兩種粒徑納米銀對Nitrosomonas europaea的毒性效應

伍玲麗,張曉雪,舒昆慧,司友斌

兩種粒徑納米銀對的毒性效應

伍玲麗,張曉雪,舒昆慧,司友斌*

(安徽農業(yè)大學資源與環(huán)境學院,農田生態(tài)保育與污染防控安徽省重點實驗室,安徽 合肥 230036)

為明晰不同粒徑納米銀(AgNPs)對歐洲亞硝化毛桿菌()的毒性效應,采用室內培養(yǎng)方式,探究10nm和50nm的AgNPs對生長、氮轉化能力、細胞結構、活性氧生成和功能基因表達的影響.結果表明,AgNPs暴露抑制生長,隨著暴露時間的延長,細菌生長抑制率增加,在4h達到最大值;培養(yǎng)基中NH4+向NO2-轉化速率減緩,的銨態(tài)氮轉化能力降低;掃描電鏡(SEM)圖像顯示AgNPs造成部分細菌表面塌陷且有孔洞,細胞膜受損嚴重;透射電鏡(TEM)圖像顯示AgNPs造成細菌內部核物質消融,細胞質膜界限模糊;流式細胞儀(FCM)檢測發(fā)現AgNPs增加細胞內活性氧的生成;qRT-PCR技術對AgNPs暴露后功能基因表達進行測定,發(fā)現AgNPs抑制功能基因的轉錄表達.綜上所述,AgNPs通過與細胞膜相互作用和產生氧化應激損傷,抑制和的表達,進而影響銨態(tài)氮轉化過程,且小粒徑AgNPs的毒性強于大粒徑.

納米銀；；氧化應激；、、基因豐度；氨氧化

納米銀(AgNPs)是尺寸范圍為1~100nm的原子簇,由約20~15000個Ag原子組成,與固相Ag相比,具有電導率和熱導率高、化學性質穩(wěn)定、催化活性強等特性[1],廣泛應用于電子器件、化妝品、生物醫(yī)學和紡織業(yè)等方面[2].AgNPs的抗菌作用已被廣泛描述,一些研究認為AgNPs釋放的Ag+是其毒性的主要原因,自身的粒子特異性可以忽略不計[3-5],也有研究表示AgNPs的毒性主要取決于粒徑大小[6].據報道,20~80nm AgNPs的毒性主要來源于Ag+的釋放,而10nm AgNPs由于尺寸較小,更易穿透細菌的細胞膜,毒性主要歸于自身的尺寸效應[7-8].AgNPs可以與細胞膜上蛋白質結合,與含有氧、磷、氮或硫原子的電子供體形成絡合物,導致膜結合酶等蛋白質失活,也可干擾呼吸鏈并阻礙能量的產生[9-10].此外,AgNPs會造成細胞膜脂肪酸組成改變,改變膜的流動性[11],導致細胞內容物的泄漏[12].AgNPs還可以與細菌的遺傳物質結合,阻斷翻譯和轉錄[13-14],也有研究表明AgNPs進入細胞內造成活性氧物質(ROS)累積導致膜損傷和基因毒性[15].

環(huán)境中的氨氧化菌群主要負責驅動NH4+-N向NO2--N的轉化,由于其獨特的生理特性對環(huán)境因子變化較為敏感[16].歐洲亞硝化毛桿菌()為氨氧化模式菌株,一直以來都作為重點研究對象[17].AgNPs已經被證明對和其他氨氧化細菌具有毒性效應[18-19].Alito等[20]在一項短期實驗中發(fā)現AgNPs能毒害氨氧化微生物,使活性污泥中的氨氧化速率降低30%左右.Wu等[21]發(fā)現在ZnONPs暴露下膜完整性受到破壞,細胞生長和硝化速率受到抑制.Yuan等[22]將暴露在不同粒徑AgNPs中,發(fā)現細胞壁遭到破壞,且與生物合成、能量產生和與氨氧化過程有關的重要蛋白質的表達受到抑制.Choi等[23]發(fā)現AgNPs對生長和氨氧化速率的抑制可能與AgNPs誘導造成細胞體內ROS累積有關.目前,已有大量研究表明AgNPs對氨氧化過程具有負面影響,但AgNPs對氨氧化微生物毒性機制仍不清楚[24].本研究以為對象,不僅從細胞水平探究兩種粒徑AgNPs對生長、細胞膜損傷、ROS生成的影響,更是從分子水平解析AgNPs對功能基因表達的影響,以期揭示AgNPs對可能的毒性機制.

