低劑量雙酚A影響哺乳動(dòng)物神經(jīng)發(fā)育研究現(xiàn)狀及爭(zhēng)議

呂　琳,董夢(mèng)琦,秦占芬*

低劑量雙酚A影響哺乳動(dòng)物神經(jīng)發(fā)育研究現(xiàn)狀及爭(zhēng)議

呂琳1,2,董夢(mèng)琦1,2,秦占芬1,2*

(1.中國科學(xué)院生態(tài)環(huán)境研究中心,環(huán)境化學(xué)與生態(tài)毒理學(xué)國家重點(diǎn)實(shí)驗(yàn)室,北京 100085；2.中國科學(xué)院大學(xué),北京 100049)

為全面認(rèn)識(shí)低劑量BPA(雙酚A)對(duì)哺乳動(dòng)物神經(jīng)發(fā)育的影響,本文從中國知網(wǎng)、PubMed和Web of Science 3個(gè)數(shù)據(jù)庫中獲取了國內(nèi)外關(guān)于低劑量BPA影響哺乳動(dòng)物神經(jīng)發(fā)育的研究報(bào)道,并使用toxR工具對(duì)其可信度進(jìn)行評(píng)估,最終篩選獲得26項(xiàng)相關(guān)研究;從美國食品藥品監(jiān)督管理局(FDA)工作網(wǎng)站上獲取最新BPA毒性報(bào)告1篇.通過比較分析這些研究,發(fā)現(xiàn)大部分研究報(bào)道低劑量BPA暴露導(dǎo)致哺乳動(dòng)物神經(jīng)行為、特定腦區(qū)內(nèi)組織學(xué)結(jié)構(gòu)和細(xì)胞特征、神經(jīng)遞質(zhì)和激素穩(wěn)態(tài)、腦中關(guān)鍵基因表達(dá)以及表觀遺傳特征發(fā)生改變.但是,就導(dǎo)致的神經(jīng)行為的改變而言,一些研究的結(jié)果并不一致甚至有諸多矛盾之處.這些不一致的結(jié)果可能與動(dòng)物實(shí)驗(yàn)設(shè)計(jì)的差異有關(guān),其中神經(jīng)行為測(cè)試的質(zhì)量控制和統(tǒng)計(jì)方法中統(tǒng)計(jì)單元的選擇對(duì)于研究結(jié)果的影響尤其值得關(guān)注.總之,從目前的文獻(xiàn)來看,低劑量BPA對(duì)哺乳動(dòng)物神經(jīng)發(fā)育的影響尚待進(jìn)一步的確認(rèn).

雙酚A；腦發(fā)育；發(fā)育神經(jīng)毒性；行為測(cè)試；質(zhì)量控制

雙酚A(BPA)為高產(chǎn)量化學(xué)品,主要用于合成聚碳酸酯、環(huán)氧樹脂等高分子材料,后者用來制造各類消費(fèi)品,如食品容器或者食品容器的內(nèi)涂層.另外,單體的BPA在熱敏紙、化妝品、個(gè)人護(hù)理品、衣物、玩具等消費(fèi)品中也有應(yīng)用.通過日常使用接觸這些消費(fèi)品、攝入被污染的食物等途徑,普通人群每天持續(xù)暴露BPA,據(jù)估算BPA的日攝入量在0.01～ 4.5μg/kg體重.因此,在血液、尿液、胎盤、母乳等人體樣品中普遍有BPA存在[1-3],血清中濃度甚至可達(dá)mmol水平[4].因此,BPA的毒性效應(yīng)和健康影響一直受到關(guān)注.

神經(jīng)毒性研究報(bào)道,哺乳動(dòng)物發(fā)育過程中暴露BPA會(huì)導(dǎo)致其在后期出現(xiàn)神經(jīng)行為異常,且伴隨關(guān)鍵腦區(qū)細(xì)胞和分子特征的改變[5-7].同時(shí),有人群流行病學(xué)調(diào)查數(shù)據(jù)顯示,兒童焦慮、注意力缺陷等神經(jīng)行為的異常與母親孕期尿液中的BPA濃度相關(guān)[8],這些人群數(shù)據(jù)為動(dòng)物實(shí)驗(yàn)結(jié)果提供了支持.為此,一些研究人員主張BPA具有神經(jīng)發(fā)育毒性,應(yīng)該加強(qiáng)監(jiān)管.但是,來自化學(xué)品監(jiān)管部門的數(shù)據(jù)認(rèn)為,低劑量的BPA沒有包括發(fā)育神經(jīng)毒性在內(nèi)的明顯的毒性效應(yīng).當(dāng)綜合評(píng)估BPA毒性效應(yīng)的已有文獻(xiàn)時(shí),監(jiān)管部門依舊堅(jiān)持認(rèn)為,雖然有數(shù)據(jù)提示BPA具有潛在的發(fā)育神經(jīng)毒性及其他毒性的風(fēng)險(xiǎn),但同時(shí)存在不一致的結(jié)果,需要進(jìn)一步的研究[9].盡管如此,本著預(yù)防的原則,目前加拿大、美國、歐盟等一些國家或地區(qū)已經(jīng)禁止了BPA在嬰兒奶瓶中使用,但更多的產(chǎn)品中仍然有BPA的大量使用.

