李建華,張亞運,孟維坤,蘇冠勇

基于可疑篩查技術(shù)研究有機磷阻燃劑V6轉(zhuǎn)化規(guī)律

李建華,張亞運,孟維坤,蘇冠勇*

(南京理工大學(xué)環(huán)境與生物工程學(xué)院,江蘇省化工污染控制與資源化重點實驗室,江蘇 南京 210094)

運用Orbitrap高分辨質(zhì)譜儀,評估了人體肝微粒體(HLM)代謝V6的轉(zhuǎn)化規(guī)律.除5種已被報道過的O-脫烷基、氧化性去磷酸化和氧化脫氯產(chǎn)物外,首次發(fā)現(xiàn)了6種新型產(chǎn)物.基于其MS/MS質(zhì)譜圖,該6種代謝產(chǎn)物被鑒定為醛基和羧基產(chǎn)物.其中,5種代謝物生成量隨孵育時間呈線性增加趨勢,具有累積性.此外,推導(dǎo)的代謝途徑表明,脫烷基化和羥基化代謝物進(jìn)一步轉(zhuǎn)化為次級代謝物,即醛和羧基代謝物.

新型有機磷酸酯；人肝微粒體；高分辨質(zhì)譜儀；體外代謝；生物轉(zhuǎn)化規(guī)律

有機磷酸酯(OPEs)是一類以磷酸基團(tuán)為基本骨架,其側(cè)鏈被烷基、芳香基或鹵烷基取代的酯類化合物.由于具有良好阻燃性能和增塑性能,OPEs目前是公認(rèn)的替代性阻燃劑之一,其生產(chǎn)量和使用量大幅攀升[1-4].2,2-雙(氯甲基)丙烷-1,3-二基四(2-氯乙基)二磷酸酯(V6)是一種新型氯代OPE,常與三(1,3-二氯-2-丙基)磷酸酯(TDCIPP),磷酸三(2-氯異丙基)酯(TCIPP)混合使用于家具、汽車泡沫、嬰兒產(chǎn)品材料聚氨酯泡沫塑料中[5-7].由于其非化學(xué)鍵合特性,V6在產(chǎn)品使用周期內(nèi)容易浸出而進(jìn)入環(huán)境.近期,V6陸續(xù)被檢出于多種環(huán)境介質(zhì)(灰塵、水體、沉積物、土壤等)[1,8-10],如Fang等[8]在室內(nèi)灰塵和汽車灰塵樣品中均檢出了V6,檢出濃度范圍分別為<5～1110ng/g(中位數(shù)為12.5ng/g)和5～6160ng/g(中位數(shù)為103.0ng/g),這增加了人體暴露于V6的風(fēng)險.有研究報道已經(jīng)發(fā)現(xiàn)V6檢出于人體樣品,如手指甲[7].歐洲化學(xué)管理署發(fā)現(xiàn)V6可顯著降低大鼠血清膽堿酯酶活性,表明其可能具有類似其他OPEs的神經(jīng)毒性[5].美國國家環(huán)境保護(hù)局(US EPA)危險化學(xué)品評估認(rèn)為V6對人類具有多種潛在生物效應(yīng),如遺傳毒性、生物毒性、神經(jīng)發(fā)育毒性等[11].因此,系統(tǒng)評估V6的人體健康風(fēng)險便成了當(dāng)下亟需解決的問題.生物轉(zhuǎn)化是外源性物質(zhì)在生物體內(nèi)毒性變化的最重要決定因素之一,在環(huán)境毒理學(xué)和風(fēng)險評估中發(fā)揮著越來越重要的作用[12].因此,研究V6的代謝轉(zhuǎn)化規(guī)律對于系統(tǒng)評估V6人體健康風(fēng)險便顯得尤為重要.