1 材料與方法

1.1 實驗材料

1.1.1 供試材料 10nm的AgNPs(Ag10)表面由聚乙烯吡咯烷酮(PVP)包被,購于南京先豐納米有限公司,平均粒徑為10±5nm,形態(tài)如圖1A所示.50nm的AgNPs(Ag50)表面無包被,購于南京埃瑞普納米材料有限公司,平均粒徑為50±5nm,形態(tài)如圖1B所示.醋酸銀(CH3COOAg,又名乙酸銀),純度399.5%,密度3.26g/cm3,分子量166.91,CAS號563-63-3,購自西亞試劑.

1.1.2 菌種與培養(yǎng)基(ATCC 19718)由中國科學院城市環(huán)境研究所于昌平教授課題組提供.培養(yǎng)基配方:1.32g/L (NH4)2SO4,0.20g/L MgSO4?7H2O,0.02g/L CaCl2?2H2O,0.087g/LK2HPO4, 2.52g/L N-2-羥乙基哌嗪-Ni3-丙磺酸(EPPS),1mg/L C10H12FeN2NaO8?3H2O,0.1mg/L Na2MoO4? 2H2O和ZnSO4?7H2O,0.172mg/L MnSO4,0.04mg/L CoCl2?6H2O, 2.5mg/L酚紅,0.25mg/L CuSO4?5H2O.KHCO3溶液調節(jié)pH值至6.9~7.5.

圖1 兩種粒徑納米銀TEM圖(A:10nm; B:50nm)

1.2 菌懸液制備

將培養(yǎng)至對數生長期后4000r/min離心收集菌體,KH2PO4緩沖液洗滌殘余培養(yǎng)基,菌體重懸于緩沖液中,4℃避光保存?zhèn)溆?

1.3 N. europaea生長及NH4+、NO2-含量測定

將兩種粒徑AgNPs和菌懸液加入高溫滅菌后的培養(yǎng)基中,使AgNPs最終濃度為5,10,20mg/L,同時控制初始OD600為0.07,于30℃、150r/min恒溫避光培養(yǎng),按時取樣,每個處理三個重復.納氏試劑比色法測定培養(yǎng)基中NH4+含量,鹽酸N-(1-萘基)-乙二胺比色法測定NO2-含量.同時用紫外分光光度計(UV-2550,菁華,中國)測定OD600,生長抑制率(%)=1－處理組OD600/空白組OD600′100%.

1.4 培養(yǎng)基中AgNPs的Ag+釋放量

將AgNPs(10nm終濃度為5,10mg/L;50nm終濃度為10,20mg/L)暴露在培養(yǎng)基中,培養(yǎng)不同時間后裝入超濾離心管(Millipore 3kDa,Amicon,美國)中,4500r/min離心過濾,電感耦合等離子發(fā)射光譜儀(ICP-MS 7500CX, Agilent,美國)測定濾液中Ag+含量,每組三個重復.

1.5 N. europaea細胞形態(tài)觀察

Ag10(10mg/L)和Ag50(20mg/L)處理12h后離心(10000r/min,4℃)收集菌體,0.1mol/L磷酸鹽緩沖液(PBS)漂洗兩次,2.5%戊二醛固定4h,PBS再漂洗3次,乙醇梯度脫水,丙酮替代20min后干燥過夜,前處理結束后樣品鍍膜進行掃描電鏡SEM(S-4800,日立,日本)觀察.透射電鏡樣品用戊二醛固定后再用1%鋨酸固定1~2h,然后脫水、浸透、包埋、烘干、切片和染色后在透射電鏡TEM(H-7650,日立,日本)下觀察,同時設置空白對照組.

