6:2氟調(diào)羧酸在蚯蚓體內(nèi)的毒理效應(yīng)及代謝轉(zhuǎn)化

宗玉璐,楊麗萍,趙淑艷*

6:2氟調(diào)羧酸在蚯蚓體內(nèi)的毒理效應(yīng)及代謝轉(zhuǎn)化

宗玉璐1,楊麗萍2,趙淑艷1*

(1.大連理工大學(xué)海洋科學(xué)與技術(shù)學(xué)院,工業(yè)生態(tài)與環(huán)境工程教育部重點(diǎn)實(shí)驗(yàn)室,遼寧 盤(pán)錦 124221；2.南開(kāi)大學(xué)環(huán)境科學(xué)與工程學(xué)院,天津 300071)

以赤子愛(ài)勝蚓()為受試生物,通過(guò)活體與離體實(shí)驗(yàn),研究6:2氟調(diào)羧酸(6:2FTCA) 在蚯蚓體內(nèi)的毒理效應(yīng)和代謝轉(zhuǎn)化機(jī)制.結(jié)果表明,6:2FTCA對(duì)蚯蚓體內(nèi)丙二醛(MDA)含量和過(guò)氧化物酶(POD)活性無(wú)顯著影響,但能夠使過(guò)氧化氫酶(CAT)活性提高,使超氧化物歧化酶(SOD)和谷胱甘肽轉(zhuǎn)移酶(GST)活性顯著升高,說(shuō)明6:2FTCA對(duì)蚯蚓產(chǎn)生了氧化脅迫效應(yīng).6:2FTCA在蚯蚓細(xì)胞色素P450(CYP450)和GST酶提取液中的降解動(dòng)力學(xué)均符合一級(jí)動(dòng)力學(xué)模型,在CYP450(0.014/h)酶液中的降解速率明顯高于GST(0.006/h),其終端全氟羧酸(PFCAs)代謝產(chǎn)物為全氟己酸(PFHxA)、全氟戊酸(PFPeA)和全氟丁酸(PFBA),說(shuō)明CYP450和GST參與了6:2FTCA在蚯蚓體內(nèi)的代謝轉(zhuǎn)化,且CYP450貢獻(xiàn)大于GST.蚯蚓腸道好氧微生物對(duì)6:2FTCA具有顯著的降解效果,終端PFCAs降解產(chǎn)物為PFHxA和PFPeA,而腸道厭氧微生物對(duì)6:2FTCA無(wú)降解作用.

6:2氟調(diào)羧酸；蚯蚓；毒理效應(yīng)；酶代謝；腸道微生物

多氟和全氟化合物(PFAS)被廣泛應(yīng)用于工業(yè)和生活產(chǎn)品生產(chǎn)中,具有環(huán)境持久性、生物累積性和生物毒性等特點(diǎn),是一類(lèi)新型持久性有機(jī)污染物(POPs)[1-2].全氟辛酸(PFOA)是全氟羧酸(PFCAs)的一種典型代表產(chǎn)品,被美國(guó)環(huán)保署(EPA)列為疑似致癌物,于2015年停止相關(guān)產(chǎn)品生產(chǎn)[2].目前,國(guó)際上普遍采用短鏈同系物,如全氟己酸(PFHxA)和全氟丁酸(PFBA),或者一些含有非氟化結(jié)構(gòu)部分的新型PFASs作為PFOA替代品[3].

6:2 氟調(diào)羧酸(6:2FTCA,C6F13CH2COOH)作為一種新型的PFOA替代品,被廣泛應(yīng)用于表面活性劑和含氟乳化劑等氟化工原料生產(chǎn)中,在生產(chǎn)和使用過(guò)程中釋放到各種環(huán)境介質(zhì)中[2,4].另外,一些PFCAs前體物質(zhì)也能夠在環(huán)境中通過(guò)生物和非生物途徑轉(zhuǎn)化為6:2FTCA,導(dǎo)致環(huán)境介質(zhì)和生物體中6:2FTCA的污染[5-8].據(jù)報(bào)道,德國(guó)廢水樣品中6:2FTCA最高濃度為8.62μg/L[9],北美地表水中濃度達(dá)到10μg/L[9],山東德州土壤和植物中檢出6:2FTCA最高濃度分別為0.12ng/g和0.57ng/g[10],黃河中游水相和顆粒相中檢測(cè)到的6:2FTCA最高濃度分別為0.254ng/L和2.88ng/g[11].6:2FTCA對(duì)小鼠肝臟的毒性要較PFOA低[3],對(duì)斑馬魚(yú)胚胎具有發(fā)育毒性且能夠抑制胚胎發(fā)育過(guò)程中紅細(xì)胞的形成[9],對(duì)大型水蚤、搖蚊、浮萍和端足蟲(chóng)等表現(xiàn)出比PFOA更強(qiáng)的毒性[12-13].但是,6:2FTCA對(duì)陸生無(wú)脊椎動(dòng)物的毒性尚不明確.雖然全氟化的PFCAs表現(xiàn)出較強(qiáng)的穩(wěn)定性,但是一些含有非氟化結(jié)構(gòu)的PFASs能夠發(fā)生降解轉(zhuǎn)化.目前,尚無(wú)以6:2FTCA作為母體化合物進(jìn)行降解轉(zhuǎn)化研究的報(bào)道,而6:2FTCA作為一些多氟物質(zhì)中間代謝產(chǎn)物的相關(guān)研究報(bào)導(dǎo)較多.6:2 氟調(diào)醇(6:2FTOH)[4-8,14-15]、6:2氟調(diào)聚物磺酰胺烷基甜菜堿(6:2FTAB)[16]和6:2氟調(diào)磺酸(6:2FTSA)[17-18]等PFCA前體物質(zhì),都能夠在環(huán)境介質(zhì)或者動(dòng)植物體內(nèi)被降解生成中間產(chǎn)物6:2FTCA,隨后6:2FTCA再進(jìn)一步轉(zhuǎn)化生成終端產(chǎn)物PFCAs.因此,推測(cè)6:2FTCA在環(huán)境中會(huì)被轉(zhuǎn)化為持久性更強(qiáng)的PFCAs,是環(huán)境中PFCAs的潛在來(lái)源,其生態(tài)環(huán)境風(fēng)險(xiǎn)不可忽視.