為全面認(rèn)識(shí)BPA發(fā)育神經(jīng)毒性研究現(xiàn)狀,探討科學(xué)界、監(jiān)管界關(guān)于BPA安全性爭(zhēng)議的原因,有必要對(duì)相關(guān)文獻(xiàn)進(jìn)行系統(tǒng)分析.本文在中國知網(wǎng)、PubMed及Web of Science數(shù)據(jù)庫中,以“雙酚A& 神經(jīng)發(fā)育毒性/腦/行為”或“bisphenol A & Developmental neurotoxicity/brain/behavior”為關(guān)鍵詞進(jìn)行檢索,以美國食品藥品監(jiān)督管理局(FDA)規(guī)定的每日容許攝入量(ADI)50μg/kg·bw/天為標(biāo)準(zhǔn)[10],篩選關(guān)注該劑量或低于該劑量的BPA哺乳動(dòng)物神經(jīng)發(fā)育毒性相關(guān)文獻(xiàn).為了確定所得文獻(xiàn)的可信度,使用ToxR工具[11]對(duì)所得文獻(xiàn)進(jìn)行評(píng)估,最終獲得26篇符合標(biāo)準(zhǔn)的文獻(xiàn)(見表1).其中,有6篇文獻(xiàn)缺少對(duì)實(shí)驗(yàn)動(dòng)物只數(shù)的表述,但在綜合評(píng)估其文獻(xiàn)內(nèi)容后,認(rèn)為其結(jié)果較為可信,可以用于文分析討論.此外,從美國食品藥品監(jiān)督管理局(FDA)工作網(wǎng)站上獲取最新BPA毒性報(bào)告1篇.在此基礎(chǔ)上,本文綜述BPA對(duì)哺乳動(dòng)物行為、腦組織中細(xì)胞行為、腦中關(guān)鍵效應(yīng)分子、腦中關(guān)鍵基因表達(dá)以及表觀遺傳修飾的影響相關(guān)報(bào)道,并對(duì)部分研究結(jié)果存在差異的原因進(jìn)行討論.

表1 低劑量雙酚A暴露對(duì)哺乳動(dòng)物神經(jīng)發(fā)育的影響

續(xù)表1

注:表中暴露劑量單位均為μg/(kg·d);GD表示妊娠天數(shù),PND表示產(chǎn)后天數(shù).

1 低劑量BPA哺乳動(dòng)物發(fā)育神經(jīng)毒性研究現(xiàn)狀

1.1 低劑量BPA對(duì)哺乳動(dòng)物神經(jīng)行為的影響

神經(jīng)行為異常是化學(xué)品神經(jīng)發(fā)育毒性的直觀效應(yīng)之一.一些動(dòng)物研究顯示,發(fā)育過程中接觸低劑量BPA會(huì)導(dǎo)致動(dòng)物行為學(xué)指標(biāo)發(fā)生改變.孕期-哺乳期是哺乳動(dòng)物腦發(fā)育的關(guān)鍵時(shí)期,所以幾個(gè)研究集中在這一時(shí)期對(duì)孕鼠進(jìn)行低劑量BPA持續(xù)暴露,待子鼠進(jìn)入青春期或成年后對(duì)其進(jìn)行行為學(xué)測(cè)試. Change及其團(tuán)隊(duì)通過灌胃方式對(duì)Sprague- Dawley大鼠進(jìn)行了40μg/(kg·d) BPA暴露,將其子鼠飼養(yǎng)至成年,發(fā)現(xiàn)BPA暴露組的子鼠在莫里斯水迷宮試驗(yàn)(MWM)中逃脫時(shí)間增長(zhǎng),指示子鼠空間記憶能力受損[12].Poimenova等[13]則令Wistar大鼠在這一時(shí)期通過飲食途徑攝入相同劑量BPA,對(duì)子鼠進(jìn)行Y迷宮訓(xùn)練試驗(yàn),發(fā)現(xiàn)其進(jìn)入新異臂的次數(shù)顯著減少,這同樣指示了空間記憶能力的損傷.除此之外,Chang的團(tuán)隊(duì)還發(fā)現(xiàn)雌性子鼠同時(shí)出現(xiàn)了焦慮樣行為,這表明BPA還使動(dòng)物出現(xiàn)了情緒異常[12].類似地,Kumar等人對(duì)小鼠進(jìn)行50μg/(kg·d)劑量的BPA灌胃,在曠場(chǎng)試驗(yàn)中發(fā)現(xiàn)雄性子鼠出現(xiàn)焦慮樣行為,雌性子鼠卻未出現(xiàn)明顯的焦慮反應(yīng)[14].Jones等[15]則在Long-Evans大鼠的食物中加入BPA,使其攝入劑量為5μg/(kg·d),然后在高架迷宮(EPM)中分別觀察雌性子鼠和雄性子鼠的表現(xiàn),發(fā)現(xiàn)BPA消除了兩性子鼠在EPM中表現(xiàn)的性別差異,這可能指示了BPA對(duì)動(dòng)物焦慮反應(yīng)性別差異的影響.另外,Dessi- Fulgheri等[16]還觀察到了雌性子鼠社交等行為的雄性化.這些研究均認(rèn)為,孕期-哺乳期持續(xù)接觸BPA會(huì)導(dǎo)致子鼠出現(xiàn)神經(jīng)行為的異常,且部分影響可能是性別相關(guān)的.