1.6 N. europaea細胞內ROS檢測

乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)作為抗氧化劑可以清除細胞內多余的ROS,降低AgNPs產生的氧化損傷[25],本實驗添加NAC驗證細胞內部ROS的產生.將預培養(yǎng)1h后10000r/min離心收集菌體,PBS洗滌后加入DCFH-DA探針,按照活性氧檢測試劑盒(碧云天S0033,上海)的說明,于30℃孵育30min后利用流式細胞儀(FCM, FACSCalibur, BD,美國)于488nm為激發(fā)波長、525nm為發(fā)射波長的條件下檢測熒光強度.試驗共5個處理:空白對照(CK)、Ag10(10mg/L)、Ag50(20mg/L)、Ag10(10mg/L)+5mmol/L NAC、Ag50(20mg/L)+ 5mmol/L NAC.

1.7 功能基因amoA、hao、merA表達分析

1.7.1 RNA提取與反轉錄 取約108個菌體,加入溶菌酶消化10min,4℃和12000r/min下離心2min,使用UNIQ-10柱式Ⅰ總RNA抽提試劑盒(生工,上海)提取總RNA,按照TransScript All-in-One First-Stand cDNA Synthesis SupriMix for qPCR(TransGen, 北京)試劑盒進行反轉錄.RT體系與條件:10μmol/L Random Primer 1μL,RNA 6μL,RNase free dH2O 5μL,混合后70℃熱激3min,冰浴5min,然后繼續(xù)加入dNTP Mixture 2μL,RNase free dH2O 0.5μL,M-MLV 1μL,5′M-MLV Buffer 4μL,RNase Inhibitor 0.5μL,總體積共20μL.將各組分混勻后置于42℃水浴1h,得到第一鏈cDNA.

1.7.2 Real-Time PCR 本實驗根據Ultra SYBR Mixture(康為世紀, 北京)試劑盒說明,采用SYBR Green I法實時熒光定量PCR(相對定量)進行mRNA表達量測定.20μL反應體系為:10μL 2×SYBR Premix EX-Taq Mi,0.5μL 10μmol/L PCR Forward Primer和PCR Reverse Primer,0.4μL ROX (50×),2μL Template cDNA,6.6μL RNase Free dH2O.、、的所用引物如表1所示,每個樣品3次重復.

Real-time PCR擴增條件: 95℃預變性10min,40個循環(huán)( 95℃變性15s, 61℃退火35s,97℃延伸10s) 最后從65℃升至97℃獲得熔解曲線,采用2-△△Ct法進行相對表達量計算.

表1 Real-time PCR實驗所用引物

1.8 數據處理與分析

試驗數據采用SPSS 18.0進行統(tǒng)計分析,并對不同處理間的數據用單因素方差分析. (ANOVA)和Duncan多重比較進行顯著性差異(<0.05)檢驗,再用Origin 9.0繪圖.

2 結果與分析

2.1 AgNPs對N. europaea生長抑制

暴露在兩種粒徑的AgNPs中,細菌生長受到顯著抑制(圖2).生長與AgNPs劑量呈負相關,且在同等劑量下,10nm AgNPs對生長的抑制作用比50nm強.生長抑制率隨著AgNPs暴露時間的延長而增加,在4h達到最大值.4h后抑制率沒有顯著變化,甚至10nm(5mg/L)和50nm(5、10mg/L)AgNPs處理組抑制率出現降低趨勢,說明開始適應AgNPs存在.