蚯蚓是典型的陸生無(wú)脊椎動(dòng)物,常被用作評(píng)價(jià)土壤外源化學(xué)污染物生態(tài)風(fēng)險(xiǎn)的指示生物[19].污染物在蚯蚓體內(nèi)發(fā)生攝食、吸收、代謝轉(zhuǎn)化和排泄等復(fù)雜的生物過(guò)程,而污染物在蚯蚓體內(nèi)的代謝轉(zhuǎn)化可能同時(shí)包括蚯蚓組織中的酶代謝和腸道中的微生物降解.細(xì)胞色素P450酶(CYP450)屬于I相代謝酶,是廣泛存在于生物體內(nèi)的血紅素酶系,催化的反應(yīng)機(jī)制包括環(huán)氧化、脫烷基、羥基化和脫硫等[20].谷胱甘肽-S-轉(zhuǎn)移酶(GST)屬于II相代謝酶,催化結(jié)合反應(yīng)使結(jié)合物更易于排出體外達(dá)到解毒的目的[21-22].研究顯示,CYP450和GST是一些PFCAs前體物質(zhì)在動(dòng)植物體內(nèi)發(fā)生代謝轉(zhuǎn)化的關(guān)鍵酶[15,23-24]. CYP450和GST能夠參與6:2FTSA在蚯蚓體內(nèi)的代謝轉(zhuǎn)化[22],CYP450是6:2FTSA在南瓜體內(nèi)降解轉(zhuǎn)化的關(guān)鍵酶[24],GST參與了6:2FTOH在大豆組織中的代謝[15].蚯蚓腸道特有的微生境中存在大量的厭氧和好氧細(xì)菌,這些細(xì)菌對(duì)一些有機(jī)污染物在蚯蚓體內(nèi)的降解具有一定的貢獻(xiàn),如六氯環(huán)己烷(HCH)[25]和硫丹[26]能被蚯蚓腸道內(nèi)分離出來(lái)的細(xì)菌所降解.然而,蚯蚓腸道微生物對(duì)全氟辛烷磺酰胺(FOSA)并未表現(xiàn)出降解能力[27].6:2FTCA能否在蚯蚓代謝酶及腸道微生物作用下發(fā)生降解,還有待進(jìn)一步研究.

本研究以赤子愛(ài)勝蚓()作為模式動(dòng)物,通過(guò)活體()和離體()實(shí)驗(yàn)相結(jié)合的方式,研究6:2FTCA在蚯蚓體內(nèi)的生態(tài)毒理效應(yīng)和代謝轉(zhuǎn)化機(jī)制.通過(guò)分析氧化應(yīng)激標(biāo)志物的變化,如丙二醛(MDA)含量變化,過(guò)氧化物酶(POD)、過(guò)氧化氫酶(CAT)、超氧化物歧化酶(SOD)和GST活性變化,闡明6:2FTCA對(duì)蚯蚓的毒理效應(yīng);通過(guò)分析代謝酶(CYP450和GST)對(duì)6:2FTCA的代謝轉(zhuǎn)化規(guī)律和蚯蚓腸道微生物對(duì)6:2FTCA的降解特性,揭示6:2FTCA在陸生動(dòng)物體內(nèi)代謝發(fā)生的內(nèi)在機(jī)制和主要控制因素.為準(zhǔn)確評(píng)估環(huán)境中6:2FTCA的生態(tài)風(fēng)險(xiǎn)和PFCAs的來(lái)源提供理論依據(jù).