目前,關(guān)于V6的代謝研究較少,僅Alves等[6]在2018年報道了人肝微粒體和S9代謝V6生成的13種代謝產(chǎn)物,包括O-脫烷基、氧化脫氯、羥基化和硫酸鹽產(chǎn)物.除此之外,V6的生物代謝研究仍有一些問題值得探索: V6代謝體系是否存在其他未知代謝產(chǎn)物,這對于準(zhǔn)確評估V6的毒性效應(yīng)極為重要; OPEs部分代謝產(chǎn)物似乎具有高于母體的生物毒性[13],如Su等[14]證實磷酸三苯酯(TPHP)的二酯產(chǎn)物磷酸二苯酯(DPHP)可以誘導(dǎo)更多基因的異常表達(dá).

高分辨質(zhì)譜儀(HRMS)以其質(zhì)量范圍寬、掃描速度快、靈敏度高等優(yōu)勢,被廣泛應(yīng)用于低濃度已知或未知化合物的定量檢測,復(fù)雜組分的痕量分析等方面,逐漸在環(huán)境樣品的可疑/未知化合物方面發(fā)揮重要的識別作用[15].基于HRMS開發(fā)的可疑和非靶向識別技術(shù)具有高通量的優(yōu)勢,拓展了傳統(tǒng)靶向分析的廣度,已廣泛應(yīng)用于生物醫(yī)藥、食品安全、環(huán)境監(jiān)測等領(lǐng)域[16-19].借助于此識別技術(shù),環(huán)境化學(xué)家在不同環(huán)境介質(zhì)中不斷發(fā)現(xiàn)新型污染物[20-23],甚至未知的代謝產(chǎn)物[24-26]等,這為全面識別生物體系中V6代謝產(chǎn)物提供了技術(shù)支撐.

本研究基于靜電場軌道阱高分辨質(zhì)譜儀(Orbitrap-HRMS),重新評估了人肝微粒體對V6的代謝規(guī)律,主要研究工作包括:探究是否存在新型代謝產(chǎn)物;基于代謝產(chǎn)物,構(gòu)制V6完整代謝路徑圖;探究新穎代謝產(chǎn)物含量隨時間變化趨勢,該研究成果將為尋找V6新生物標(biāo)志物和評估其毒性效應(yīng)提供數(shù)據(jù)支撐.

1 材料與方法

1.1 實驗藥品

圖1 V6的化學(xué)結(jié)構(gòu)

V6(化學(xué)結(jié)構(gòu)見圖1)和內(nèi)標(biāo)d15-TDCIPP購自于AK Scientific公司(Union city,CA,USA),50供體混合性別人肝微粒體(HLM)來自于BioreclamationIVT公司(Westbury,NY,USA),其酶活見表1.磷酸一氫鉀、磷酸二氫鈉、氯化鈉、煙酰胺腺嘌呤二核苷酸磷酸(NADPH)和乙酸銨均購自Sigma-Aldrich公司(St. Louis, MO, USA).實驗中使用的所有溶劑均為色譜純,購自Tedia公司(Fairfield,OH,USA).

表1 HLM中CYP450s酶活

1.2 V6孵育實驗

孵育體系包括563μL的0.01mol/L磷酸鹽緩沖液(PBS,pH=7.4)、30 μL的20mg/mL HLM蛋白(反應(yīng)濃度1mg/mL)和1μL的30mmol/L V6儲備液(反應(yīng)濃度1mmol/L,含有<1%的二甲基亞砜(DMSO)),該混合液在恒溫水浴箱中37℃、120r/min下預(yù)孵育5min,然后加入6μL的100mmol/L NADPH(反應(yīng)濃度1mmol/L)啟動反應(yīng).反應(yīng)1h后,向體系中補充等體積NADPH以充分反應(yīng).在0, 0.5, 1.0, 1.5和2.0h間隔時間點取樣,每次取樣100μL,反應(yīng)液移至干凈玻璃管中,然后加入400μL含有12.5ng內(nèi)標(biāo)的冰冷乙腈以終止反應(yīng).然后將混合液渦旋15s后3500r/min離心10min,取400μL上清液至另一個干凈玻璃管中,氮吹濃縮至100μL,經(jīng)0.2μm離心過濾器(COSTAR, CN:8169)過濾后待上樣分析.

