趙金玉, 鐘玉芳, 王藝陪, 王楊君, 安 靜, 盛國英, 傅家謨

(上海大學(xué)環(huán)境與化學(xué)工程學(xué)院環(huán)境污染與健康研究所,上海 200444)

6-羥基-多溴聯(lián)苯醚 47對(duì) A549細(xì)胞抗氧化系統(tǒng)的影響

趙金玉, 鐘玉芳, 王藝陪, 王楊君, 安 靜, 盛國英, 傅家謨

(上海大學(xué)環(huán)境與化學(xué)工程學(xué)院環(huán)境污染與健康研究所,上海 200444)

多溴聯(lián)苯醚(polybrominated diphenyl ethers,PBDEs)是一類具有內(nèi)分泌干擾和神經(jīng)毒性的持久性有機(jī)污染物,但對(duì)其代謝產(chǎn)物的毒性研究還較少.從細(xì)胞毒性和氧化應(yīng)激的角度,探討 2,2′,4,4′-四溴聯(lián)苯醚 (2,2′,4,4′-tetrabromodiphenyl ether,BDE47)的羥基代謝物 6-羥基 -多溴聯(lián)苯醚 47(6-hydroxylated-2,2′,4,4′-tetrapolybrominated diphenyl ethers,6-OH-BDE47)對(duì)人肺癌細(xì)胞系 A549的作用.研究結(jié)果發(fā)現(xiàn),6-OH-BDE47對(duì) A549細(xì)胞具有明顯的細(xì)胞毒性,隨著染毒劑量增加和時(shí)間延長(zhǎng),A549細(xì)胞存活率逐漸下降.急性染毒實(shí)驗(yàn)證實(shí),6-OH-BDE47對(duì) A 549細(xì)胞的氧化應(yīng)激系統(tǒng)有嚴(yán)重的影響.當(dāng)細(xì)胞培養(yǎng)體系中 6-OH-BDE47濃度≥0.5μmol/L時(shí),24 h急性暴露能誘導(dǎo)A549細(xì)胞的總超氧化物歧化酶 (total superoxide dismutase,T-SOD)活力升高 1.03倍以上;而 6-OH-BDE47濃度≥2.0 μmol/L時(shí),還原性谷胱甘肽 (reduced the glutathione,GSH)含量減少 71.2%.由此可見,6-OH-BDE47有一定的細(xì)胞毒性,能刺激A549細(xì)胞發(fā)生氧化應(yīng)激,誘導(dǎo)抗氧化酶升高和內(nèi)源性還原劑耗竭.

多溴聯(lián)苯醚;持久性有機(jī)污染物;氧化應(yīng)激;多溴聯(lián)苯醚 47

Abstract:Polybrominated diphenyl ethers(PBDEs),a classof persistentorganic pollutants,wereproved to be endocrine disrup tors and neurotoxic agents.However,there are few reports about the toxic effectsof their hydroxylated metabolites.Our objectivewas to investigate the cytotoxicity of 6-hydroxylated-2,2′,4,4′-tetrapolybrominated diphenyl ethers(6-OH-BDE47)and its effectson anti-oxidative systems in A549 cells after acute exposure.The results show that6-OH-BDE47 hasobviouscytotoxicity,and the A549 cell survival gradually decrease after exposure in a dose-and time-dependent manner. Acute exposure experiment confirmed that the oxidative stress system of A549 cells was seriously impaired by 6-OHBDE47.When the final concentration of 6-OH-BDE47 in culture medium was higher than 0.5μmol/L,the total superoxide dismutase(T-SOD)content in treatment group cells increased by 1.03 times compared to the control group cells;and≥2.0μmol/L of 6-OH-BDE47 reduced the glutathione content by 71.2%.These results suggest that 6-OH-BDE47 has certain cytotoxicity on A 549 cells and can impair oxidative stress responses as evidenced by increased SOD activity and decreased GSH.

