評價飲水DBPs致HepG2細(xì)胞損傷的2種試驗方法

張紹慧,陳 釗,廖 靜,魏 巍,劉愛林,魯文清

(華中科技大學(xué)同濟醫(yī)學(xué)院,勞動衛(wèi)生與環(huán)境衛(wèi)生學(xué)系,環(huán)境與健康教育部重點實驗室,湖北 武漢 430030)

評價飲水DBPs致HepG2細(xì)胞損傷的2種試驗方法

張紹慧,陳 釗,廖 靜,魏 巍,劉愛林,魯文清*

(華中科技大學(xué)同濟醫(yī)學(xué)院,勞動衛(wèi)生與環(huán)境衛(wèi)生學(xué)系,環(huán)境與健康教育部重點實驗室,湖北 武漢 430030)

應(yīng)用結(jié)晶紫染色法細(xì)胞毒性試驗和胞質(zhì)分裂阻滯法微核試驗(CBMNT)評價飲水消毒副產(chǎn)物(DBPs)的HepG2細(xì)胞毒性和遺傳毒性,探討該2種試驗方法納入飲水安全性評價體系的可行性.根據(jù)遺傳毒性和致癌性選用了5種DBPs,分別對HepG2細(xì)胞進(jìn)行染毒,結(jié)果表明,5種DBPs的細(xì)胞毒性大小排序為3-氯-4-二氯甲基-5-羥基-2(5氫)-呋喃酮(MX)>二氯乙腈>二溴乙酸>二氯乙酸>三氯乙酸,其細(xì)胞毒性潛力(%C?值)分別為89.74,283.81,1217.02,5066.81,9335.38μmol/L,該5種DBPs在一定濃度下均可致HepG2細(xì)胞微核率顯著增加,表現(xiàn)出明顯的遺傳毒性,其遺傳損傷能力排序與細(xì)胞毒性大小相同.可考慮將這兩種實驗方法納入到飲水安全性評價體系中.

消毒副產(chǎn)物；細(xì)胞毒性試驗；微核試驗

Abstract：Cytotoxicity and genotoxicity of drinking water disinfection by-products (DBPs) in HepG2 cells was to evaluate using crystal violet-colorimetric assay and the cytokinesis-block micronucleus test, and the feasibility of introducing these assays into evaluation system for drinking water safty was explored. HepG2 cells were treated with five DBPs which were chosen based on their genotoxicity and carcinogenicity. The descending rank order in cytotoxicity was 3- chloro -4 - (dichloromethyl) -5 hydroxy -2 (5 H) – furanone (MX) > dichloroacetonitrile (DCAN) > dibromoacetic acid (DBA) > dichloroacetic acid ( DCA ) > trichloroacetic acid (TCA), with a value of cytotoxic potency (% C ? values) ranged from 89.74 (MX) to 9335.38 (TCA) μmol/L. Micronuclei occurring frequency significantly increased in HepG2 cells treated with five DBPs. The rank order of their genotoxicity among the five DBPs was the same as that of their cytotoxicity. The results indicated that these two assays are feasible for assessing the drinking water safety.

Key words：disinfection by-products；cytotoxicity assay；micronucleus assay

在飲水消毒處理過程中,消毒劑與水中的有機物,如腐殖酸、富里酸、藻類等易發(fā)生取代、加成和氧化反應(yīng),能生成鹵代烴類等消毒副產(chǎn)物(DBPs).有報道的DBPs已達(dá)600多種[1],其中一些 DBPs已被證實具有遺傳毒性,某些甚至有致癌性[2].因此對飲水可能導(dǎo)致的潛在遺傳毒性進(jìn)行檢測對飲水安全性評價具有重要意義.現(xiàn)有關(guān)于飲水 DBPs暴露的遺傳毒性評價方法中所采用的受試生物種類較少且遺傳學(xué)觀察終點單一,不能準(zhǔn)確鑒別遺傳毒物與非遺傳毒物.建立一套多受試系統(tǒng)和多觀察終點的遺傳毒理學(xué)組合實驗用于評價飲水安全性顯得尤為迫切.本研究選取健康風(fēng)險較高的5種DBPs[1],以人類來源的肝腫瘤細(xì)胞HepG2為靶細(xì)胞,應(yīng)用結(jié)晶紫染色法和胞質(zhì)分裂阻滯法微核試驗評價 DBPs的細(xì)胞毒性和遺傳毒性, 為將該2種試驗方法納入到飲水安全性評價體系的可行性提供依據(jù).