為確保實驗數(shù)據(jù)準(zhǔn)確可靠,本實驗設(shè)置了陽性對照、陰性對照和空白對照.陽性對照體系中,以備受關(guān)注的磷酸三苯酯(TPHP)替代V6,以檢查代謝系統(tǒng)的穩(wěn)健性;陰性對照以等體積的PBS替代HLM或NADPH;而空白對照是以等量的DMSO替代V6;其他實驗條件同上.

1.3 儀器條件

V6及其代謝產(chǎn)物分析儀器為超高效液相色譜-Orbitrap 高分辨質(zhì)譜聯(lián)用儀(Thermo Fisher Scientific公司,Waltham,MA),分離柱為Eclipse Plus C18(50mm×2.1mm×3.5μm,Agilent,USA),柱溫為35℃.流動相為水(A)和甲醇(B),兩相均含有2mmol/L乙酸銨,流動相流速為0.45mL/min.洗脫梯度為:初始5% B,保持1min;2min內(nèi)增加至50% B;然后7min內(nèi)增加至100% B,保持8min;然后在0.1min內(nèi)回到初始條件,保持到23min.進(jìn)樣體積為5μL.Orbitrap-HRMS質(zhì)譜條件如下:離子源采用電噴霧電離(ESI),正離子模式,噴淋電壓+3500V,鞘氣壓35任意單位(arb),輔助氣壓10arb,毛細(xì)管溫度320℃,輔助氣加熱溫度425℃,離子掃描模式Full MS-AIF、選擇離子監(jiān)測(SIM)和平行反應(yīng)監(jiān)測(PRM),模式參數(shù)如下:掃描范圍50～750/,分辨率35000;碰撞能量(NCEs)10, 20, 40eV.

1.4 V6及其代謝產(chǎn)物篩查策略

圖2 V6及其代謝產(chǎn)物的靶向-可疑篩查策略流程

分為兩個步驟(圖2):1)基于標(biāo)準(zhǔn)品V6,運用Orbitrap-HRMS的SIM模式進(jìn)行定量分析;2)基于已報道OPEs的代謝路徑,即羥基化、O-脫烷基,氧化脫氯,氧化作用[6],列出V6所有可疑代謝產(chǎn)物名單.可疑篩查基本流程:1)Full MS-AIF模式下獲得V6代謝前后色譜圖和質(zhì)譜圖;2)用TraceFinder軟件分析其色譜圖和質(zhì)譜圖,找出可疑產(chǎn)物的化學(xué)式,并與可疑數(shù)據(jù)庫中化合物進(jìn)行匹配,將相應(yīng)化合物列為候選物質(zhì).此步驟中,需要遵守代謝產(chǎn)物篩查標(biāo)準(zhǔn):a)信噪比/>10;b)測定離子質(zhì)荷比(/)和目標(biāo)性離子/的質(zhì)量誤差(Δmass)<10′10-6;c)陰性對照中相同保留時間無明顯峰;d)所有的離子碎片是合乎邏輯且可解釋的;3)運用Orbitrap-HRMS的PRM模式獲得二級碎片信息,進(jìn)一步推測潛在代謝產(chǎn)物的化學(xué)結(jié)構(gòu).

1.5 數(shù)據(jù)分析

采用Xcalibur v2.2和TraceFinder v3.3軟件(Thermo Fisher Scientific,Bremen, German)定性定量分析V6及其代謝產(chǎn)物.采用ChemDraw 2004軟件繪制代謝產(chǎn)物的化學(xué)結(jié)構(gòu)和代謝路徑圖.代謝產(chǎn)物隨孵育時間的相對響應(yīng)強度采用EXCEL 2010進(jìn)行處理,所得數(shù)據(jù)用GraphPad Prism 5軟件進(jìn)行繪制.不同時間點的代謝產(chǎn)物相對響應(yīng)強度差異采用Student’s-test進(jìn)行分析.