Key words:polybrominated diphenyl ethers;persistent organic pollutants;oxidative stress;BDE47

多溴聯(lián)苯醚 (polybrominated diphenyl ethers,PBDEs)是一類廣泛應(yīng)用于電子、化工、建材等領(lǐng)域的溴代阻燃劑,具有環(huán)境穩(wěn)定性和親脂性等特征,容易在環(huán)境中積累并通過食物鏈富集.隨著 PBDEs應(yīng)用的增長(zhǎng),其對(duì)生態(tài)環(huán)境的危害也日益凸顯.有研究預(yù)測(cè),在未來的 15～30年內(nèi),PBDEs將成為主要的環(huán)境持久性有機(jī)污染物.目前,在許多環(huán)境介質(zhì)和生物體內(nèi)均可以檢測(cè)到 PBDEs的存在,如大氣、水、沉積物、魚類、淡水貝類、母乳等[1-5],人體樣品中以BDE47,BDE99,BDE153和 BDE183為主要同系物[6-7].PBDEs有兩種類型的代謝產(chǎn)物,即羥基化PBDEs和甲氧基 PBDEs,在人體內(nèi)也檢測(cè)到了這兩類代謝物[8].研究表明,PBDEs具有內(nèi)分泌干擾效應(yīng)、神經(jīng)發(fā)育毒性等特性[9-11].目前,對(duì) PBDEs代謝產(chǎn)物的毒性研究還較少,本實(shí)驗(yàn)選用 BDE-47的羥基代謝物 6-OH-BDE47,觀察其對(duì)人肺癌細(xì)胞 A549的細(xì)胞毒性以及氧化應(yīng)激相關(guān)的超氧化物歧化酶SOD和谷胱甘肽 GSH的影響.

1 試劑與儀器

1.1 材料與試劑

人肺癌細(xì)胞系 A 549,購自中國科學(xué)院典型培養(yǎng)物保藏委員會(huì)細(xì)胞庫;噻唑藍(lán) (methyl thiazolyl tetrazolium,MTT),購自上海前塵生物公司;二甲基亞砜 (DMSO),美國 Amresco公司生產(chǎn);超氧化物歧化酶 SOD檢測(cè)試劑盒和谷胱甘肽 GSH檢測(cè)試劑盒,購自南京建成生物工程公司;其余試劑為國產(chǎn)分析純.

1.2 儀器

實(shí)驗(yàn)儀器有 Thermo Labsystems MK3酶標(biāo)儀、Thermo全波長(zhǎng)熒光 /比色掃描讀數(shù)儀、國產(chǎn)恒溫水浴鍋等.

2 實(shí)驗(yàn)方法

2.1 實(shí)驗(yàn)分組與染毒處理

本實(shí)驗(yàn)設(shè) 1個(gè)溶劑對(duì)照組和 3個(gè)劑量 6-OHBDE47染毒組.溶劑對(duì)照組用體積分?jǐn)?shù)為 0.1%的DMSO處理,6-OH-BDE47溶于 DMSO,分 0.1,0.5和 2.0μmol/L 3個(gè)濃度組.

2.2 測(cè)定細(xì)胞存活率的M TT試驗(yàn)

MTT試驗(yàn)是通過測(cè)定活細(xì)胞代謝噻唑藍(lán)的藍(lán)紫色產(chǎn)物的量來反映細(xì)胞存活率或細(xì)胞活力的細(xì)胞毒性檢測(cè)方法,并通過該試驗(yàn)篩選受試物的作用時(shí)間.稱取一定量的MTT溶于磷酸鹽緩沖液 (phosphate buffer saline,PBS)中 ,配成 5 mg/mL MTT/PBS溶液,0.22μm濾膜過濾后于 4℃避光保存.取對(duì)數(shù)生長(zhǎng)期的 A549細(xì)胞接種于 4塊 96孔培養(yǎng)板中,每孔2×103個(gè)細(xì)胞,每組 5個(gè)平行樣.培養(yǎng)過夜后換無血清培養(yǎng)基,分別加入體積分?jǐn)?shù)為 0.1%的 DMSO,終濃度分別為 0.1,0.5和 2.0μmol/L的 6-OH-BDE47(DMSO濃度控制在 0.1%).分別染毒 6,24,48,72 h后加入終濃度 0.5 mg/mL的MTT,37℃孵育4 h,吸去上清后加 DMSO溶解,用酶標(biāo)儀于 492 nm處測(cè)其光密度.

