5種鹵代乙酰胺類消毒副產(chǎn)物對(duì)小鼠淋巴瘤細(xì)胞Tk基因致突變性研究

袁婷婷，王新亮，董慧峪

1.北京醫(yī)院檢驗(yàn)科，北京100730

2.天津市城市規(guī)劃設(shè)計(jì)研究院，天津300201

3.中國(guó)科學(xué)院生態(tài)環(huán)境研究中心，北京100085

5種鹵代乙酰胺類消毒副產(chǎn)物對(duì)小鼠淋巴瘤細(xì)胞Tk基因致突變性研究

袁婷婷1，王新亮2，董慧峪3,*

1.北京醫(yī)院檢驗(yàn)科，北京100730

2.天津市城市規(guī)劃設(shè)計(jì)研究院，天津300201

3.中國(guó)科學(xué)院生態(tài)環(huán)境研究中心，北京100085

鹵代乙酰胺類(HAcAm)消毒副產(chǎn)物(DBPs)是飲用水中新興含氮類DBPs之一。為探究HAcAms的致突變性,選擇小鼠淋巴瘤細(xì)胞作為受試細(xì)胞,分別考察了一氯代乙酰胺、二氯代乙酰胺、三氯代乙酰胺、一碘代乙酰胺、二碘代乙酰胺(DIAcAms)共5種HAcAms對(duì)小鼠淋巴瘤細(xì)胞Tk基因突變的影響。結(jié)果表明,在0.05～20μmol·L-1暴露濃度下,5種HacAms類DBPs對(duì)小鼠淋巴瘤細(xì)胞呈現(xiàn)出一定的細(xì)胞毒性,隨著暴露濃度的升高,細(xì)胞毒性增強(qiáng)。暴露4 h后,5種HacAms中只有DIAcAms呈現(xiàn)出顯著的致突變性(暴露劑量高于5μmol·L-1),且以小集落為主。上述研究結(jié)果為研究HacAms對(duì)哺乳動(dòng)物細(xì)胞的致突變作用提供了基礎(chǔ)數(shù)據(jù)。

鹵代乙酰胺；小鼠淋巴瘤細(xì)胞；突變分析

飲用水消毒副產(chǎn)物(DBPs)是飲用水加氯消毒過程生成的微量污染物。部分DBPs對(duì)人或哺乳動(dòng)物具有致癌、致畸形、致突變“三致”作用,三者之間緊密聯(lián)系,其中突變包括基因突變和染色體畸變,是癌癥和畸形產(chǎn)生的重要危險(xiǎn)因素?；贒BPs在飲用水中檢出率、檢出水平和毒理學(xué)研究結(jié)果,世界衛(wèi)生組織、美國(guó)環(huán)境環(huán)保署分別對(duì)飲用水中的14種、11種DBPs提出限量控制要求,我國(guó)《生活飲用水衛(wèi)生標(biāo)準(zhǔn)》也將13種DBPs納入控制范圍(包括三鹵甲烷類、鹵乙酸類、鹵酸鹽、鹵乙腈等)。近年來隨著污染物解析技術(shù)的發(fā)展和毒理學(xué)研究的深入,發(fā)現(xiàn)未受控DBPs可能對(duì)人體健康造成危害的風(fēng)險(xiǎn)更大,由此未受控DBPs日益受到關(guān)注。

作為一種新型的鹵代含氮DBPs,鹵代酰胺類(HAcAms)DBPs已在歐美國(guó)家得到廣泛重視[1]。一氯乙酰胺(CAcAm)、一溴乙酰胺、二氯乙酰胺(DCA-cAm)、二溴乙酰胺和三氯乙酰胺(TCAcAm)均已在美國(guó)飲用水出廠水中檢出,最高濃度可達(dá)幾十μg·L-1[2]。已有毒理學(xué)研究表明,HAcAms的細(xì)胞毒性是鹵乙酸(HAAs)的102倍左右,遺傳毒性是HAAs的20倍左右[3],HAcAms細(xì)胞毒性顯著高于受控類三鹵甲烷(THMs)、HAAs,然而HAcAms對(duì)哺乳動(dòng)物細(xì)胞的毒性與致突變性尚未有文獻(xiàn)報(bào)道,開展HAcAms對(duì)哺乳動(dòng)物細(xì)胞突變的研究對(duì)全面了解其“三致”風(fēng)險(xiǎn),保障人民群眾安全用水具有重要的意義[4-5]。