2 結(jié)果與分析

2.1 V6代謝體系中新型代謝產(chǎn)物的發(fā)現(xiàn)及鑒定

基于Orbitrap-HRMS,在HLM孵育體系中,一共發(fā)現(xiàn)了11種V6代謝產(chǎn)物(表2).其中,5種被標(biāo)為M1～M5的代謝產(chǎn)物已有報道.本研究發(fā)現(xiàn)了6種從未被報道過的代謝產(chǎn)物,即M6 (/578.9252,RT= 4.97min,Δmass=-3.215′10-6),M7(/516.9329,RT= 3.60min,Δmass=-3.082′10-6),M8(/562.9303, RT= 5.96min,Δmass=-0.286′10-6),M9(/500.9380, RT= 4.51min,Δmass=4.104′10-6),M10(/578.9252,RT= 4.75min,Δmass=1.014′10-6)和M11(/542.9486, RT= 5.12min,Δmass=-1.973′10-6).

基于6種新型代謝產(chǎn)物的MS/MS質(zhì)譜圖和母體V6的化學(xué)結(jié)構(gòu)(圖1),進(jìn)一步推測了其具體化學(xué)結(jié)構(gòu),結(jié)果如圖3所示.M6有3個特征離子,即/358.9527 [C9H16Cl4PO4]+,354.9658 [C9H15Cl3PO6]+和234.9684 [C5H10Cl2PO4]+.碎片離子/358.9527可分為連接兩個磷酸基團(tuán)的鏈(下稱連接鏈) [C5H8Cl2]+和一個含有兩個烷基鏈的磷酸[C4H8Cl2PO4]+;離子/234.9684為連接鏈[C5H8Cl2]+和磷酸[H2PO4]+;離子/354.9658可分解為[C5H8Cl2]+和[C4H7ClPO6]+,表明了磷酸烷基鏈中的一個氯原子被羧基取代.基于此,M6被推測為羧基化合物.M7的分子量為516.9329,比M6少62Da,證明了M6通過O-脫烷基作用轉(zhuǎn)化為M7.M7的二級碎片離子/296.9610 [C7H13Cl3PO4]+比M6的碎片離子/354.9658少[C2H4Cl]+,進(jìn)一步表明了磷酸基團(tuán)上的一個烷基鏈被去除.同M6,M7也產(chǎn)生了碎片離子/234.9684和354.9670,證明一個羧基基團(tuán)可能位于另一個磷酸基團(tuán)的烷基鏈上.因此,M7為羧基產(chǎn)物,其羧基位于一個磷酸基團(tuán)上,而另一個磷酸基團(tuán)上缺少一個烷基鏈.M8能生成/338.9713 [C9H15Cl3PO5]+、/276.9790 [C7H12Cl2PO5]+和/214.9868 [C5H9ClPO5]+3個特征碎片.碎片離子/338.9713進(jìn)一步可分為[C4H8Cl2PO4]+和[C5H7ClO]+,其中[C4H8Cl2PO4]+為含有2個烷基鏈的磷酸結(jié)構(gòu),而[C5H7ClO]+為連接鏈[C5H8Cl2]+上的一個氯原子被氧化為醛基.碎片/276.9790和214.9868也含有碎片[C5H7ClO]+,進(jìn)一步印證了連接鏈上醛基的存在.基于此,M8被鑒定為醛基產(chǎn)物,其醛基位于連接鏈上.M9的特征碎片/140.9945 [C2H6PO5]+可分為[C2H4O]+和[H2PO4]+,表明了含有兩個烷基的磷酸上的一個氯代烷基鏈氧化脫氯,并脫去另一個烷基鏈.因此,M9被推測為醛基產(chǎn)物,其磷酸上一個氯原子被氧化為醛基,另一個烷基通過O-脫烷基作用被去除.M10的分子量和M6相同,表明這2個化合物可能是同分異構(gòu)體.M10的碎片離子/230.9816可分為[C5H7ClO2]+和[H2PO4]+,表明了連接鏈[C5H8Cl2]+的一個氯原子被氧化為羧基基團(tuán).M10的另一個碎片/354.9661也含有碎片[C5H7ClO2]+,也印證了這個推測.因此,M10為羧基基團(tuán)位于連接鏈上的氧化產(chǎn)物.M11的二級碎片/195.0051能分解成[C5H6O2]+和[H2PO4]+.與連接鏈[C5H8Cl2]相比, [C5H6O2]+結(jié)構(gòu)少了2個[HCl],多了2個[O],表明了連接鏈上的2個氯原子被氧化為醛基.另一個碎片/256.9966可分為[C2H5ClPO4]+和[C5H6O2]+,進(jìn)一步印證了這個推測.因此,M11被推測為二醛基產(chǎn)物,其兩個醛基均位于連接鏈上.