除孕期-哺乳期持續(xù)暴露外,僅在發(fā)育過程中某一階段接觸BPA的子鼠也出現(xiàn)了行為學(xué)指標(biāo)的改變.Gioiosa等[17]在圍產(chǎn)期通過飲食途徑對(duì)母鼠進(jìn)行10μg/(kg·d)的BPA暴露,發(fā)現(xiàn)其子鼠在新異性探索行為測(cè)試、曠場(chǎng)測(cè)試以及EPM中的性別差異消失. Zhang等[18]則在哺乳期對(duì)母鼠進(jìn)行了0.5μg/(kg·d)的BPA皮下注射后,同樣觀察到子鼠在Y迷宮中表現(xiàn)出空間記憶能力的下降.另外,有四項(xiàng)研究分別在青春期對(duì)子鼠進(jìn)行了40μg/(kg·d) BPA暴露.其中, Della Seta等[19]觀察了雄性子鼠性成熟后的生殖行為,發(fā)現(xiàn)其嗅探行為減少,Bowman團(tuán)隊(duì)[20]通過對(duì)象放置試驗(yàn)發(fā)現(xiàn)子鼠的空間記憶能力下降,Tandon等[21]通過條件及被動(dòng)回避測(cè)試得到了同樣的結(jié)論. Xu等[22]則在曠場(chǎng)試驗(yàn)、EPM和MWM中觀察到了子鼠多種行為的性別差異消除[22].這些研究均顯示,BPA在關(guān)鍵發(fā)育階段暴露對(duì)動(dòng)物神經(jīng)行為產(chǎn)生了影響.

值得注意的是,盡管上述研究中都出現(xiàn)了某些行為學(xué)測(cè)試的陽性結(jié)果,但是也存在某些陰性的結(jié)果,尤其在這些研究之間,存在諸多矛盾之處(表2).Ferguson等[23]在孕期哺乳期對(duì)母鼠進(jìn)行了一系列濃度的BPA灌胃,在曠場(chǎng)試驗(yàn)、迷宮試驗(yàn)中均未觀察到BPA對(duì)子鼠行為產(chǎn)生任何顯著影響;Rebuli團(tuán)隊(duì)[24]以同樣的方案進(jìn)行暴露,在EPM中也未觀察到BPA的影響.同樣使用MWM和EPM進(jìn)行測(cè)試時(shí),使用灌胃方法進(jìn)行暴露的Chang團(tuán)隊(duì)[13]觀察到BPA影響了鼠在MWM中的表現(xiàn),但未影響其在EPM中的行為,而使用飲食暴露的Jones[15]卻觀察到了完全相反的現(xiàn)象.Poimenova等[12]的實(shí)驗(yàn)中,雌性小鼠出現(xiàn)焦慮樣行為,Kumar等[14]的研究卻顯示雄性小鼠出現(xiàn)焦慮、雌性小鼠無顯著性差異.另外, Xu報(bào)道了青春期BPA暴露會(huì)導(dǎo)致鼠出現(xiàn)社交行為改變[22],但Della Seta等[19]卻未觀察到這一現(xiàn)象.

表2 低劑量BPA暴露對(duì)哺乳動(dòng)物行為影響的實(shí)驗(yàn)結(jié)果差異

總體來說,已有研究顯示BPA對(duì)動(dòng)物神經(jīng)行為存在潛在的影響,但目前并未得到一致的結(jié)論,其具體效應(yīng)仍需進(jìn)一步研究.