1 材料與方法

1.1 試劑與材料

6:2FTCA(98%)、全氟庚酸(PFHpA,98%)、全氟戊酸(PFPeA,97%)購(gòu)自北京百靈威科技有限公司,全氟己酸(PFHxA,98%)購(gòu)自Matrix Science公司,三氟乙酸(TFA,99%)、五氟丙酸(PFPrA,97%)和全氟丁酸(PFBA,98%)購(gòu)自上海麥克林生化科技有限公司.煙酰胺腺嘌呤二核苷酸(NADH)和-煙酰胺腺嘌呤二核苷酸磷酸(NADP+)購(gòu)自Biosharp.葡萄糖-6-磷酸脫氫酶(G6PDH)和葡萄糖-6-磷酸鈉(GLC-6-PO4)購(gòu)自Sigma-Aldrich(中國(guó)).高效液相色譜(HPLC)級(jí)甲醇(99.9%)和甲基叔丁基醚(MTBE)等購(gòu)自大連博諾生化試劑有限公司.

1.2 活體暴露實(shí)驗(yàn)

供試土壤采自遼寧盤(pán)錦農(nóng)田(種植蔬菜,周邊無(wú)污染源)0～10cm的表層土壤,自然風(fēng)干14d后過(guò)2mm篩,將配好的6:2FTCA甲醇儲(chǔ)備液加入土壤中,混勻后放入通風(fēng)櫥中于室溫下平衡4d[23]. 測(cè)得實(shí)驗(yàn)土壤中6:2FTCA的初始暴露濃度為62.0ng/g干重(dw).赤子愛(ài)勝蚓()購(gòu)自遼寧華電環(huán)?？萍加邢薰掘球攫B(yǎng)殖場(chǎng)(沈陽(yáng)).室溫(22～27℃)下馴化培養(yǎng)14d,挑取帶有生殖環(huán)帶的成熟蚯蚓(10條),清腸24h后放入裝有100g染毒土壤(含水率為30%)的燒杯中,同時(shí)設(shè)置空白對(duì)照組(暴露于未染毒土壤中),室溫下分別培養(yǎng)1,2,4,6,8,12,16和20d,每個(gè)處理設(shè)3個(gè)重復(fù)(=3)[19].取樣后,將蚯蚓清腸、清洗后,立即放入冷水浴中處理.

1.3 蚯蚓粗酶提取液的制備

將蚯蚓樣品(約0.3g)放入手動(dòng)勻漿器中,再以1:9的比例加入磷酸鹽緩沖液(0.05mol/L,pH 7.5),在冰水浴中研磨成勻漿[22].將蚯蚓勻漿在4℃、12000r/min下離心10min,收集上清液,于-80℃保存.用硫代巴比妥酸(TBA)法測(cè)定MDA含量,以牛血清蛋白為標(biāo)準(zhǔn)用考馬斯亮藍(lán)染色法測(cè)定蛋白質(zhì)含量,氮藍(lán)四唑(NBT)光化還原法測(cè)定SOD活性,愈創(chuàng)木酚法測(cè)定POD活性,紫外吸收法測(cè)定CAT活性,GST活性采用1-氯-2,4-二硝基苯(CDNB)比色法測(cè)定[22,28-29].MDA含量用nmol/g蛋白質(zhì)表示,GST用nmol/min/mg蛋白質(zhì)表示,其他酶活性(SOD、POD和CAT)用U/mg蛋白質(zhì)表示.

1.4 體外暴露實(shí)驗(yàn)

將已清腸24h的蚯蚓放入甘油溶液中(15min, 0℃,20%)實(shí)施安樂(lè)死,然后用0.15mmol/L KCl溶液沖洗蚯蚓表面黃色液體后放入玻璃勻漿器中,以蚯蚓:緩沖液(0.1mol/L,pH=7.4,1mol/L EDTA, 0.15mmol/L KCl)=4:1的比例研磨成蚯蚓組織勻漿液.根據(jù)Kunze等[30]的方法稍作改動(dòng)進(jìn)行GST酶液提取(操作溫度為0～4℃).取一部分勻漿于10000離心20min,上清液再于100000離心50min后過(guò)0.22μm濾膜,然后過(guò)GST瓊脂糖純化樹(shù)脂進(jìn)行凈化后得到GST酶提取液.GST酶液保存在0.05mol/L磷酸鹽和0.5μmol GSH(內(nèi)含有10mmol/L KNO3+ 0.05mmol/L EDTA+ 2mmol/L NADH+3mM Na2MoO4)中,放入-80℃冰箱保存.根據(jù)Yang等[31]的實(shí)驗(yàn)方法提取CYP450粗酶液(0～4℃).另取一部分勻漿,于15000離心20min,上清液再于150000離心90min得到CYP450酶液粗提物.使用蚯蚓CYP450和GST ELISA試劑盒在酶標(biāo)儀(美國(guó)Molecular Device)上測(cè)定蚯蚓勻漿和粗酶提取液中CYP450總量和GST含量.