小鼠淋巴瘤細(xì)胞Tk基因突變?cè)囼?yàn)是一種具有較高的檢出靈敏度、較廣的檢測(cè)譜且簡(jiǎn)便易行的體外短期致突變?cè)囼?yàn)方法[6]。該方法可檢出發(fā)生于Tk位點(diǎn)的點(diǎn)突變、小缺失,同時(shí)還能檢出包括Tk位點(diǎn)在內(nèi)的大缺失、易位、重組甚至染色體非整倍性等廣泛的遺傳學(xué)改變。歐美國(guó)家均已將其列為一組必選的遺傳毒性測(cè)試,我國(guó)亦將其列為保健食品、藥品、化妝品等安全性毒理學(xué)評(píng)價(jià)中的必選實(shí)驗(yàn)之一。本研究采用小鼠淋巴瘤細(xì)胞Tk基因突變?cè)囼?yàn)檢驗(yàn)5種HAcAms類DBPs的致突變性,為水體中HAcAms類DBPs引起的健康風(fēng)險(xiǎn)提供毒理學(xué)依據(jù),同時(shí)亦可為Tk基因突變?cè)囼?yàn)檢驗(yàn)在水體中微量污染物風(fēng)險(xiǎn)評(píng)估中的應(yīng)用積累數(shù)據(jù)資料。

1 材料與方法(Materials and methods)

1.1 實(shí)驗(yàn)材料

實(shí)驗(yàn)用小鼠淋巴瘤細(xì)胞L178Ytk+/-3.7.2C由日本國(guó)立衛(wèi)生研究所提供。5種受試HAcAms詳細(xì)信息如表1所示。實(shí)驗(yàn)用陽(yáng)性對(duì)照甲磺酸甲酯(MMS,98%)購(gòu)自Sigma Aldrich(美國(guó))公司,二甲基亞砜(DMSO,99%)購(gòu)自中國(guó)國(guó)藥有限公司。

表1 5種鹵代乙酰胺類消毒副產(chǎn)物(DBPs)信息Table 1 Detailed information of the five investigated haloacetamides(HacAms)

表2 5種鹵代乙酰胺類DBPs的毒性與致突變性Table 2 Toxicity and mutagenicity of five HAcAms in mouse lymphoma cells

1.2 實(shí)驗(yàn)方法

基礎(chǔ)培養(yǎng)基(RPMI-0)由RPMI 1640中添加200 g·mL-1丙酮酸鈉,100 U·mL-1青霉素和100μg·mL-1鏈霉素；處理用培養(yǎng)基(RPMI-5)、常規(guī)培養(yǎng)用培養(yǎng)基(RPMI-10)和集落生長(zhǎng)用培養(yǎng)基(RPMI-20)分別有基礎(chǔ)培養(yǎng)基入5%、10%或20%的馬血清(56℃、30 min滅活)制成。L5178Y細(xì)胞液氮下保存,復(fù)蘇后常規(guī)開放式懸浮培養(yǎng)于RPMI-10,在37℃、5%CO2條件下培養(yǎng)至穩(wěn)定的對(duì)數(shù)增長(zhǎng)狀態(tài),細(xì)胞密度維持在1×106·mL-1以下,使用時(shí)傳代不應(yīng)超過10代。在與正式試驗(yàn)相同條件下處理細(xì)胞,表達(dá)培養(yǎng)2 d后測(cè)定相對(duì)懸浮增長(zhǎng)率(relative suspension growth, RSG)。在20%～100%RSG范圍內(nèi)以等比或等差設(shè)定4個(gè)劑量。另設(shè)溶劑對(duì)照(DMSO)和陽(yáng)性對(duì)照(MMS,10μg·mL-1)。受試物處理細(xì)胞用RPMI-5調(diào)整細(xì)胞密度為106·mL-1(每組20mL)。按1%體積加入受試物、溶劑對(duì)照或陽(yáng)性對(duì)照,振蕩處理4 h。處理結(jié)束后,1 000 r·min-1離心5 min,棄上清液,用PBS洗滌細(xì)胞一次,重懸于RPMI-10中,并調(diào)整細(xì)胞密度至2×105·mL-1。