2.2 AgNPs對N. europaea銨態(tài)氮轉化的影響

隨著培養(yǎng)時間延長,無銀對照組中銨態(tài)氮由于向硝酸鹽氮轉化,其含量逐漸降低.不同粒徑、不同劑量AgNPs暴露下銨態(tài)氮轉化能力受到抑制(圖3).AgNPs刺激8h后,對照組中NH4+含量減少了0.10mg/mL.Ag10(5,10,20mg/L)處理組NH4+濃度分別降低0.09,0.07,0.06mg/mL,而Ag50(5,10, 20mg/L)處理組NH4+濃度分別減低0.10,0.09, 0.07mg/mL.由于NH4+的轉化,培養(yǎng)液中NO2-含量逐漸增加,與對照組相比,AgNPs暴露組中NO2-含量明顯降低.其中,Ag10(10,20mg/L),Ag50(20mg/L)處理下,培養(yǎng)液中NO2-含量幾乎不變,而Ag10(5mg/L)和Ag50(5,10mg/L)處理組NO2-含量略有增加,但顯著低于對照組.

圖2 兩種粒徑納米銀暴露下N. europaea生長抑制率變化

2.3 AgNPs的Ag+ 釋放

暴露在環(huán)境中的AgNPs容易游離出Ag+,本研究選取10nm(5,10mg/L)和50nm(10,20mg/L)的AgNPs進行Ag+釋放量的測定(圖4).前6h內培養(yǎng)基中Ag+含量隨著培養(yǎng)時間的延長而增多,6h后趨于穩(wěn)定.暴露12h后,Ag10(5,10mg/L)Ag+釋放量占1.09%、0.75%;Ag50(10、20mg/L)的Ag+釋放量高達5.41%、4.56%.粒徑為50nm的AgNPs釋放的Ag+量顯著高于10nm的AgNPs,且50nm的AgNPs初始游離出的Ag+較高.

圖4 培養(yǎng)基中AgNPs的Ag+釋放量

2.4 AgNPs對N.europaea細胞形態(tài)及結構的影響

通過SEM和TEM觀察AgNPs處理12h后細胞形態(tài)與內部結構的變化,其中圖A、C、E放大40000倍,圖B、F放大12000倍,圖D放大15000倍.空白組(圖5A、B)中細胞飽滿且表面光滑,結構清晰完整,細胞內部物質分布均勻.掃描電鏡圖像(圖5C、E)顯示AgNPs處理造成細胞膜表面出現塌陷,且有大小不一的孔洞,細胞內容物流出.透射電鏡圖像(圖5D、F)顯示AgNPs處理造成細胞內核解體且中央出現大片空白區(qū)域,細胞器聚集于細胞邊緣,細胞質壁界限模糊.

A、B:空白對照; C、D:10mg/LAg10; E、F:20mg/LAg50

2.5 AgNPs對N.europaea的氧化應激作用

暴露在兩種粒徑AgNPs中細胞內ROS含量顯著上升(圖6).空白組中細胞內平均熒光強度為6.7,Ag10(10mg/L)和Ag50(20mg/L)處理組中細胞內平均熒光強度升高到35.2和28.1,添加NAC后熒光強度又顯著降低為12.7和12.5.說明AgNPs能刺激內部ROS的產生和累積,AgNPs氧化應激作用可能是對產生毒性效應的原因之一.

圖6 兩種粒徑納米銀暴露后N. europaea細胞內ROS含量變化

Ag10為10mg/L;Ag50為20mg/L;NAC為5mmol/L

圖中標有不同小寫字母者為Duncan檢測下差異顯著(<0.05),下同

2.6 AgNPs對N. europaea功能基因表達的影響

負責編碼氨氧加氧酶,主要調控NH4+到NH2OH的轉化,負責編碼羥氨氧化還原酶,主要調控NH2OH到NO2-轉化過程[26],merA調控重金屬應激[27].本研究以的16S RNA為參照基因,以無銀處理為對照組,AgNPs和Ag+暴露12h后功能基因、、表達如圖7所示.基因表達下調為負性調控(抑制表達),表達上調則為正性調控(促進表達).Ag+處理組中、、表達與對照相比分別下調23.26、1.96、12.82倍;Ag10(10mg/L)處理組、表達分別下調13.33、8.06倍,表達上調1.46倍;Ag50(20mg/L)處理組、表達分別下調1.37、2.79倍,則上調了1.90倍.說明Ag+能顯著抑制、、表達,AgNPs處理下、表達下調,輕微上調.