表2 HLM代謝體系中V6的潛在代謝產(chǎn)物

綜上所述,M6、M7和M10為羧基產(chǎn)物,而M8、M9和M11為醛基產(chǎn)物,這可能由肝微粒體含有乙醇脫氫酶和醛類脫氫酶作用生成的[27].這與以前的研究結(jié)果相一致,如Van den Eede等[27]發(fā)現(xiàn)TDCIPP、TCIPP和磷酸三(2-丁氧基)乙酯(TBOEP)可以被HLM轉(zhuǎn)化為相應(yīng)的羧基和/或醛基代謝物.Alves等[6]推測V6的氧化脫氯代謝物可能被轉(zhuǎn)化為醛中間體和羧酸,但未得到證實.Wang等[28]研究了6種典型的OPEs在成年斑馬魚體內(nèi)的代謝及其主要代謝物分布,其中5種可代謝為酮/醛代謝物和羧基代謝物.

圖3 V6的6種新型代謝產(chǎn)物結(jié)構(gòu)鑒定

2.2 新型代謝產(chǎn)物生成路徑的推導(dǎo)

結(jié)合Alve等[6]推導(dǎo)的V6I相和II相代謝路徑及Van den Eede等[27]推導(dǎo)的幾種常見OPEs(如與V6結(jié)構(gòu)相似的TDCIPP)的代謝路徑,本研究推導(dǎo)了6種新型代謝產(chǎn)物的生成路徑(圖4).路徑I,O-脫烷基化反應(yīng)導(dǎo)致烷基鏈C-O鍵斷裂,生成代謝物M1;隨后M1發(fā)生氧化脫氯,生成M3;M3一方面可以進(jìn)一步氧化為羧基代謝物(M7),M3的羥基也可能進(jìn)一步脫氫,生成M9等醛類代謝物.路徑II,V6首先通過氧化脫氯生成羥基化代謝產(chǎn)物M4;然后M4可以進(jìn)一步氧化為羧基化合物M6;M6的O-脫烷基反應(yīng)生成M7.路徑III,V6的氧化脫氯導(dǎo)致M8的形成;隨后M8可以進(jìn)一步氧化為羧基代謝物M10或二醛代謝物M11.

結(jié)合Alve推導(dǎo)的V6代謝路徑[6],可知V6首先被代謝為羥基化產(chǎn)物,隨后羥基化產(chǎn)物被氧化為醛基產(chǎn)物,進(jìn)一步被氧化為羧基產(chǎn)物.據(jù)報道,許多有機污染物的羥基化產(chǎn)物具有比母體更大的毒性[29-30];當(dāng)羧基存在時,可顯著降低物質(zhì)毒性[31],因此,轉(zhuǎn)化產(chǎn)物對V6毒性變化有重要影響.后續(xù)研究應(yīng)在關(guān)注V6的同時,也要關(guān)注其代謝產(chǎn)物.