在上述蚯蚓GST酶提取液(1mL)、CYP450酶提取液(0.5g)中分別加入50μL 6:2FTCA及經(jīng)預(yù)培養(yǎng)的500μL NADPH再生系統(tǒng)(1.6mmol/L NADP+, 3.3mmol/LGlc-6-PO4,0.4U/mL,G6PDH,3.3mmol/L MgCl2+3.95mL磷酸鹽緩沖液)在水浴搖床中(室溫25℃、120rpm)進(jìn)行體外培養(yǎng)實(shí)驗(yàn),培養(yǎng)容器聚丙烯(PP)管用錫箔紙包裹.分別于0,2,6,8,12,24和32h進(jìn)行取樣.實(shí)驗(yàn)同時(shí)設(shè)置空白對(duì)照組Ⅰ(酶液),對(duì)照組Ⅱ(6:2FTCA+沸水于100℃中煮5min滅活后的酶液)和對(duì)照組Ⅲ(6:2FTCA),分別培養(yǎng)2h和32h后進(jìn)行取樣.所有實(shí)驗(yàn)均設(shè)置3組平行(=3).取樣時(shí),PP管中加入1/2體積甲醇并漩渦混勻1min以停止孵育反應(yīng),置于-20℃保存,留待PFASs提取與分析.

1.5 蚯蚓腸道微生物對(duì)6:2FTCA的降解實(shí)驗(yàn)

采用以往實(shí)驗(yàn)方法并稍加改進(jìn)[32].將在6:2FTCA染毒土壤中馴化20d后的蚯蚓清腸并清洗干凈,用75%的酒精麻醉蚯蚓后,取下蚯蚓胃以下的腸道.將0.1g(鮮重, ww)腸道放入滅菌手動(dòng)勻漿器中,加入1mL經(jīng)滅菌的預(yù)冷PBS(0.1mol/L,pH=7.0)均勻研磨至無(wú)明顯顆粒.

好氧降解實(shí)驗(yàn):在無(wú)菌操作情況下,取1mL的蚯蚓腸道勻漿液加入到100mL LB液體培養(yǎng)基進(jìn)行混菌富集培養(yǎng)(水浴搖床,100r/min),取已培養(yǎng)至對(duì)數(shù)期的一代菌群于LB液體培養(yǎng)基中再馴化,二代菌群培養(yǎng)至對(duì)數(shù)期.取50mL培養(yǎng)到二代對(duì)數(shù)期的懸浮液加到離心管中進(jìn)行離心(300r/min,10min)去掉其中的液體LB,取0.2mL腸道微生物懸浮液加入到裝有6:2FTCA染毒處理的MSM培養(yǎng)基的PP瓶中,混合均勻后在水浴搖床中(25℃,100r/min)于好氧條件下進(jìn)行培養(yǎng).

開(kāi)幕式由中國(guó)煤炭工業(yè)協(xié)會(huì)副會(huì)長(zhǎng)兼秘書(shū)長(zhǎng)姜志敏主持，中國(guó)煤炭工業(yè)協(xié)會(huì)副會(huì)長(zhǎng)梁嘉琨致辭。全國(guó)政協(xié)常委、中國(guó)煤炭工業(yè)協(xié)會(huì)會(huì)長(zhǎng)王顯政，國(guó)家安監(jiān)總局副局長(zhǎng)、國(guó)家煤礦安全監(jiān)察局局長(zhǎng)付建華，國(guó)家能源局煤炭司副司長(zhǎng)李豪峰，中國(guó)工業(yè)經(jīng)濟(jì)聯(lián)合會(huì)常務(wù)副會(huì)長(zhǎng)、中國(guó)煤炭工業(yè)協(xié)會(huì)副會(huì)長(zhǎng)路耀華，中國(guó)煤炭加工利用協(xié)會(huì)理事長(zhǎng)呂英，印度和烏克蘭的選煤協(xié)會(huì)主席以及山東、陜西一些地方政府領(lǐng)導(dǎo)和神華、中煤能源、鞍山重機(jī)等企業(yè)的領(lǐng)導(dǎo)出席了開(kāi)幕式。

厭氧降解實(shí)驗(yàn):在厭氧手套箱內(nèi),另取1mL的蚯蚓腸道勻漿液加入到脫氧的LB液體培養(yǎng)基中,移出厭氧手套箱后置于水浴搖床(25℃,100r/min)中培養(yǎng)至二代對(duì)數(shù)期,離心后取0.2mL懸浮液加入到脫氧的MSM培養(yǎng)基(6:2FTCA)中于厭氧手套箱中進(jìn)行降解實(shí)驗(yàn).