1.2 BPA對(duì)哺乳動(dòng)物特定腦區(qū)內(nèi)組織學(xué)結(jié)構(gòu)和細(xì)胞特征的影響

除神經(jīng)行為學(xué)指標(biāo)外,腦組織形態(tài)以及其中細(xì)胞特征也是指示腦發(fā)育的重要指標(biāo).神經(jīng)元突觸是神經(jīng)系統(tǒng)中的功能單位,其數(shù)量與形態(tài)的正常與否指示了腦發(fā)育過程是否受到干擾.海馬是腦中負(fù)責(zé)記憶、學(xué)習(xí)功能的主要結(jié)構(gòu),其中神經(jīng)元形態(tài)是評(píng)價(jià)化學(xué)品神經(jīng)發(fā)育毒性的指標(biāo)之一.Kimura等[25]在GD8-18對(duì)孕鼠進(jìn)行40μg/(kg·d) BPA暴露后,觀察到子鼠海馬中突觸密度下降、分支減少,且這一改變?cè)谄涑赡旰笠廊淮嬖?如前所述,Zhang等[18]通過行為學(xué)測(cè)試觀察到BPA損傷鼠的空間記憶能力,與此同時(shí),該研究也觀察到了海馬中突觸密度的改變[18]. Bowman等[20]報(bào)道了類似的發(fā)現(xiàn),并認(rèn)為這二者是相關(guān)的.

神經(jīng)干細(xì)胞(NSCs)的增殖及分化異常可能說明腦發(fā)育過程受到了干擾.一些研究使用BrdU、Ki67等標(biāo)記正在增殖的NSCs,結(jié)果顯示,發(fā)育過程中低劑量BPA暴露會(huì)抑制腦中神經(jīng)干細(xì)胞的增殖[26-29],Nakamura等[29]的研究顯示這種抑制出現(xiàn)在子鼠接觸藥物一段時(shí)間后.BPA還可能影響NSCs的命運(yùn)決定,Tiwari的團(tuán)隊(duì)進(jìn)行了兩項(xiàng)研究,發(fā)現(xiàn)BPA造成子鼠海馬和腦室下區(qū)(SVZ)中神經(jīng)源性標(biāo)記物含量降低,認(rèn)為BPA抑制了NSCs向神經(jīng)譜系的分化[26-27].Tandon等[21]通過標(biāo)記髓磷脂等髓鞘標(biāo)記物發(fā)現(xiàn),BPA干擾了海馬中髓鞘形成過程.Kumar等[14]則通過標(biāo)記不同類型神經(jīng)突觸發(fā)現(xiàn),BPA造成子鼠大腦皮層中興奮性和抑制性突觸比例改變,他們認(rèn)為這是子鼠出現(xiàn)焦慮樣行為的原因.

從已有研究來看,低劑量BPA在組織、細(xì)胞層面影響腦發(fā)育過程,主要表現(xiàn)為突觸形成改變和NSCs增殖及分化受到干擾.

1.3 BPA對(duì)哺乳動(dòng)物神經(jīng)遞質(zhì)和激素的影響

神經(jīng)遞質(zhì)的合成及代謝穩(wěn)態(tài)是評(píng)估腦功能的指標(biāo)之一,有研究報(bào)道了BPA對(duì)這一過程產(chǎn)生了干擾.幾項(xiàng)研究分別對(duì)處于重要發(fā)育時(shí)期的鼠進(jìn)行低劑量BPA暴露后,在其青春期或成年后檢測(cè)了其血清或腦組織中幾種重要神經(jīng)遞質(zhì)的水平,發(fā)現(xiàn)BPA會(huì)影響動(dòng)物體內(nèi)的神經(jīng)遞質(zhì)水平,例如多巴胺[30]、5-羥色胺[18,31]、谷氨酸[18]、乙酰膽堿[18]等.部分研究還發(fā)現(xiàn),這種遞質(zhì)水平的變化同時(shí)伴隨著其與其代謝產(chǎn)物比例的改變,因此,BPA可能干擾了腦中神經(jīng)遞質(zhì)的代謝穩(wěn)態(tài)[30-31].另外,也有研究顯示了這種干擾的性別相關(guān)性[18].

目前,BPA被認(rèn)為具有內(nèi)分泌干擾作用,其對(duì)腦中重要激素的作用也是神經(jīng)發(fā)育毒性關(guān)注的指標(biāo).Fahrenkopf和Wagner的研究顯示,孕期BPA暴露會(huì)以ER依賴的方式誘導(dǎo)小鼠大腦視前內(nèi)側(cè)核中細(xì)胞的孕激素受體表達(dá),說明BPA干擾了腦中雌激素的作用過程[32].Fernandez等[33]則發(fā)現(xiàn)BPA會(huì)影響子鼠血清中的甲狀腺素(T4)水平,但對(duì)三碘甲狀腺原氨酸(T3)水平無顯著影響.