圖7 納米銀和銀離子脅迫下N. europaea基因表達量變化

3 討論

3.1 AgNPs對N. europaea生長抑制與其釋放的Ag+量相關

.對環(huán)境因子的變化較為敏感.本研究發(fā)現AgNPs抑制生長,培養(yǎng)基中NH4+向NO2-轉化過程受阻,氨氧化過程減緩,且10nm比50nm的AgNPs影響更大. Radniecki等[28]得出類似結論,并發(fā)現對小粒徑AgNPs的敏感性增加是由于其表面積與體積比較大,AgNPs的毒性與其顆粒大小有關.納米材料與細胞直接接觸被廣泛認為是其生物毒性來源之一[29],也有大量文獻證實AgNPs對氨氧化微生物的毒性與釋放的Ag+密切相關[30-31].本研究中50nm的AgNPs釋放的Ag+高于10nm的AgNPs,12h后50nm(20mg/L)的AgNPs釋放的Ag+高達1mg/L,而10nm的AgNPs Ag+釋放量只有幾十μg/L,可能是因為10nm的AgNPs表面由PVP包被,在液體環(huán)境中較為穩(wěn)定,而50nm的AgNPs表面無包被,更易釋放Ag+,這與Arnaout和Gunsch[32]的研究結果相近.

3.2 AgNPs造成N. europaea的細胞結構損傷及細胞內活性氧的累積

掃描電鏡(SEM)和透射電鏡(TEM)圖像顯示,AgNPs造成細胞表面塌陷且有孔洞,細菌內部核物質消融且質膜界限不明顯,AgNPs與細菌表面直接作用可能是AgNPs導致死亡的原因之一.通過染色進行電鏡觀察已成為一種普遍方法,本研究得出在銀脅迫下細胞膜遭到破壞與他人在文獻中描述的一樣[9].AgNPs不僅可以直接破壞細胞結構損傷細胞,還可以誘導細胞體內ROS的累積,對造成氧化損傷.ROS主要包括羥基自由基(·OH)、超氧陰離子(·O2-)、單線態(tài)氧(1O2)、過氧化氫(H2O2)等具有殺菌作用的自由基[33].電子自旋共振光譜(EPR)被認為是證明ROS存在的有效工具,我們前期工作已檢測到AgNPs產生的自由基為·OH[34].當微生物體內ROS含量超過自身抗氧化防御能力會導致生物體谷胱甘肽(GSH)的耗竭以及多種抗氧化酶活性的改變,是AgNPs毒性作用的可能機制之一[35].AgNPs暴露下內部ROS含量測定結果顯示兩種粒徑AgNPs暴露后細胞內ROS含量均明顯增高,NAC處理后細胞內ROS顯著降低,進一步印證了AgNPs的氧化應激作用.氧化應激是AgNPs毒性機制中報道最多的,ROS的產生會導致細胞壁損傷、細胞膜破壞、蛋白質損傷和電子傳遞中斷[36].