1 材料與方法

1.1 材料

HepG2細(xì)胞由荷蘭萊登大學(xué)Darroudi博士惠贈;5種DBPs(表1),苯并(a)芘[B(a)P],細(xì)胞松弛素B和二甲基亞砜(DMSO)均為Sigma公司產(chǎn)品; DMEM高糖培養(yǎng)基、新生牛血清和胰蛋白酶均為Gibco公司產(chǎn)品.

表1 5種DBPs在水中的濃度、遺傳毒性和致癌性Table 1 Summary of occurrence, genotoxicity, and carcinogenicity of five DBPs

1.2 細(xì)胞毒性試驗

處于對數(shù)生長期細(xì)胞經(jīng) 0.25%胰蛋白酶消化后,接種于 96孔細(xì)胞培養(yǎng)板.預(yù)實驗可知每孔接種4000個細(xì)胞、經(jīng)72h培養(yǎng)后細(xì)胞可基本單層鋪滿孔底.三氯乙酸、二氯乙酸和二溴乙酸溶于D-hank’s液中;二氯乙腈和MX溶于DMSO中.溶劑的濃度不超過1%.各DBPs設(shè)8～10個染毒濃度組、1個陰性對照組(含有細(xì)胞+培養(yǎng)基+血清)和 1個空白對照組(僅含有培養(yǎng)基+血清),每組設(shè)5個平行孔.各孔總體積為200μL.接種后置于37℃、5%CO2培養(yǎng)箱中.培養(yǎng)72h后,細(xì)胞經(jīng)PBS清洗2次,甲醇固定,1%結(jié)晶紫染液染色.去離子水沖洗后晾干,每孔加 10%的冰乙酸.于595nm的波長下檢測吸光度值,結(jié)果以相對陰性對照組的生長率表示[3].

1.3 胞質(zhì)分裂阻滯法微核試驗(CBMNT)

細(xì)胞接種于6孔細(xì)胞培養(yǎng)板(2×105個/孔)中,補加培養(yǎng)基至2mL,于37℃,5%CO2培養(yǎng)箱中培養(yǎng)24h后染毒.所有受試物均設(shè)4個濃度,陽性對照為B(a)P(100μmol/L),陰性對照同細(xì)胞毒性試驗.染毒之前用PBS沖洗細(xì)胞2次,加入新鮮培養(yǎng)基.各受試物的染毒時間均為24h.染毒結(jié)束后更換新鮮培養(yǎng)基,加入 4μL細(xì)胞松弛素B(3μg/mL),混勻后孵育24h,將細(xì)胞轉(zhuǎn)入離心管,離心后加入5mL預(yù)冷的KCl.再經(jīng)多次固定后制片.以10%Giemsa染色 10min,沖洗晾干后封片.鏡下觀察計數(shù)細(xì)胞,計算核分裂指數(shù)(NDI)和微核率(MN‰)[4].

1.4 統(tǒng)計分析

試驗結(jié)果輸入 EXCEL表格后,應(yīng)用 SPSS 11.5統(tǒng)計分析軟件.采用χ2檢驗對微核率進(jìn)行統(tǒng)計分析;核分裂指數(shù)使用Dunnett’t檢驗.

2 結(jié)果

2.1 5種DBPs致HepG2的72h細(xì)胞毒性

由圖1可見,在各DBPs的劑量反應(yīng)曲線中,隨著染毒濃度的增加,相對陰性對照組的生長率逐漸下降.

圖1 DBPs致HepG2細(xì)胞72h細(xì)胞毒性Fig.1 The cytotoxic effect of DBPs on HepG2 cells after 72h treatment

2.2 5種 DBPs誘導(dǎo)的 HepG2細(xì)胞微核率和NDI

由表2可見,隨著DBPs染毒濃度的增加,各染毒濃度水平出現(xiàn)的微核率均高于同組的陰性對照值,同時NDI值均出現(xiàn)下降趨勢,即反映該染毒劑量致細(xì)胞毒性損傷在增強.且微核率與NDI值這兩項指標(biāo)與DBPs染毒濃度具有一定的劑量-反應(yīng)關(guān)系.各DBPs誘導(dǎo)HepG2細(xì)胞微核率與陰性對照相比出現(xiàn)統(tǒng)計學(xué)意義的最低濃度值相差較大,具體的數(shù)值為:100μmol/L(MX),200μmol/L(DCN),500μmol/L (DBA),1000μmol/L (DCA),5000μmol/L (TCA).