實(shí)驗(yàn)全程避光,分別設(shè)置6:2FTCA為唯一碳源組(6:2FTCA+腸道微生物,F+M)、排除非生物干擾組(6:2FTCA+煮沸腸道,C)、6:2FTCA和外加碳源組(6:2FTCA+0.5%葡萄糖+腸道微生物,F+M+G),同時(shí)設(shè)置空白對(duì)照組.實(shí)驗(yàn)進(jìn)行48h后,將與降解培養(yǎng)實(shí)驗(yàn)相同體積的甲醇加入到PP瓶中,旋渦震蕩至混合均勻,-20℃保存,留待PFASs提取與分析.

1.6 樣品前處理和儀器分析方法

土壤、蚯蚓和腸道微生物樣品提取與純化參照以往方法[27].使用Waters 超高效液相色譜(ACQUITY-UPLC)-質(zhì)譜(MS, XEVO-TQS)聯(lián)用儀,在負(fù)電噴霧電離模式下(ESI)對(duì)樣品中PFASs含量進(jìn)行定量分析.色譜柱為Waters UPLC C18 (1.7μm, 2.1mm×50mm),柱溫為38℃.流動(dòng)相為甲酸銨水溶液(A,2mmol/L)和甲醇(B),流速為0.45mL/min,進(jìn)樣體積為10μL,梯度洗脫條件:0～ 0.5min,25%B;0.5～5.0min,25%～85%B;5.0～5.1min, 85%～100%B;5.1～8.0min,100%B;8.0～10.0min,100%～ 25%B.質(zhì)譜條件:毛細(xì)管電壓-2.2kV,霧化氣流速7.00bar,脫溶劑氣流800L/h,錐孔氣流150L/h,離子源溫度150℃,去溶劑溫度400℃.各PFASs的定量參數(shù)列于表1.

表1 PFASs的定量離子、錐孔電壓和碰撞能

1.7 質(zhì)量保證與質(zhì)量控制

回收率采用空白基質(zhì)加標(biāo),降解實(shí)驗(yàn)采用相同的步驟制備方法空白樣,采用添加基質(zhì)的外標(biāo)法定量,方法檢出限(MDL)信噪比為3:1.各基質(zhì)中PFASs的回收率范圍為81.4%～117.5%,所測(cè)目標(biāo)物不進(jìn)行回收率校正.PFASs在酶液和腸道微生物培養(yǎng)基中的MDLs分別為0.0017～0.0187pmol/g ww和0.0015～ 0.0029pmol/mL.

1.8 數(shù)據(jù)處理與分析

6:2FTCA在蚯蚓酶液中的降解速率常數(shù)(,1/h)由如下公式計(jì)算:

式中:C和分別為(h)時(shí)刻和孵育開(kāi)始時(shí)蚯蚓酶液中6:2FTCA的濃度(nmol/g ww).

6:2FTCA暴露組與空白對(duì)照組毒理指標(biāo)的差異顯著性用單因素方差分析(ANOVA)和配對(duì)樣本T檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)分析,腸道微生物對(duì)6:2FTCA降解影響的組間差異用Tukey’s檢驗(yàn)其顯著性(IBM SPSS Software, 22.0).<0.05認(rèn)為有顯著差異.

2 結(jié)果與討論

2.1 6:2FTCA對(duì)蚯蚓的毒理效應(yīng)