1.4 BPA對(duì)哺乳動(dòng)物腦中關(guān)鍵基因表達(dá)及重要信號(hào)通路的影響

有研究在孕期及哺乳期對(duì)孕鼠進(jìn)行環(huán)境劑量BPA暴露后將其子鼠飼養(yǎng)至成年,然后檢測(cè)腦中關(guān)鍵基因的表達(dá)水平.據(jù)報(bào)道,BPA對(duì)多種基因的表達(dá)水平產(chǎn)生了干擾.Malloy等[34]發(fā)現(xiàn)小鼠在孕期-哺乳期通過飲食攝入BPA后,其大腦中關(guān)鍵基因如鉀電壓門控通道蛋白Kcnq1、DNA甲基轉(zhuǎn)移酶Dnmt1、tet甲基胞嘧啶雙加氧酶Tet1/2的表達(dá)水平上調(diào),而這些基因的表達(dá)水平可能與膠質(zhì)細(xì)胞分化相關(guān). Tandon及其團(tuán)隊(duì)對(duì)大鼠進(jìn)行BPA灌胃后檢查了其大腦基因表達(dá)水平,發(fā)現(xiàn)其中少突膠質(zhì)細(xì)胞標(biāo)記物PLP、Olig1、MBP等分子表達(dá)水平降低[21].除大腦外,Cao等[35-36]還發(fā)現(xiàn)BPA對(duì)下丘腦雌激素受體相關(guān)基因表達(dá)水平產(chǎn)生影響.

發(fā)育過程中,不同時(shí)空位置、不同種類的細(xì)胞中的基因表達(dá)受到多種信號(hào)通路的調(diào)節(jié).在腦發(fā)育過程中,較為重要的信號(hào)通路包括Wnt、Notch、BMP等.研究人員推測(cè),如果這些信號(hào)通路受到干擾,腦發(fā)育就可能受到影響.Tiwari團(tuán)隊(duì)[26-27]的研究顯示,BPA對(duì)腦中的Wnt信號(hào)通路產(chǎn)生干擾,改變了其靶基因的表達(dá)水平,且這種干擾并非只是分子層面的,其最終造成了海馬神經(jīng)干細(xì)胞的異常增殖;當(dāng)使用外源物質(zhì)逆轉(zhuǎn)這種干擾時(shí),這種異常增殖也隨之消失;研究認(rèn)為,BPA確實(shí)影響了腦細(xì)胞中的Wnt信號(hào)通路,并由此干擾了腦的發(fā)育.Tandon等[21]則在觀察到BPA影響鼠行為以及髓鞘形成的同時(shí)發(fā)現(xiàn), BPA改變了大腦中Notch信號(hào)通路相關(guān)基因的表達(dá)水平,如Notch受體Notch1、配體Jagged1、靶基因Hes1等,這說明BPA干擾了腦中的Notch信號(hào)通路.

綜合來看,在基因表達(dá)調(diào)控層面,BPA對(duì)腦發(fā)育產(chǎn)生了一定的影響.

1.5 BPA對(duì)哺乳動(dòng)物腦細(xì)胞中表觀遺傳修飾的影響

腦發(fā)育過程中,DNA甲基化、組蛋白修飾等表觀遺傳修飾機(jī)制能夠影響神經(jīng)干細(xì)胞命運(yùn)決定等過程[37],還參與了腦的性二態(tài)分化[38].Yaoi等[39]對(duì)鼠進(jìn)行低劑量BPA暴露后對(duì)其腦細(xì)胞進(jìn)行了限制性標(biāo)記基因組掃描(RLGS),發(fā)現(xiàn)其中多個(gè)基因啟動(dòng)子CpG島甲基化水平改變.Kumar等[40]也關(guān)注了BPA對(duì)大腦表觀遺傳修飾的改變,發(fā)現(xiàn)BPA不僅降低了大腦皮層和海馬中的DNA甲基化水平,還升高了組蛋白乙酰化水平.由此可見,發(fā)育過程中的BPA接觸會(huì)導(dǎo)致動(dòng)物腦中表觀遺傳修飾水平改變.