d0平板接種效率(plating efficiency,PE0):將處理后的細(xì)胞用RPMI-20梯度稀釋至8個(gè)·mL-1,25 mL(陰性對(duì)照為50 mL)。以200μL·孔-1(1.6個(gè)細(xì)胞·孔-1)接種到一塊96孔培養(yǎng)板(陰性對(duì)照接種2塊)。培養(yǎng)12 d后計(jì)數(shù)每塊培養(yǎng)板有集落生長(zhǎng)的孔數(shù)。d2平板接種效率(plating efficiency,PE2):剩余細(xì)胞作表達(dá)培養(yǎng)2 d。每天計(jì)數(shù)細(xì)胞密度,調(diào)整至2×105·mL-1,并計(jì)算每日細(xì)胞增長(zhǎng)率(daily cell growth, DCG),據(jù)此求算2 d內(nèi)RSG。PE2接種方法同PE0。

TFT抗性突變頻率(mutant frequency,MF)的測(cè)定:表達(dá)培養(yǎng)結(jié)束后,取適量細(xì)胞,用RPMI-20調(diào)整密度至1×104·mL-1,50 mL(陰性對(duì)照組為100 mL),加入三氟胸甘(TFT),使其濃度為2μg·mL-1。以200μL·孔-1(2 000個(gè)細(xì)胞·孔-1)接種2塊96孔培養(yǎng)板(陰性對(duì)照組接種4塊)。培養(yǎng)12 d后分別計(jì)數(shù)含有大集落(large colony,LC)的孔數(shù)、只含小集落(small colony,SC)的孔數(shù)、同時(shí)含有LC和SC的孔數(shù)。大小集落的區(qū)分標(biāo)準(zhǔn):LC大小超過每孔直徑的1/4,呈薄層分布；SC大小在每孔直徑的1/4以下,呈塊狀,結(jié)構(gòu)致密。

1.3 數(shù)據(jù)統(tǒng)計(jì)與分析

數(shù)據(jù)統(tǒng)計(jì)分析使用SPSS 19.0(IBM,USA)進(jìn)行。采用ANOVA方法分析HAcAms暴露組與空白對(duì)照組之間的差異,P＜0.05表示差異顯著；P＜0.01表示差異極顯著。

圖1 不同HAcAms劑量作用下Tk基因總突變頻率及小集落相對(duì)含量Fig.1 Effect of HAcAms dose on the mutant frequency ofTkgene and relative amount of small colony

2 結(jié)果(Results)

2.1 細(xì)胞毒性

當(dāng)HAcAms的暴露劑量高于50μmol·L-1時(shí),部分RTG低于本測(cè)試方法最低限定值(20%)[7],因此本文中5種HAcAms的暴露劑量均控制在0.05～20μmol·L-1范圍內(nèi)。如表2所示,小鼠淋巴瘤細(xì)胞相對(duì)懸浮生長(zhǎng)率(RSG)、RTG均隨著HAcAms劑量的增大而降低。當(dāng)CAcAm、DCAcAm、TCAcAm、IA-cAm、DIAcAm的劑量為20μmol·L-1時(shí),小鼠淋巴瘤細(xì)胞的RSG分別為55%、53%、58%、47%、41%, RTG值分別為48%、42%、35%、38%、28%,顯示出較強(qiáng)的細(xì)胞毒性,其中IAcAm、DIAcAm的RSG、RTG值均低于氯代 HAcAms的值,表明碘代HAcAms的細(xì)胞毒性高于氯代HAcAms。