2.3 SOD活力檢測(cè)

SOD是細(xì)胞啟動(dòng)氧化應(yīng)激效應(yīng)和清除超氧陰離子的重要角色.本實(shí)驗(yàn)采用黃嘌呤氧化酶法,通過測(cè)定超氧陰離子 O2-氧化羥胺生成的亞硝酸鹽含量可反映 SOD的活力.高等動(dòng)物細(xì)胞內(nèi)有兩種 SOD,即銅鋅 SOD(CuZn-SOD)和錳 SOD(Mn-SOD).本實(shí)驗(yàn)將對(duì)細(xì)胞內(nèi)總 SOD(T-SOD)和分型 SOD分別進(jìn)行檢測(cè),以觀察不同亞型 SOD在 6-OH-BDE47氧化應(yīng)激效應(yīng)中的作用.取對(duì)數(shù)生長(zhǎng)期的 A549細(xì)胞,接種于 75 mL細(xì)胞培養(yǎng)瓶,每瓶約 1×106個(gè)細(xì)胞.細(xì)胞培養(yǎng)過夜后按上述分組染毒,每組 3個(gè)平行樣,根據(jù)MTT試驗(yàn)篩選的染毒時(shí)間,染毒 24 h后收獲細(xì)胞,超聲破碎,其余具體操作步驟按說明書進(jìn)行.SOD活力 (U/mg pro)=(OD對(duì)照管-OD測(cè)定管)×反應(yīng)總體積 /(OD對(duì)照管×50% ×取樣量 ×待測(cè)樣本蛋白含量).

2.4 GSH含量檢測(cè)

GSH是抗氧化系統(tǒng)中含量豐富的抗氧化劑,測(cè)定其含量可反映細(xì)胞抗氧化水平.利用二硫代二硝基苯甲酸與 GSH上的巰基反應(yīng),生成的黃色化合物可進(jìn)行比色定量.取對(duì)數(shù)生長(zhǎng)期的 A549細(xì)胞,接種于 75 mL細(xì)胞培養(yǎng)瓶,每瓶約 1×106個(gè)細(xì)胞.細(xì)胞培養(yǎng)過夜后按上述分組染毒,24 h后收獲細(xì)胞,超聲破碎,其余具體操作步驟按說明書進(jìn)行.GSH含量(mg/g pro)=(OD測(cè)定管-OD空白管) ×標(biāo)準(zhǔn)管 GSH濃度 ×GSH分子量 307/[(OD標(biāo)準(zhǔn)管-OD空白管)×待測(cè)樣本蛋白含量].

2.5 Bradford法定量蛋白

參照文獻(xiàn)[12]中的方法進(jìn)行.

2.6 統(tǒng)計(jì)學(xué)方法

本研究采用 SPSS10.0進(jìn)行 One-way ANOVA分析,均數(shù)以 x-±s表示.

3 實(shí)驗(yàn)結(jié)果

3.1 6-OH-BDE47對(duì) A549細(xì)胞存活率的影響

由圖1可見,A 549細(xì)胞的存活率與 6-OHBDE47之間存在著明顯的劑量和時(shí)間效應(yīng)關(guān)系.低濃度 (0.1μmol/L)6-OH-BDE47作用 24 h后,細(xì)胞仍保持 90%的存活率 (后續(xù)實(shí)驗(yàn)選擇 24 h作為染毒時(shí)間),72 h后降至 80%;而高濃度 (2.0μmol/L)6-OH-BDE47作用 6 h后,細(xì)胞的存活率下降至 80%左右,72 h后只有 30%的細(xì)胞存活.