綜上所述,現(xiàn)有低劑量BPA神經(jīng)發(fā)育毒性研究顯示,特定腦區(qū)內(nèi)組織學(xué)結(jié)構(gòu)和細(xì)胞特征、神經(jīng)遞質(zhì)和激素穩(wěn)態(tài)、腦中關(guān)鍵基因表達(dá)以及表觀遺傳特征等非經(jīng)典神經(jīng)毒性指標(biāo)均受到BPA影響.但是,經(jīng)典的神經(jīng)毒性指標(biāo),即神經(jīng)行為的相關(guān)研究所得結(jié)論存在較大分歧.因此從目前的文獻(xiàn)來看,低劑量BPA對(duì)神經(jīng)發(fā)育的影響仍需進(jìn)一步研究明確.

2 BPA研究結(jié)果的不一致性分析

盡管眾多科學(xué)研究均報(bào)道了BPA對(duì)動(dòng)物的腦發(fā)育過程具有干擾作用,但這一觀點(diǎn)并未完全被化學(xué)品監(jiān)管部門認(rèn)可.歐盟化學(xué)品管理局(ECHA)、美國食品藥品監(jiān)督管理局(FDA)等化學(xué)品監(jiān)管機(jī)構(gòu)分別綜合審查了BPA神經(jīng)發(fā)育毒性的相關(guān)研究報(bào)道后,在BPA毒性報(bào)告中都陳述了其潛在的神經(jīng)發(fā)育毒性風(fēng)險(xiǎn),但并未因此提升BPA的管控力度.目前,FDA在官方網(wǎng)站上標(biāo)稱BPA是“安全(SAFE)”的.這與大部分科學(xué)研究得到的結(jié)論相反.化學(xué)品監(jiān)管機(jī)構(gòu)得出這一結(jié)論的主要原因是,現(xiàn)有的BPA神經(jīng)發(fā)育毒性研究結(jié)果之間存在分歧.很多因素可能會(huì)導(dǎo)致這種差異性結(jié)果的出現(xiàn),具體分析如下:

第一,實(shí)驗(yàn)動(dòng)物品系可能是影響因素之一.首先,不同品系的鼠之間認(rèn)知能力存在差異,這導(dǎo)致其在迷宮試驗(yàn)等行為學(xué)測(cè)試中的表現(xiàn)差異較大,會(huì)影響神經(jīng)毒理學(xué)實(shí)驗(yàn)的結(jié)果[41].另外,不同品系鼠的腦發(fā)育過程對(duì)于不同物質(zhì)的敏感性也有所不同.例如,美國國家環(huán)境健康科學(xué)研究所(NIEHS)和FDA共同發(fā)起了“學(xué)術(shù)界和監(jiān)管機(jī)構(gòu)對(duì)雙酚A毒性的聯(lián)合研究”(CLARITY-BPA)項(xiàng)目,并與毒理學(xué)研究人員開展了合作研究.與先前的研究不同,這些研究未觀察到BPA對(duì)動(dòng)物非生殖行為產(chǎn)生影響.但同時(shí),他們也未在所設(shè)置的對(duì)照組中觀察到預(yù)期的性別差異[42].經(jīng)過分析,研究人員認(rèn)為美國國家毒理學(xué)中心(NCTR)供給的Sprague-Dawley大鼠在行為學(xué)上與其他品系大鼠存在差異,其可能對(duì)性別相關(guān)的干擾不敏感.可見,動(dòng)物品系的選擇會(huì)影響得到的結(jié)果[42-43].在本文綜述的26篇低劑量BPA神經(jīng)發(fā)育毒性相關(guān)研究中,有16篇使用大鼠作為研究對(duì)象[11-12,14-15,17-20,22-23,25,27,31-32,34-35],10篇使用小鼠進(jìn)行研究[13,16,21,24,28-30,33,38-39],大鼠、小鼠分別包含不同的品系,這可能帶來結(jié)果的分歧.

第二,不同的暴露方式可能會(huì)影響毒理學(xué)實(shí)驗(yàn)的結(jié)果.本文綜述研究中,有8篇[17,19,28-30,32,34,38]采取皮下注射的方式進(jìn)行暴露,而有18篇通過口服途徑進(jìn)行暴露.這兩種不同的暴露途徑可能導(dǎo)致實(shí)際攝入BPA的量不同.因此,即使暴露相同劑量的BPA,動(dòng)物腦組織最終的BPA及其代謝產(chǎn)物水平可能不一致,這可能導(dǎo)致動(dòng)物受到的影響產(chǎn)生差異.在口服暴露的研究中,使用灌胃方法[12-13,15,18,20-25,27,35,39]的研究有13項(xiàng),通過飲食暴露[11,14,16,31,33]的有5項(xiàng).這3種方法中,食物暴露和飲水暴露不易確定動(dòng)物實(shí)際接觸的藥物量,可能會(huì)導(dǎo)致結(jié)果受到影響.而灌胃暴露已經(jīng)被證明會(huì)給動(dòng)物造成強(qiáng)大的壓力,這種壓力對(duì)動(dòng)物行為的影響甚至可能超過BPA的影響[43-44].這可能是采用該種暴露方式的研究報(bào)道陰性結(jié)果的原因之一.