3.3 AgNPs對N. europaea功能基因amoA、hao和merA表達的影響

qRT-PCR測定結果顯示兩種粒徑AgNPs均抑制和表達,輕微上調.其中負責調控氨氧化過程第一步的基因受AgNPs影響最大,這可能是AgNPs影響銨態(tài)氮轉化的主要原因,與Michels等[26]研究結果類似.也有文獻報道AgNPs雖然抑制硝化作用,的表達卻沒有改變,AgNPs不影響的轉錄[32].用于NADPH還原,在重金屬存在時傾向于上調[37],本研究發(fā)現AgNPs暴露下表達受抑制,說明AgNPs對的影響與一般重金屬應激機制不同.10nm的AgNPs對功能基因表達的抑制效果比50nm的AgNPs強,可能是小粒徑AgNPs更易進入細胞內部,與細胞內物質(包括核酸)結合,破壞細胞內部結構[38].Choi等[39]同樣發(fā)現小粒徑AgNPs對氨氧化細菌基因的損傷較嚴重.根據nAg50(20mg/L)12h釋放的Ag+量,選擇1mg/L Ag+暴露下對功能基因表達的影響,發(fā)現Ag+對、、表達抑制較明顯.10nm的由PVP包被的AgNPs釋放的Ag+較少但對基因的影響依舊很大,其中PVP作為一種涂層材料是無毒的[40],說明AgNPs對基因的表達與粒徑大小有關.50nm的AgNPs的毒性可能部分來自釋放的Ag+,部分由于本身的特異抗菌性,10nm AgNPs的毒性主要是自身作用的結果[41].AgNPs的毒性大小與其濃度及粒徑有關.

雖然本文利用兩種不同粒徑的AgNPs探討了對的毒性作用,并發(fā)現AgNPs通過直接破壞細胞結構、產生氧化應激和影響和基因表達進而影響銨態(tài)氮的轉化,但是關于AgNPs對毒性作用及其影響機理還有待進一步的研究.

4 結論

4.1 AgNPs抑制了生長,導致銨態(tài)氮轉化過程延緩,且粒徑越小抑制作用越強.

4.2 AgNPs對的毒性作用主要有兩條途徑:AgNPs能直接作用于表面,破壞細胞膜,導致胞內物質流出,核物質消融且質膜界限不明顯;刺激細菌體內ROS生成,ROS累積造成氧化損傷.

4.3 實時熒光定量PCR結果顯示,AgNPs抑制和表達,對的表達影響較小.

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Toxicity of two sizes of silver nanoparticles to.

WU Ling-li, ZHANG Xiao-xue, SHU Kun-hui, SI You-bin*

(Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University, Hefei 230036, China)., 2019,39(10)：4401~4408

The laboratory incubation experiments were conducted to study the toxicity of silver nanoparticles with different particle sizes on, and the effects of two sizes of nanosilver (10nm and 50nm) on the bacterial growth, nitrogen transformation, cellular structure, reactive oxygen generation and gene expression were investigated. The results showed that nanosilver inhibited the growth of. With the extension of exposure time, the inhibition rate of bacterial growth activity increased and reached to maximum at 4h. In the medium, the transformation rate of NH4+to NO2-was slowed down, and the nitrogen transformation ability bywas reduced. Scanning electron microscopy (SEM) images showed that nanosilver heavily damaged the cell membrane by causing holes on the surface of bacteria. Transmission electron microscope (TEM) images showed that the nuclear material inside the bacteria was disappeared and the boundary of the cytoplasmic membrane was blurred. Flow cytometry (FCM) was employed to detect that nanosilver could generate intracellular reactive oxygen (ROS) in the cells. qRT-PCR technology was used to determine the expression ofandofafter the exposure to nanosilver, and it was found that nanosilver inhibited the expression of the functional genes. In conclusion, nanosilver could interact with cell membrane, generated oxidative stress damage and inhibited the expression of functional genesandof, which further affected the transformation process of ammonium nitrogen. In addition, the toxicity of nanosilver with small particle size was stronger than that of large particle size.

nanosilver；；oxidative stress；gene expression of,, and；ammoxidation

X171.5

A

1000-6923(2019)10-4401-08

伍玲麗(1994-),女,安徽蕪湖人,安徽農業(yè)大學碩士研究生,主要研究方向為微生物毒理.

2019-03-11

國家自然科學基金重點項目(41430752);安徽農業(yè)大學研究生創(chuàng)新基金(2019ysj-67)

* 責任作者, 教授, youbinsi@ahau.edu.cn