圖1 6-OH-BDE47對(duì) A549細(xì)胞存活率的影響Fig.1 Survival rate of A549 cells after treatment by 6-OH-BDE47

3.2 6-OH-BDE47對(duì) A 549 SOD活力的影響

由表 1可見,0.1μmol/L組與對(duì)照組細(xì)胞 TSOD活力無統(tǒng)計(jì)學(xué)差異,而 0.5μmol/L組和 2.0 μmol/L組細(xì)胞的 T-SOD活力與對(duì)照組相比分別升高了 1.03倍和 1.15倍,差異有統(tǒng)計(jì)學(xué)意義.Mn-SOD活力在各染毒組均表現(xiàn)出升高的趨勢(shì),0.1μmol/L組升高 47.1%,0.5μmol/L組升高 2倍,2.0μmol/L處理組升高2.96倍,但僅2.0μmol/L組與對(duì)照組的差異有統(tǒng)計(jì)學(xué)意義.

表 1 不同濃度 6-OH-BDE47對(duì) A549總 SOD活力和分型SOD活力的影響Table 1 Changes of T-SOD,CuZn-SOD and M n-SOD activities in A549 cells after the treatment by d ifferent doses of 6-OH-BDE47(U/m g pro,x—±s,n=3)

3.3 6-OH-BDE47對(duì) A549 GSH含量的影響

由表 2可見,隨著染毒濃度的升高,各處理組GSH含量逐漸下降,0.1,0.5和 2.0μmol/L組細(xì)胞中GSH含量分別下降 21.6%,34.8%和 71.2%,其中2.0μmol/L組與對(duì)照組的差異有統(tǒng)計(jì)學(xué)意義.

表 2 不同濃度 6-OH-BDE47對(duì) A549 GSH含量的影響Table 2 Changes of GSH concentration in A549 cells after the treatment by d ifferent doses of 6-OH-BDE47

表 2 不同濃度 6-OH-BDE47對(duì) A549 GSH含量的影響Table 2 Changes of GSH concentration in A549 cells after the treatment by d ifferent doses of 6-OH-BDE47

注:與對(duì)照組相比,＊P<0.05

組別 對(duì)照組 0.1μmol/L組0.5μmol/L組2.0μmol/L組GSH含量 8.06±0.36 6.31±0.46 5.26±2.94 2.32±1.90＊

4 結(jié) 束 語

在生理狀態(tài)下,細(xì)胞會(huì)產(chǎn)生一定量的自由基,同時(shí)細(xì)胞內(nèi)的抗氧化系統(tǒng)維持在一定水平以清除這些自由基,避免其與核酸、蛋白等生物大分子反應(yīng)而損傷細(xì)胞.在外來刺激作用下,細(xì)胞會(huì)產(chǎn)生大量自由基,此時(shí)抗氧化系統(tǒng)也會(huì)被調(diào)動(dòng)起來,其中 SOD作為一種金屬酶可催化超氧陰離子自由基O2-發(fā)生歧化反應(yīng),從而清除氧自由基,隨著 O2-的產(chǎn)生量增加,SOD會(huì)相應(yīng)大量表達(dá).GSH作為抗氧化系統(tǒng)中含量豐富的抗氧化劑,參與許多氧化物質(zhì)如 H2O2,ROOH等的解毒過程.從本工作的研究結(jié)果可見,在高于 0.5μmol/L 6-OH-BDE47作用下,A549細(xì)胞SOD活力可增加 2～3倍,這說明 6-OH-BDE47可能使細(xì)胞產(chǎn)生大量O2-,從而誘導(dǎo) SOD的大量表達(dá),且主要是Mn-SOD含量升高;同時(shí),2.0μmol/L 6-OHBDE47使細(xì)胞產(chǎn)生了相當(dāng)數(shù)量的 H2O2,ROOH等自由基,促進(jìn) GSH的消耗而使其含量下降.氧化應(yīng)激效應(yīng)可能導(dǎo)致細(xì)胞的損傷甚至死亡,本研究觀察到隨著 6-OH-BDE47劑量的增加,細(xì)胞存活率有逐漸下降的趨勢(shì),推測(cè)與細(xì)胞氧化應(yīng)激水平有一定關(guān)系,但其具體機(jī)理有待進(jìn)一步研究.