表2 5種DBPs對HepG2細(xì)胞微核率和NDI的影響Table 2 NDI and MN in HepG2 cells induced by 5 DBPs

表2 5種DBPs對HepG2細(xì)胞微核率和NDI的影響Table 2 NDI and MN in HepG2 cells induced by 5 DBPs

注:a.微核率的統(tǒng)計方法為χ2檢驗;b.NDI的統(tǒng)計方法為Dunnett’t 檢驗;n.d.細(xì)胞毒性過大,細(xì)胞大量死亡而無法計數(shù)1000個雙核細(xì)胞;*與陰性對照相比較,微核率變化具有統(tǒng)計學(xué)意義, P<0.05;#與陰性對照相比較,NDI變化具有統(tǒng)計學(xué)意義,P<0.05

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3 討論

HepG2細(xì)胞作為一種代謝完全的人類來源細(xì)胞,保留了一系列生物轉(zhuǎn)化過程中的I相和II相酶,已經(jīng)成為評價化合物遺傳毒性的有力工具[5].結(jié)晶紫染色觀察細(xì)胞相對活性具有操作簡便且靈敏、準(zhǔn)確等特點[6].微核試驗可對人類、嚙齒類動物的致癌、致突變物進(jìn)行有效鑒別, Galloway等[7]對49種化學(xué)物應(yīng)用小鼠骨髓細(xì)胞微核試驗進(jìn)行致癌性篩查,只有4種致癌物得不到陽性結(jié)果.Tinwell等[8]研究后發(fā)現(xiàn),大部分人類致癌物和經(jīng)典的致突變物都會引起小鼠骨髓細(xì)胞的微核率顯著增加.由微核試驗發(fā)展而來的CBMNT可有效去除因受試物或不良的細(xì)胞培養(yǎng)狀態(tài)所致的細(xì)胞分裂動力學(xué)改變而引起的假陰性結(jié)果,因而與傳統(tǒng)的微核測定法相比,具有更好的精確度[9].

在結(jié)晶紫染色試驗的基礎(chǔ)上,采用細(xì)胞毒性潛力對 DBPs的相對毒性大小進(jìn)行定量比較.細(xì)胞毒性潛力不僅可反映受試物對細(xì)胞的致死性,還綜合考慮了細(xì)胞生長及分裂周期受到的延遲和干擾作用[3],可用于在多類型靶細(xì)胞間進(jìn)行多種受試物細(xì)胞毒性大小的定量比較.本研究結(jié)果表明,5種DBPs對HepG2細(xì)胞毒性大小與文獻(xiàn)報道的該 5種 DBPs對靶細(xì)胞 CHO[3,10]和 S. typhimurium[11]的細(xì)胞毒性大小排序基本一致,進(jìn)一步驗證了本研究結(jié)果的可靠性.

在遺傳毒性試驗研究中,5種DBPs誘導(dǎo)的微核率隨染毒濃度的增大均呈上升趨勢,并最終達(dá)到使 HepG2細(xì)胞遺傳毒性明顯增強的效應(yīng). 其中DCA在1000μmol/L、TCA在5000μmol/L的濃度下即可獲得致HepG2細(xì)胞的陽性遺傳毒性結(jié)果.Plewa等[3]和Mark等[10]應(yīng)用SCGE/CHO試驗體系檢測DBPs的遺傳毒性時, DCA和TCA在 染 毒 劑 量 分 別 達(dá) 到 25000μmol/L 和5000μmol/L時也未發(fā)現(xiàn)其遺傳毒性作用,本研究采用的CBMNT/ HepG2試驗體系對檢出DBPs的遺傳毒性似乎更為靈敏.在以往其他一些遺傳毒性試驗中呈現(xiàn)陰性結(jié)果的 TCA[1]在本次試驗中也獲得陽性結(jié)果,表明以HepG2細(xì)胞為靶細(xì)胞的試驗方法可能會提高 DBPs遺傳毒性的檢出效率.根據(jù)5種DBPs出現(xiàn)陽性結(jié)果時的最小染毒濃度,結(jié)合出現(xiàn)陽性結(jié)果時的微核率與各自陰性對照的微核率的比值的大小,對該 5種 DBPs致HepG2細(xì)胞的遺傳毒性潛力進(jìn)行排序,由大到小依次為: MX>DCN>DBA>DCA>TCA,同細(xì)胞毒性大小順序相同.可見 DBPs的細(xì)胞毒性大小與其對靶細(xì)胞造成的遺傳損傷能力成正相關(guān).

