(guān),提示本研究中高濃度(32和64mg/L)GO對(duì)細(xì)胞的毒害作用可能是由于ROS升高造成的.

2.2.3 GO對(duì)四膜蟲(chóng)AchE活性的影響 GO暴露下四膜蟲(chóng)AchE活性變化見(jiàn)圖5,當(dāng)GO濃度大于8mg/L時(shí),隨暴露濃度升高,AchE活性呈下降趨勢(shì).由圖4可知,8mg/L GO作用下ROS活性最高,已有研究證實(shí)H2O2作為ROS的一種對(duì)AchE具有調(diào)節(jié)作用,高濃度H2O2抑制AchE活性[21],因此猜測(cè)8mg/L GO誘導(dǎo)ROS活性升高,導(dǎo)致AchE活性受抑.一般而言,當(dāng)AchE活性抑制率大于20%時(shí),說(shuō)明污染物對(duì)生物體有暴露毒性作用的存在;抑制率大于50%時(shí),認(rèn)為污染物對(duì)生物生存產(chǎn)生危害[22].本實(shí)驗(yàn)中GO高濃度(32和64mg/L)處理下,AchE活性抑制率超過(guò)50%,表明GO對(duì)四膜蟲(chóng)造成毒害作用,威脅生物生存.另外,考慮到乙酰膽堿作為神經(jīng)遞質(zhì)在運(yùn)動(dòng)行為控制中起重要作用[23],四膜蟲(chóng)的運(yùn)動(dòng)能力也受到一定程度的抑制.這也驗(yàn)證了2.2.2中GO濃度高于32mg/L時(shí)四膜蟲(chóng)難以存活的結(jié)果.周夢(mèng)媛等[24]關(guān)于Al2O3納米顆粒對(duì)梨形四膜蟲(chóng)細(xì)胞毒性的研究亦顯示,高濃度(500mg/L)納米Al2O3抑制四膜蟲(chóng)AchE活性.

圖5 GO暴露下四膜蟲(chóng)AchE活性變化Fig.5 Variation of acetylcholinesterase (AchE) activity of T.thermophila after GO exposure與CK組相比有顯著差異,* P<0.05

2.2.4 GO對(duì)線粒體內(nèi)膜、細(xì)胞膜的影響 SDH位于線粒體內(nèi)膜,參與三羧酸循環(huán)和電子傳遞鏈,能反映細(xì)胞功能狀態(tài)的糖代謝過(guò)程,其濃度可以表征四膜蟲(chóng)細(xì)胞線粒體活性的高低及內(nèi)膜完整程度[24].GO的存在使四膜蟲(chóng)SDH活性受到抑制,暴露濃度越高,抑制作用越明顯(圖6A).與對(duì)照組相比,各處理組SDH活力均顯著降低(<0.05).Zhou等[25]對(duì)暴露于石墨烯的各種類(lèi)型細(xì)胞SDH活性進(jìn)行檢測(cè),發(fā)現(xiàn)在所有測(cè)試細(xì)胞中,石墨烯以劑量-依賴(lài)方式直接抑制SDH活性,與本實(shí)驗(yàn)結(jié)果相符.線粒體是ROS產(chǎn)生的重要場(chǎng)所,也是ROS攻擊的主要部位.相關(guān)研究已證實(shí)納米材料能誘導(dǎo)氧化應(yīng)激產(chǎn)生ROS,破壞線粒體膜完整性[26].本實(shí)驗(yàn)中,GO抑制SDH活性,推測(cè)GO使四膜蟲(chóng)細(xì)胞線粒體活性降低,內(nèi)膜受損,細(xì)胞能量代謝受阻,從而抑制細(xì)胞生長(zhǎng).結(jié)合GO對(duì)四膜蟲(chóng)的生長(zhǎng)抑制作用(圖3),進(jìn)一步證實(shí)SDH活性變化可能與細(xì)胞增殖有關(guān)[24].