2.2 5種HAcAms作用下Tk基因總突變頻率及小集落相對(duì)含量

5種HAcAms作用下Tk基因總突變頻率及小集落相對(duì)含量變化如表2、圖1所示。MMS陽(yáng)性對(duì)照的結(jié)果表明,當(dāng)MMS暴露劑量為10μg·mL-1Tk基因總突變頻率顯著提升且具有統(tǒng)計(jì)學(xué)意義(P＜0.001)。隨著5種HAcAms暴露劑量的增大,Tk基因總突變頻率均呈現(xiàn)出升高的趨勢(shì)。但只有DIA-cAm暴露劑量達(dá)到5μmol·L-1、20μmol·L-1時(shí),Tk基因總突變頻率與溶劑對(duì)照差異才呈現(xiàn)出統(tǒng)計(jì)學(xué)意義(P＜0.05),其誘發(fā)突變頻率分別達(dá)到自發(fā)突變頻率的4倍及9倍。對(duì)DIAcAm各暴露劑量與總突變頻率做線性關(guān)系回歸分析,有較好的劑量響應(yīng)關(guān)系(P＜0.05)。在觀察到大集落(LC)、與小集落(SC)2種表型的突變集落中,小SC的豐度多高于LC豐度。其中DIAcAm暴露劑量為5μmol·L-1、20μmol·L-1時(shí),SC的相對(duì)豐度可分別達(dá)到 60.14%、67.14%,與Liviac等[8]觀測(cè)到DBPs暴露下小鼠淋巴瘤細(xì)胞大小集落豐度分布結(jié)果一致。

3 討論(Discussion)

飲用水中DBPs的長(zhǎng)期暴露可能帶來的健康風(fēng)險(xiǎn)已成為飲用水安全保障的研究熱點(diǎn)之一。已有流行病學(xué)的研究調(diào)查了DBPs長(zhǎng)期暴露與癌癥、生殖健康狀況(先天性畸形、死胎、流產(chǎn)、出生體重、成熟度和精液質(zhì)量)等的關(guān)聯(lián)情況[9-11]。其中已發(fā)現(xiàn)了DBPs的濃度水平與膀胱癌發(fā)病概率的相關(guān)性,但生殖健康與DBPs的關(guān)聯(lián)尚未有定論[13]。本文采用哺乳動(dòng)物細(xì)胞為受體細(xì)胞,考察了5種HAcAms暴露下的Tk基因總突變頻率,發(fā)現(xiàn)5種HAcAms均呈現(xiàn)出一定的細(xì)胞毒性,其中DIAcAm具有統(tǒng)計(jì)學(xué)意義的致突變性。在高劑量(20μmol·L-1)暴露下, Tk基因總突變頻率為DIAcAm＞IAcAm＞CAcAm＞TCAcAm≈DCAcAm,Plewa等[14]曾報(bào)道過13種HAcAms的慢性細(xì)胞毒性(以中華倉(cāng)鼠卵巢細(xì)胞為受體),其中DIAcAm＞IAcAm＞CAcAm＞DCA-cAm＞TCAcAm,與本文毒性水平分布總體一致。

MX(3-chloro-4-(dichloromethyl)-5-hydroxy-2 (5H)-furanone)是一種氯化呋喃酮類物質(zhì),美國(guó)、英國(guó)、日本、中國(guó)均從氯化消毒的自來水中檢出MX[15]。盡管MX在氯化飲水中濃度一般為在幾十個(gè)ng·L-1級(jí)別,但可占氯化飲水有機(jī)提取物總誘變性的50%左右[16]。本研究根據(jù)美國(guó)環(huán)境保護(hù)署基于QSAR計(jì)算的T.E.S.T.數(shù)據(jù)庫(kù)中的5種HAcAms與MX的致突變數(shù)據(jù)進(jìn)行比較,所得相對(duì)毒性數(shù)據(jù)如圖2所示。其中DIAcAm的相對(duì)致突變性最高(0.87),CAcAm最低(0.39),總體趨勢(shì)與本文實(shí)驗(yàn)結(jié)果相一致。