4 結(jié)語

結(jié)晶紫染色法細(xì)胞毒性實驗可對5種DBPs的細(xì)胞毒性大小作出很好的定量評價,而CBMNT也可對該5種DBPs造成的遺傳損傷進(jìn)行有效的檢出.據(jù)此,可考慮將以 HepG2細(xì)胞為靶細(xì)胞的這 2種試驗方法納入對飲水安全性進(jìn)行評價的遺傳毒理學(xué)組合實驗中,但還需應(yīng)用到采集的水樣中以獲得進(jìn)一步的驗證.

[1] Susan D R, Michael J P, Elizabeth D W, et al. Occurrence, genotoxicity, and carcinogenicity of emerging disinfection by-products in drinking water: A review and roadmap for research [J]. Mutation Research, 2007,636:178-242.

[2] Komulainen H. Experimental cancer studies of chlorinated by-products [J]. Toxicology, 2004,198:239-248.

[3] Plewa M J, Kargalioglu Y, Vankerk D, et al. Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection by-products [J]. Environmental and Molecular Mutagenesis, 2002,40:134-142.

[4] Lu W Q, Chen X N, Yue F, et al. Studies on the in vivo and in vitro mutagenicity and the lipid peroxidation of chlorinated surface (drinking) water in rats and metabolically competent human cells [J]. Mutation Research, 2002,513:151-157.

[5] Laura Marabini, Silvia Frigerio, Enzo Chiesara, et al. Toxicity evaluation of surface water treated with different disinfectants in HepG2 cells [J]. Water Research, 2006,40:267-272.

[6] 楊 浩,鞠冬陽,程希平,等.3種方法檢測體外神經(jīng)細(xì)胞存活的技術(shù)探討 [J]. 生命科學(xué)研究, 2005,9:327-330.

[7] Galloway S M. The micronucleus test and NTP rodent carcinogens: not so many false negatives [J]. Mutation Research, 1996,352:185-188.

[8] Tinwell H, Ashby J. Comparative activity of human carcinogens and NTP rodent carcinogens in the mouse bone marrow micronucleus assay: An integrative approach to genetic toxicity data assessment [J]. Environmental Health Perspectives, 1994, 102:758-762.

[9] Fenech M. The in vitro micronucleus technique [J]. Mutat. Res., 2000,455:81-95.

[10] Mark G M, Elizabeth D W, Kristin M, et al. Haloacetonitriles vs. regulated haloacetic acids: Are nitrogen-containing DBPs more toxic? [J]. Environ. Sci. Technol., 2007,41:645-651.

[11] Kargalioglu Y, McMillan B J, Minear R A, et al. An analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium [J]. Teratog. Carcinog. Mutagen., 2002,22:113-128.

Application of two assays for damage assessment of damage caused by drinking water disinfection by-products in HepG2 cells.

ZHANG Shao-hui, CHEN Zhao, LIAO Jing, WEI Wei, LIU Ai-lin, LU Wen-qing*

(Key Laboratory of Environment and Health, Ministry of Education, Department of Occuptional and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China). China Environmental Science, 2010,30(2)：275～278

X503,X824

A

1000-6923(2010)02-0275-04

2009-07-14

“十一五”國家科技支撐計劃(2006BAI19B02)

* 責(zé)任作者, 教授, luwq@mails.tjmu.edu.cn

張紹慧(1976-),男,遼寧錦州人,華中科技大學(xué)同濟醫(yī)學(xué)院博士研究生,主要從事環(huán)境遺傳毒理學(xué)研究.