LDH作為胞質(zhì)酶穩(wěn)定存在于細(xì)胞質(zhì)中,當(dāng)細(xì)胞凋亡或裂解造成細(xì)胞膜結(jié)構(gòu)破壞時(shí)即被釋放到細(xì)胞外,因此該酶是細(xì)胞膜完整性的重要指標(biāo),可作為細(xì)胞死亡的信號(hào).不同濃度GO處理后對(duì)四膜蟲(chóng)LDH活性的影響如圖6(B)所示.低濃度(0.5mg/L)條件下,GO對(duì)LDH的釋放無(wú)影響;當(dāng)GO濃度高于8mg/L時(shí),處理組LDH活性較對(duì)照組顯著升高(<0.05),說(shuō)明細(xì)胞膜損傷嚴(yán)重,胞內(nèi)組分釋放,四膜蟲(chóng)的生命活動(dòng)受到影響.Zhang等[27]研究了石墨烯和碳納米管對(duì)PC12細(xì)胞的毒性作用,發(fā)現(xiàn)石墨烯和碳納米管均能誘導(dǎo)細(xì)胞內(nèi)LDH表達(dá)水平升高.另有研究證實(shí),GO能誘導(dǎo)HeLa細(xì)胞LDH的釋放[28],與本實(shí)驗(yàn)結(jié)果一致.

目前,有關(guān)納米材料細(xì)胞毒性的研究有3種主要機(jī)制:氧化應(yīng)激、金屬毒性和物理刺穿導(dǎo)致細(xì)胞破裂[29].針對(duì)石墨烯家族納米材料(GFNs)的毒性機(jī)制,有研究提出,GFNs通過(guò)不同方式進(jìn)入細(xì)胞,誘導(dǎo)ROS產(chǎn)生,使LDH增加,隨后引起各種細(xì)胞損傷(如細(xì)胞膜損傷、線粒體損傷、細(xì)胞凋亡或壞死等),是GFNs細(xì)胞毒性的主要可能機(jī)制[30].在本實(shí)驗(yàn)中,GO濃度為0~8mg/L時(shí),GO與細(xì)胞作用產(chǎn)生的ROS呈濃度依賴(lài)性(圖4A),LDH亦呈上升趨勢(shì),推測(cè)由于GO誘導(dǎo)細(xì)胞產(chǎn)生ROS,ROS水平過(guò)高引發(fā)脂質(zhì)過(guò)氧化反應(yīng),損傷細(xì)胞膜,此時(shí)可能是ROS對(duì)細(xì)胞毒性起貢獻(xiàn)作用;高濃度GO(32和64mg/L)暴露下,ROS低于正常水平,而胞外LDH活性顯著升高,推測(cè)四膜蟲(chóng)細(xì)胞受到破壞,從而產(chǎn)生強(qiáng)毒害作用.

2.2.5 GO對(duì)四膜蟲(chóng)細(xì)胞凋亡形態(tài)學(xué)分析 以上結(jié)果已證實(shí)GO能引起細(xì)胞膜及線粒體的損傷,為進(jìn)一步檢驗(yàn)其毒性,利用流式細(xì)胞儀對(duì)GO暴露下的四膜蟲(chóng)細(xì)胞凋亡形態(tài)學(xué)進(jìn)行分析(圖7).PI是一種DNA染料,由于其不能透過(guò)活細(xì)胞膜,但當(dāng)細(xì)胞膜破損后可進(jìn)入細(xì)胞,因此常用于鑒定死細(xì)胞.前向散射光(FSC)的強(qiáng)度與細(xì)胞體積大小和活力有關(guān),側(cè)向散射光(SSC)能反映細(xì)胞的復(fù)雜程度.隨著GO濃度的上升,四膜蟲(chóng)的細(xì)胞趨向于細(xì)胞結(jié)構(gòu)復(fù)雜程度上升和體積變大,表明細(xì)胞發(fā)生了凋亡現(xiàn)象. Kang等[31]的研究證實(shí)GO以劑量依賴(lài)性方式誘導(dǎo)PC12細(xì)胞凋亡.Hu等[29]認(rèn)為GO對(duì)細(xì)胞膜的物理?yè)p傷作用是導(dǎo)致細(xì)胞凋亡的主要原因.Wang等[32]研究發(fā)現(xiàn),氧化應(yīng)激是人胚肺成纖維細(xì)胞暴露于GO后細(xì)胞凋亡和DNA損傷的關(guān)鍵原因.另有研究表明,GO可直接通過(guò)影響細(xì)胞線粒體活性從而導(dǎo)致細(xì)胞凋亡[29].本研究中,64mg/L GO能明顯引起細(xì)胞凋亡,但GO對(duì)四膜蟲(chóng)細(xì)胞的具體凋亡途徑及其機(jī)制有待進(jìn)一步研究.