圖1 6:2FTCA對(duì)蚯蚓體內(nèi)MDA含量(A),POD(B)、SOD(C)、CAT(D)和GST(E)酶活性的影響

*<0.05

污染物進(jìn)入機(jī)體后會(huì)激活機(jī)體的抗氧化防御系統(tǒng)以防止氧化損傷,包括酶(SOD、CAT、和POD等)和非酶抗氧化系統(tǒng)(脂質(zhì)過(guò)氧化指標(biāo)MDA等).在受到外源物質(zhì)脅迫時(shí),生物體內(nèi)抗氧化系統(tǒng)為清除氧自由基或活性氧簇(ROS)會(huì)被誘導(dǎo)[28,33].如圖1A所示,在暴露期間,6:2FTCA染毒組蚯蚓體內(nèi)MDA含量與對(duì)照組相比無(wú)顯著變化(>0.05).本課題組前期研究顯示,相同及更短全氟碳鏈長(zhǎng)度的PFCAs(TFA、PFPrA、PFBA、PFPeA、PFHxA、PFHpA)也未對(duì)蚯蚓體內(nèi)MDA含量產(chǎn)生顯著影響[34],這說(shuō)明在6:2FTCA染毒土壤(62.0ng/g dw)中暴露20d后,6:2FTCA及其終端代謝產(chǎn)物對(duì)蚯蚓細(xì)胞均無(wú)明顯的脂質(zhì)過(guò)氧化作用.在暴露實(shí)驗(yàn)中(4～20d),處理組與對(duì)照組相比,SOD(圖1B)活性顯著提高41.4%～ 74.3%(<0.05),CAT(圖1C)活性增加37.2%～44.4% (>0.05),而POD(圖1D)活性基本無(wú)變化(>0.05).研究報(bào)道,6:2FTCA同系物8:2FTCA能夠引起水生生物部分抗氧化應(yīng)激標(biāo)志物呈現(xiàn)出不同程度的氧化應(yīng)激響應(yīng),如MDA含量增加、SOD活性增強(qiáng),而CAT活性變化不顯著[33].SOD活性變化用來(lái)表示遭受外源化學(xué)物污染后引起生物機(jī)體氧化脅迫的大小,是生物機(jī)體抗氧化系統(tǒng)的第一道防線(xiàn),通過(guò)歧化反應(yīng)將O2-催化為H2O2和O2,再被CAT和POD等H2O2清除酶捕獲[28,35].SOD被顯著誘導(dǎo),說(shuō)明蚯蚓已經(jīng)受到氧化脅迫.6:2FTCA處理組中CAT活性與空白對(duì)照組相比,雖然數(shù)理統(tǒng)計(jì)分析顯示不顯著(>0.05),但其活性增加了37.2%～44.4%(4～20d),整體呈現(xiàn)一定的誘導(dǎo)效應(yīng),而POD活性基本無(wú)變化,說(shuō)明在清除蚯蚓因暴露于6:2FTCA染毒土壤中而引起的H2O2的過(guò)程中,CAT比POD發(fā)揮了更為重要的作用.

2.2 CYP450和GST酶提取液對(duì)6:2FTCA的代謝轉(zhuǎn)化

圖2 CYP450(A, nmol/g ww)和GST(B, nmol/g ww)酶液中PFASs含量隨時(shí)間的變化

粗酶提取液中CYP450總量和GST含量約是蚯蚓整體勻漿中含量的160倍.為了證實(shí)代謝酶在6:2FTCA生物轉(zhuǎn)化中的作用,將蚯蚓的粗酶提取物(CYP450和GST)體外暴露于含有6:2FTCA的輔酶再生系統(tǒng)中.在空白對(duì)照組Ⅰ中未檢測(cè)到PFASs,對(duì)照組Ⅱ中6:2FTCA沒(méi)有明顯的消耗,對(duì)照組Ⅲ中除6:2FTCA外未檢測(cè)到其他PFASs,說(shuō)明6:2FTCA在體外試驗(yàn)過(guò)程中無(wú)背景干擾,且微生物和光降解可忽略不計(jì).在培養(yǎng)實(shí)驗(yàn)結(jié)束后,CYP450和GST粗酶液中PFASs總物質(zhì)的量回收率為初始加入6:2FTCA物質(zhì)的量的73.2%和89.7%,可能是由于部分物質(zhì)揮發(fā)、容器吸附或其他未進(jìn)行檢測(cè)的中間代謝物(6:2FTUCA、5:3FTUCA、5:2sFTOH、5:3FTCA等)所致.

如圖2所示,體外培養(yǎng)2h后, CYP450和GST酶液中母體化合物6:2FTCA的濃度開(kāi)始下降,并且隨著培養(yǎng)時(shí)間延長(zhǎng)而逐漸降低.體外培養(yǎng)32h后,6: 2FTCA在CYP450和GST酶液中轉(zhuǎn)化率分別為47.4%和20.7%.體外降解動(dòng)力學(xué)符合一級(jí)衰減動(dòng)力學(xué)模型,在CYP450和GST酶液中降解速率常數(shù)分別為0.014/h (2=0.890,<0.01)和0.006/h (2=0.941,<0.01).以上結(jié)果說(shuō)明蚯蚓體內(nèi)CYP450和GST在6:2FTCA生物轉(zhuǎn)化中起關(guān)鍵作用,且6:2FTCA在CYP450酶溶液中的生物轉(zhuǎn)化率和轉(zhuǎn)化速率都比在GST酶溶液中要高,表明CYP450對(duì)6:2FTCA在蚯蚓體內(nèi)生物轉(zhuǎn)化的貢獻(xiàn)要大于GST.此現(xiàn)象與其前體物質(zhì)6:2FTSA在蚯蚓體內(nèi)的酶代謝轉(zhuǎn)化規(guī)律相似[27].