[1] STRANDBERG B,DODEDR N G,BASU I,et al.Concentrations and spatial variations of polybrominated diphenyl ethers and other organohalogen compounds in Great Lakes Air[J]. Environmental Science and Technology,2001,35(6):1078-1083.

[2] CYNTHIA A W,MEHRAN A,DEREK C G.Levels and trendsof brominated fame retardants in the Arctic[J].Chemosphere,2006,64(2):209-233.

[3] SHE J C,XIAO J L,ZHEN L,et al.Time trends of polybrominated diphenyl ethers in sediment cores from the Pearl River Estuary, South China [J].Environmental Science and Technology,2007,41(16):5595-5600.

[4] JIAN Y G,FENG CW,BIX M,et al.Polybrominated diphenyl ethers in seafood products of South China[J].Agriculture Food Chemistry,2007,55(22):9152-9158.

[5] AKIO K,TAKEO Y,KOU J H,et al.Assessment of human exposure to polychlorinated biphenyls and polybrominated diphenyl ethers in Japan using archived samp les from the early 1980s and mid-1990s[J].Environmental Research,2005,99(1):31-39.

[6] MENESESM,W INGFORS H,SCHUMACHER M,et al.Polybrominated diphenyl ethers detected in human adipose tissue from Spain[J].Chemosphere,1999,39(13):2271-2278.

[7] SJOD IN A,HAGMAR L,KLASSON E,et al.Flame retardant exposure:polybrominated diphenyl ethers in blood from Swedish workers[J].Environmental Health Perspectives,1999,107(8):643-648.

[8] MARIA A, STEVEN N C, GORAN M, et al.Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDEmetabolites in young humans from Managua,Nicaragua[J].Environmental Health Perspective,2008,116(3):400-408.

[9] RENFANG S,YUHE H,MARGARET B,et al.Effectsof fifteen PBDE metabolites,DE71,DE79 and TBBPA on steroidogenesis in the H295R cell line [J].Chemosphere,2008,71(10):1888-1894.

[10] LEMA S,D ICKEY J,SCHULTZ I,et al.Dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether(PBDE-47)alters thyroid status and thyroid hormone-regulated gene transcription in the pituitary and brain [J].Environmental Health Perspective,2008,116(12):1694-1699.

[11] D INGEMANSM M L,DE GROOTA,VAN KLEEF R GD M, et al. Hydroxylation Increases the neurotoxic potential of BDE-47 to Affect exocytosis and calcium Homeostasis in PC12 Cells[J].Environmental Health Perspectives,2008,116(5):637-643.

[12] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the p rincip le of p rontein-dye binding[J].Analytical Biochemistry,1976,72(7):248-254.

(編輯:劉志強(qiáng))

Effects of 6-hydroxylated Polybrom inated D iphenyl Ether s-47 on Anti-Oxidative System s of A549 Cells

ZHAO Jin-yu, ZHONG Yu-fang, WANG Yi-pei, WANG Yang-jun,
AN Jing, SHENG Guo-ying, FU Jia-mo(Institute of Environmental Pollution and Health,School of Environmental and Chemical Engineering,Shanghai University,Shanghai200444,China)

R 994

A

1007-2861(2010)03-0302-04

10.3969/j.issn.1007-2861.2010.03.017

2009-02-13

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973計(jì)劃)資助項(xiàng)目(2008CB418205);上海市重點(diǎn)學(xué)科建設(shè)資助項(xiàng)目(S30109)

安 靜 (1976～),女,副研究員,博士,研究方向?yàn)榄h(huán)境毒理學(xué).E-mail:peace74839@shu.edu.cn