碘代DBPs的細(xì)胞毒性與遺傳毒性通常均高于氯代DBPs與溴代DBPs[17],本文研究結(jié)果發(fā)現(xiàn)碘代HAcAms的致突變性顯著高于氯代HAcAms,進(jìn)一步證實(shí)了碘代DBPs的高“三致”作用。已有研究證明,濾后水中的碘離子、含碘微量有機(jī)物及食鹽中的碘離子均有可能成為碘代DBPs的碘源,進(jìn)而生成高毒性碘代DBPs[18][19]。飲用水中碘代DBPs的生成規(guī)律、濃度分布及毒性評(píng)估需引起足夠重視,以有效控制其帶來的飲用水健康風(fēng)險(xiǎn)。

圖2 基于QSAR計(jì)算的5種HAcAms致突變相對(duì)值(數(shù)據(jù)源自USEPA T.E.S.T.數(shù)據(jù)庫(kù)，以3-氯-4 -(二氯甲基)-5-羥基-2(5H)-呋喃(MX)為基準(zhǔn)物)Fig.2 Mutagenicity of five HAcAms based on the calculation of QSAR (Relative mutagenicity value is calculated using USEPA T.E.S.T. database values and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone is selected as the reference compound).

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Mutagenic Analysis of Five Haloacetamides Disinfection By-products in the Tk Gene of Mouse Lymphoma Cells

Yuan Tingting1,Wang Xinliang2,Dong Huiyu3,*
1.LaboratoryDepartment,Beijing Hospital,Beijing 100730,China
2.Tianjin Urban Planning&Design Institute,Tianjin 300201,China
3.ResearchCenter for Eco-environmental Sciences,Chinese Academy of Sciences,Beijing 100085,China

30 November 2015 accepted 11 January 2016

Haloacetamide(HAcAm)is one of the emerging nitrogeneous disinfection by-products(DBPs)in drinking water.To evaluate the mutagenic potential of HAcAm,effects of 5 HAcAms including chloroacetamide,dichloroacetamide,trichloroacetamide,iodoacetamide and diiodoacetamide(DIAcAm)on thymidine kinase(Tk)gene in the well-validated mouse lymphoma assay were investigated in this study.The results showed that with the exposure levels from 0.05 to 20μmol·L-1,all the 5 HAcAms investigated exhibited cytotoxicity,which increased with the elevated exposure level.After 4 h of exposure,only DIAcAm increased the frequency of mutant colonies,with a higher proportion of small colonies.These results of HAcAms provided basic data for the evaluation of mutagen-ic potential to mammalian cells.

haloacetamide;mouse lymphoma cells;mutation assay

2015-11-30 錄用日期:2016-01-11

1673-5897(2016)1-153-06

X171.5

A

10.7524/AJE.1673-5897.20151130022

袁婷婷,王新亮,董慧峪.5種鹵代乙酰胺類消毒副產(chǎn)物對(duì)小鼠淋巴瘤細(xì)胞Tk基因致突變性研究[J].生態(tài)毒理學(xué)報(bào),2016,11(1):153-158

Yuan T T,Wang X L,Dong H Y.Mutagenic analysis of five haloacetamides disinfection by-products in theTkgene of mouse lymphoma cells[J].Asian Journal of Ecotoxicology,2016,11(1):153-158(in Chinese)

國(guó)家自然科學(xué)基金課題(51408590)

袁婷婷(1985-),女,檢驗(yàn)醫(yī)師,研究方向?yàn)榧?xì)胞生物毒理學(xué),E-mail:ttyuan.ok@163.com；

),E-mail:hydong@rcees.ac.cn

簡(jiǎn)介:董慧峪(1983-)，男，環(huán)境工程博士，助理研究員，主要研究方向水體安全消毒與輸配，發(fā)表學(xué)術(shù)論文10余篇。