圖3 不同實(shí)驗(yàn)組終端PFCA代謝產(chǎn)物物質(zhì)的量比例

體外培養(yǎng)過(guò)程中,在粗酶液(CYP450和GST)中檢測(cè)到了3個(gè)PFCA代謝終產(chǎn)物,包括PFBA、PFPeA和PFHxA(圖2).隨著培養(yǎng)時(shí)間延長(zhǎng),PFBA、PFPeA和PFHxA的含量逐漸升高,說(shuō)明6:2FTCA能夠在蚯蚓體內(nèi)I相和II相代謝酶的作用下代謝轉(zhuǎn)化生成3種不同碳鏈長(zhǎng)度的PFCAs終產(chǎn)物.6:2FTCA在CYP450和GST粗酶液中代謝產(chǎn)物的物質(zhì)的量百分比分別如下: PFHxA(40.2%)>PFBA(31.3%)>PFPeA (28.5%),PFBA(49.3%)>PFPeA(27.0%)>PFHxA(23.7mol%)(圖3).PFBA和PFHxA分別是CYP450和GST粗酶液中主要的PFCA終端代謝產(chǎn)物.根據(jù)6:2FTCA終端降解產(chǎn)物的組成,推測(cè)6:2FTCA在蚯蚓粗酶液中的代謝轉(zhuǎn)化路徑如下: 6:2FTCA脫-HF后生成6:2FTUCA,再通過(guò)-氧化途徑發(fā)生CH2-CH2, CH2-CF2和CF2-CF2鍵斷裂,最終生成穩(wěn)定的PFCA終端產(chǎn)物.然而,在蚯蚓體內(nèi)未檢測(cè)到PFHpA,說(shuō)明6:2FTCA在蚯蚓體內(nèi)并未發(fā)生-氧化.此代謝轉(zhuǎn)化路徑與6:2FTOH在植物體內(nèi)的降解有些類(lèi)似,即6:2FTOH通過(guò)連續(xù)脫氫和氧化作用生成6:2FTCA,然后再通過(guò)氧化和脫除一或者兩個(gè)碳生成PFPeA和PFHxA[15].

2.3 蚯蚓腸道微生物對(duì)6:2FTCA的降解

暴露于有機(jī)污染物的蚯蚓腸道中含有大量的好氧和厭氧降解微生物.為了進(jìn)一步研究蚯蚓腸道微生物對(duì)6:2FTCA的降解能力,模擬蚯蚓腸道環(huán)境分別進(jìn)行好氧和厭氧降解實(shí)驗(yàn).在好氧實(shí)驗(yàn)過(guò)程中,與初始加入培養(yǎng)基中的6:2FTCA濃度(F)相比,非生物干擾組(C)無(wú)顯著變化,說(shuō)明本實(shí)驗(yàn)過(guò)程中非生物干擾可忽略(圖4).在以6:2FTCA為唯一碳源組(F+M)和外加碳源組(F+M+G)中,6:2FTCA濃度顯著降低(<0.05),檢測(cè)到PFHxA和PFPeA兩種PFCAs終端降解產(chǎn)物.PFCA在F+M和F+M+G微生物實(shí)驗(yàn)組中的物質(zhì)的量百分比分別如下:PFHxA(91.4%) >PFPeA (8.6%)和PFHxA(90.2%)>PFPeA (9.8%)(圖3).另外,外加碳源組(F+M+G)降解效果明顯強(qiáng)于以6:2FTCA為唯一碳源組(F+M),說(shuō)明添加葡萄糖作為共代謝底物后,可能對(duì)好氧微生物共生網(wǎng)絡(luò)產(chǎn)生了影響,從而促進(jìn)了6:2FTCA的降解.而在厭氧環(huán)境中,各實(shí)驗(yàn)組之間無(wú)顯著差別,且未檢測(cè)到任何PFCAs產(chǎn)物.以上結(jié)果說(shuō)明,蚯蚓腸道微生物在好氧環(huán)境下對(duì)6:2FTCA有明顯的降解作用,而在厭氧環(huán)境下幾乎無(wú)降解作用.

蚯蚓腸道微生物能夠降解一些有機(jī)污染物,如六氯環(huán)己烷(HCH)[25]和硫丹[26].但是對(duì)殺蟲(chóng)劑克線(xiàn)磷[34]和FOSA等的降解較差[27].6:2FTCA前體物質(zhì)在好氧情況下能夠被微生物降解生成包括6:2FTCA在內(nèi)的一系列中間產(chǎn)物,然后再進(jìn)一步降解轉(zhuǎn)化生成終端產(chǎn)物PFCAs.6:2FTOH能被污泥好氧堆肥微生物[14]、土壤好氧微生物[37]、烷烴降解菌和氟乙酸降解菌[38]等降解轉(zhuǎn)化,6:2FTSA能被土壤[39]和沉積物[18]中的有氧微生物降解,生成6:2FTCA及其它中間產(chǎn)物,再經(jīng)過(guò)脫除HF和-CF2-生成PFBA、PFPeA和PFHxA.另外,也有一些研究表明,6:2FTCA前體物質(zhì)在厭氧環(huán)境下降解較為困難,如6:2FTSA[17]在土壤厭氧微生物作用下不發(fā)生降解,6:2FTOH在厭氧污泥中能降解為包括6:2FTCA在內(nèi)的一些中間產(chǎn)物,但是不能再將6:2FTCA進(jìn)一步降解生成PFCAs[38].因此,普遍認(rèn)為,在厭氧產(chǎn)甲烷的環(huán)境下,缺少能將6:2FTCA等PFCA前體物質(zhì)進(jìn)一步氧化脫羧生成PFCAs的生化機(jī)制.