圖7 不同濃度GO暴露下的凋亡形態(tài)學(xué)分析 Fig.7 Flow cytometry analysis under different GO concentrationsP2代表不同取樣的粒子區(qū)位,P1表示發(fā)生變化的粒子區(qū)位

3 結(jié)論

3.1 GO濃度高于32mg/L時(shí),嗜熱四膜蟲(chóng)生長(zhǎng)受到顯著抑制(<0.05),細(xì)胞存活率低于50%.

3.2 在實(shí)驗(yàn)濃度范圍內(nèi),隨GO暴露濃度增加, ROS和SOD水平呈現(xiàn)先升后降的趨勢(shì),AchE活性降低,細(xì)胞毒性增強(qiáng).GO抑制位于線粒體內(nèi)膜的SDH活性,促進(jìn)細(xì)胞質(zhì)中LDH的釋放,破壞細(xì)胞膜和線粒體內(nèi)膜完整性.64mg/L GO能引起四膜蟲(chóng)細(xì)胞明顯凋亡.

3.3 中、低濃度(0~8mg/L)GO暴露下,氧化損傷對(duì)細(xì)胞毒性起主要貢獻(xiàn)作用;高濃度(32和64mg/L)GO暴露下,四膜蟲(chóng)凋亡現(xiàn)象的產(chǎn)生可能是GO抑制其生長(zhǎng)作用導(dǎo)致的,具體凋亡途徑及其機(jī)制仍需進(jìn)一步研究.

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Toxicity effects of graphene oxide to

LIAO Yuan-chen1, CHANG Ye-qian1, XU Chen-ke1, CUI Yi-bin2, LI Mei1*

(1.State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China；2.Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment, Nanjing 210042, China)., 2019,39(3)：1299~1305

Effects of graphene oxide (GO) on the growth, acetylcholinesterase (AchE) activity, oxidative stress, membrane damage and apoptosis ofwere investigated. The results showed that the concentrations higher than 32mg/L GOsignificantly inhibited the growth of(<0.05), and the cell survival rate was less than 50%. With the increase of GO concentration (0~64mg/L), the levels of reactive oxygen species (ROS) and superoxide dismutase (SOD) incells initially also increased but then decreased while the activity of AchE decreased. GO inhibited the activity of succinate dehydrogenase (SDH) and promoted the release of lactate dehydrogenase (LDH). Highest concentration of GO (64mg/L) caused cell apoptosis. Based on the results, it can be concluded that oxidative stress contributed to cytotoxicity at low and medium concentrations of GO (0~8mg/L) while at higher concentrations (32 and 64mg/L) of GO, apoptosis ofmay be causedby GO inhibiting its growth.

graphene oxide (GO)；；toxic mechanism；oxidative stress

X172

A

1000-6923(2019)03-1299-07

廖苑辰(1996-),女,福建長(zhǎng)汀人,南京大學(xué)碩士研究生,主要研究方向?yàn)榄h(huán)境毒理學(xué).

2018-08-19

國(guó)家自然科學(xué)基金資助項(xiàng)目(41571468,41773115),江蘇省科技支撐項(xiàng)目(BE2016736)

* 責(zé)任作者, 教授, meili@nju.edu.cn