第三,在毒理學(xué)研究中,對(duì)不同處理組的動(dòng)物進(jìn)行指標(biāo)檢測(cè)及數(shù)據(jù)統(tǒng)計(jì)時(shí)應(yīng)以“窩”為單位,并在同一處理組保證足夠的窩數(shù),以此排除遺傳背景帶來的誤差.但是,在現(xiàn)有的BPA神經(jīng)發(fā)育毒性研究中,僅有11項(xiàng)明確統(tǒng)計(jì)單元為“窩”[15-16,18,22-23,30-32,34-35,38],其余研究均以“只”[11-12,14,17,19,21,24,28-29]為單元或描述不清[13,20,25,27,39].不同遺傳背景的動(dòng)物可能在行為學(xué)測(cè)試中表現(xiàn)不同,不合適的統(tǒng)計(jì)方法可能會(huì)造成假陽性或是假陰性的結(jié)果出現(xiàn).

第四,本文在綜述現(xiàn)有相關(guān)研究后發(fā)現(xiàn),即使是不存在上述問題的研究得到的結(jié)果依然存在差異,尤其是行為學(xué)測(cè)試的結(jié)果.這可能是由于行為學(xué)測(cè)試受到的影響因素較為復(fù)雜,本身就不易得到高度一致的重復(fù).現(xiàn)有研究顯示,動(dòng)物飼養(yǎng)條件的細(xì)微不同、實(shí)驗(yàn)時(shí)環(huán)境噪音的不同、實(shí)驗(yàn)人員數(shù)量及相對(duì)位置的不同均會(huì)影響動(dòng)物在行為學(xué)測(cè)試中的表現(xiàn).這些細(xì)節(jié)一般不會(huì)在研究報(bào)告中完整體現(xiàn),而兩項(xiàng)實(shí)驗(yàn)設(shè)計(jì)看似完全相同的研究可能因此得到不同的結(jié)果[42].另外,現(xiàn)有的行為學(xué)測(cè)試往往被認(rèn)為指示了動(dòng)物的某一項(xiàng)認(rèn)知功能.例如,鼠在莫里斯水迷宮中的逃生時(shí)間增加被認(rèn)為是空間記憶能力損害的表現(xiàn).但事實(shí)上,這些測(cè)試中的指標(biāo)并非僅由一項(xiàng)認(rèn)知功能決定.Tanila在綜合評(píng)估鼠的行為學(xué)試驗(yàn)報(bào)道后發(fā)現(xiàn),在莫里斯水迷宮試驗(yàn)中,如果鼠出現(xiàn)焦慮情緒,其游泳速度會(huì)加快、逃生時(shí)間變短,而這可能被誤認(rèn)為是空間記憶能力較好的表現(xiàn)[42].一些非認(rèn)知功能受到影響也會(huì)影響鼠在行為學(xué)試驗(yàn)中的表現(xiàn),例如嗅覺、運(yùn)動(dòng)能力等.Karl等人在綜述了鼠神經(jīng)毒理學(xué)相關(guān)研究后認(rèn)為,毒理學(xué)中不一致的行為測(cè)試結(jié)果可能來自未被關(guān)注到的健康指標(biāo)[44].

總體來說,現(xiàn)有的發(fā)育神經(jīng)毒性測(cè)試方法受到的影響因素非常復(fù)雜,包括動(dòng)物品系、暴露方式、統(tǒng)計(jì)方式等,行為學(xué)測(cè)試本身易受干擾的特點(diǎn)也是因素之一.這些都可能是BPA神經(jīng)發(fā)育毒性研究結(jié)論不一致的原因.

3 結(jié)論與展望

3.1 結(jié)論

大部分研究顯示,發(fā)育過程中低劑量BPA暴露會(huì)導(dǎo)致哺乳動(dòng)物神經(jīng)發(fā)育受損,但其中行為學(xué)結(jié)果存在差異,其可能與實(shí)驗(yàn)設(shè)計(jì)的差異相關(guān),尤其是統(tǒng)計(jì)單元的選擇和行為學(xué)測(cè)試質(zhì)量控制.