圖4 蚯蚓腸道好氧微生物(A)和厭氧微生物(B)對(duì)6:2FTCA的降解作用.不同字母表示各值之間差異顯著性

<0.05

雖然蚯蚓腸道好氧微生物對(duì)6:2FTCA降解產(chǎn)生的主要PFCA終端產(chǎn)物(PFPeA和PFHxA)與上述文獻(xiàn)中報(bào)道的微生物降解(PFBA、PFPeA和PFHxA)有所區(qū)別,但是均證明了6:2FTCA在厭氧微生物作用下不能發(fā)生降解轉(zhuǎn)化,而能夠在好氧微生物作用下發(fā)生降解,且降解過(guò)程中只發(fā)生-氧化而沒(méi)有發(fā)生-氧化.綜上,6:2FTCA能夠在蚯蚓腸道好氧微生物作用下通過(guò)-氧化、脫除-HF和碳鍵斷裂等作用生成不同碳鏈長(zhǎng)度的PFPeA和PFHxA,而在蚯蚓腸道厭氧菌作用下不發(fā)生降解.

3 結(jié)論

3.1 在6:2FTCA(62.0ng/g dw)染毒土壤中暴露20d后,蚯蚓體內(nèi)雖未顯示明顯的脂質(zhì)過(guò)氧化,但是部分關(guān)鍵抗氧化酶出現(xiàn)了一定的氧化應(yīng)激效應(yīng).

3.2 CYP450和GST能夠參與6:2FTCA在蚯蚓體內(nèi)的代謝轉(zhuǎn)化,最終降解產(chǎn)物為PFBA、PFPeA和PFHxA,且CYP450對(duì)6:2FTCA在蚯蚓體內(nèi)生物轉(zhuǎn)化的貢獻(xiàn)要明顯高于GST.

3.3 蚯蚓腸道好氧微生物對(duì)6:2FTCA具有明顯的降解作用,終端PFCAs降解產(chǎn)物為PFPeA和PFHxA,而腸道厭氧微生物對(duì)6:2FTCA基本無(wú)降解作用.

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Toxicological effects and biotransformation mechanism of 6:2fluorotelomer carboxylic acid (6:2FTCA) in earthworms ().

ZONGYu-lu1, YANG Li-ping2, ZHAO Shu-yan1*

(1.Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, School of Ocean Science and Technology, Dalian University of Technology, Panjin 124221, China；2.School of Environmental Science and Engineering, Nankai University, Tianjin 300071, China)., 2022,42(6)：2886～2893

Earthworms () were exposed to the soil spiked with 6:2 fluorotelomeric carboxylic acid (6:2 FTCA) to investigate the toxicological effects and biotransformation mechanisms of 6:2 FTCA in earthworms afterandexposure. Compared to the controls, no significant effects were observed in malondialdehyde (MDA) contents and peroxidase (POD) activities, while catalase (CAT) activities were increased, and superoxide dismutase (SOD) and glutathione-s-transferase (GST) activities were significantly induced in 6:2 FTCA treatments. This suggested that 6:2 FTCA induced oxidative stress in the earthworm cells. Biodegradation of 6:2 FTCA in the earthworm cytolchrome P450 (CYP450) enzyme extracts and GST enzyme extracts fitted well with the first order kinetics. The biodegradation rate in CYP450 extracts (0.014/h) was much higher than that in GST extracts (0.006/h), indicating CYP450 and GST were involved in the enzymatic transformation and CYP450 contributed more than GST to 6:2 FTCA biotrans formation in earthworms. Three terminal perfluorocarboxylic acid (PFCA) metabolites, including perfluorohexanoic acid (PFHxA), perfluoropentanoic acid (PFPeA) and perfluorobutanoic acid (PFBA) were observed in the enzyme extracts. The results of gut microbial degradation test showed that aerobic microorganisms contributed to 6:2 FTCA biodegradation in earthworms significantly, and the terminal PFCA metabolites were PFHxA and PFPeA, but anaerobic microorganisms didn’t contribute to 6:2 FTCA biotransformation in earthworms.

6:2 FTCA；earthworm；toxicological effects；enzyme metabolism；gut microorganisms

X171.5

A

1000-6923(2022)06-2886-08

宗玉璐(1995-),女,河南洛陽(yáng)人,大連理工大學(xué)碩士研究生,主要從事研究方向?yàn)橛袡C(jī)污染物環(huán)境行為.

2021-11-22

國(guó)家自然科學(xué)基金項(xiàng)目(41603106,42177374);中央高校基本科研業(yè)務(wù)費(fèi)專(zhuān)項(xiàng)(DUT21JC42)

* 責(zé)任作者, 副教授, zhaoshuyan@dlut.edu.cn