圖4 6種新型代謝產(chǎn)物在HLM中的可能生成路徑

M1,M3和M4為已報道過的代謝產(chǎn)物

2.3 新型代謝產(chǎn)物隨時間變化趨勢

如圖5所示, M6含量在2.0h孵育時間內(nèi),呈現(xiàn)穩(wěn)步增加趨勢;在反應(yīng)1.5h后,M6的生成量與0h相比,增加量明顯(<0.05).在0.5h可以明顯觀察到M6的含量,而與此同時M7幾乎沒有產(chǎn)生,說明M6的產(chǎn)生早于M7,這與之前推測M6是M7的前驅(qū)物是一致的.M7的含量從0.5h到1.0h緩慢增加,然后從1.0h到1.5h暫時下降,可能是由于M7進(jìn)一步代謝所致,隨后在1.5～2.0h的孵育時間內(nèi),M7呈現(xiàn)快速增加的趨勢,可能是因為大量的前體(M6)加速次級代謝產(chǎn)物(M7)的積累.與M7相似,M9的生成量在后孵育階段1.5～2h也比前孵育階段0～1.5h明顯加快(<0.05),這種變化趨勢與M3相似,證實了M3與M9之間可能存在轉(zhuǎn)化.M8的含量從0h到1.5h成線性增加,隨后從1.5～2.0h產(chǎn)量顯著降低,這與本研究推導(dǎo)的M8可以被進(jìn)一步轉(zhuǎn)化為次生代謝物M10和M11結(jié)論一致.M10和M11的含量在孵育時間2h內(nèi)均呈現(xiàn)增加趨勢,表明M8向M10和M11的轉(zhuǎn)化明顯.M10在0.5h開始有檢出,而M11在0.5h有明顯的生成量,這說明醛中間體(M8)更容易轉(zhuǎn)化為羧基產(chǎn)物(M11).

綜上所述,除醛基產(chǎn)物M8外,其余5種新型代謝產(chǎn)物在2.0h內(nèi)均有所富集.事實上,醛類、羧酸類化合物也是其他OPEs的常見代謝產(chǎn)物[27,32-33].另外,目前關(guān)于OPEs代謝路徑表明羧基產(chǎn)物是可發(fā)現(xiàn)的末端產(chǎn)物[27,32],因此,OPEs的羧基產(chǎn)物可能是其暴露于人體的新型生物標(biāo)志物.基于這一點,人體樣品中V6羧基代謝產(chǎn)物含量及其在人體內(nèi)歸趨需要加強研究.

圖5 6種新型代謝產(chǎn)物相對響應(yīng)隨孵育時間變化趨勢

基于0h初始值分析不同時間點顯著性差異(<0.05, *;<0.01, **); 相對響應(yīng)由代謝產(chǎn)物的峰面積除以內(nèi)標(biāo)d15-TDCIPP的峰面積計算所得

3 結(jié)論

3.1 本研究首次發(fā)現(xiàn)了V6的6種醛基和羧基代謝產(chǎn)物,其由初級代謝產(chǎn)物(脫烷基和羥基化產(chǎn)物)進(jìn)一步轉(zhuǎn)化而成.

3.2 5種新識別代謝產(chǎn)物含量在孵育時間穩(wěn)定增加,這為尋找V6新生物標(biāo)志物提供借鑒.

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Biotransformation of an organophosphate flame retardant V6 based on suspect screening technique.

LI Jian-hua, ZHANG Ya-yun, MENG Wei-kun, SU Guan-yong*

(Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China)., 2022,42(9)：4409～4415

This study studied metabolism of V6 in human liver microsomes (HLM) using Orbitrap high-resolution mass spectrometer (Orbitrap-HRMS). With the exception of 5 reported metabolites including O-dealkylated, oxidative de-phospherylated and O- dechlorinated compounds, 6 novel products were firstly observed. Based on their MS/MS spectra, these newly discovered metabolites were identified as aldehyde and carboxyl products. Of them, 5 metabolites can be accumulative according to their linearly increasing tendency inexperiment. In addition, the proposed metabolic pathway demonstrated the primarily metabolites, i.e. dealkylated and hydroxylated metabolites were further transformed into secondary metabolites, i.e., aldehyde and carboxyl metabolites.

organophosphate flame retardant of emerging concern；human liver microsomes；high-resolution mass spectrometer；metabolism；biotransformation rule

X503.2

A

1000-6923(2022)09-4409-07

2022-02-25

國家自然科學(xué)基金資助項目(22006068,21976088);江蘇省杰出青年基金項目(BK20211521);中國博士后科學(xué)基金項目(2021M690079);江蘇省博士后科研資助(2021K174B);中央高?；究蒲袠I(yè)務(wù)費專項資金資助(30920021115)

*責(zé)任作者, 教授, sugy@njust.edu.cn

李建華(1986-),女,河南許昌人,副教授,理學(xué)博士,主要研究方向為新型環(huán)境污染物的識別及其在生物體內(nèi)的遷移轉(zhuǎn)化.發(fā)表論文20多篇.