3.2 展望

目前,低劑量BPA神經(jīng)發(fā)育毒性仍需進(jìn)一步明確.未來,在研究BPA對(duì)神經(jīng)系統(tǒng)發(fā)育的影響時(shí)應(yīng)著重關(guān)注實(shí)驗(yàn)設(shè)計(jì),例如使用“窩”為統(tǒng)計(jì)單元以排除遺傳背景的影響,使用飲食途徑等對(duì)動(dòng)物行為影響較小的暴露方式等,以明確低劑量BPA對(duì)腦發(fā)育的不良效應(yīng)及其產(chǎn)生效應(yīng)的濃度.在進(jìn)行行為學(xué)測(cè)試時(shí),應(yīng)盡量使用自動(dòng)化設(shè)備以排除實(shí)驗(yàn)人員的影響,并綜合評(píng)估包括動(dòng)物本身健康狀況在內(nèi)的多項(xiàng)指標(biāo)以得出更準(zhǔn)確的結(jié)論.

[1] Micha?owicz J. Bisphenol A – Sources, toxicity and biotransformation [J]. Environmental Toxicology and Pharmacology, 2014,37(2):738- 758.

[2] Vandenberg L N, Hauser R, Marcus M, et al. Human exposure to bisphenol A (BPA) [J]. Reproductive Toxicology, 2007,24(2):139-177.

[3] Andra S S, Charisiadis P, Arora M, et al. Biomonitoring of human exposures to chlorinated derivatives and structural analogs of bisphenol A [J]. 2015,85:352-379.

[4] Yang J, Wang H, Du H, et al. Serum Bisphenol A, glucose homeostasis, and gestational diabetes mellitus in Chinese pregnant women: A prospective study [J]. Environmental Science and Pollution Research, 2020.https://doi.org/10.1007/s11356-020-11263-4.

[5] Itoh K, Yaoi T, Fushiki S. Bisphenol A, an endocrine-disrupting chemical, and brain development [J]. Neuropathology, 2012,32(4): 447-457.

[6] Mhaouty-Kodja S, Belzunces L P, Canivenc M-C, et al. Impairment of learning and memory performances induced by BPA: Evidences from the literature of a MoA mediated through an ED [J]. Molecular and Cellular Endocrinology, 2018,475:54-73.

[7] Wolstenholme J T, Rissman E F, Connelly J J. The role of bisphenol A in shaping the brain, epigenome and behavior [J]. Hormones and Behavior, 2011,59(3):296-305.

[8] Grohs M N, Reynolds J E, Liu J, et al. Prenatal maternal and childhood bisphenol a exposure and brain structure and behavior of young children [J]. Environmental Health, 2019,18(1):85.

[9] FDA. Updated Review of Literature and Data on Bisphenol A [R/OL]. https://www.fda.gov/food/food-additives-petitions/bisphenol-bpa, 2021.

[10] Beronius A, Johansson N, Ruden C, et al. The influence of study design and sex-differences on results from developmental neurotoxicity studies of bisphenol A, implications for toxicity testing [J]. Toxicology, 2013,311(1/2):13-26.

[11] Segal D, Makris S L, Kraft A D, et al. Evaluation of the ToxRTool's ability to rate the reliability of toxicological data for human health hazard assessments [J]. Regulatory Toxicology and Pharmacology, 2015,72(1):94-101.

[12] Chang H, Wang M, Xia W, et al. Perinatal exposure to low-dose bisphenol A disrupts learning/memory and DNA methylation of estrogen receptor alpha in the hippocampus [J]. Toxicology Research, 2016,5(3):828-835.

[13] Poimenova A, Markaki E, Rahiotis C, et al. Corticosterone-regulated actions in the rat brain are affected by perinatal exposure to low dose of bisphenol A [J]. Neuroscience, 2010,167(3):741-749.

[14] Kumar D, Thakur M K. Anxiety like behavior due to perinatal exposure to Bisphenol-A is associated with decrease in excitatory to inhibitory synaptic density of male mouse brain [J]. Toxicology, 2017, 378:107-113.

[15] Jones B A, Watson N V. Perinatal BPA exposure demasculinizes males in measures of affect but has no effect on water maze learning in adulthood [J]. Hormones and Behavior, 2012,61(4):605-610.

[16] Dessi-Fulgheri F, Porrini S, Farabollini F. Effects of perinatal exposure to bisphenol A on play behavior of female and male juvenile rats [J]. Environmental Health Perspectives, 2002,110:403-407.

[17] Gioiosa L, Fissore E, Ghirardelli G, et al. Developmental exposure to low-dose estrogenic endocrine disruptors alters sex differences in exploration and emotional responses in mice [J]. Hormones and Behavior, 2007,52(3):307-316.

[18] Zhang H, Kuang H, Luo Y, et al. Low-dose bisphenol A exposure impairs learning and memory ability with alterations of neuromorphology and neurotransmitters in rats [J]. Science of the Total Environment, 2019,697:134036.

[19] Della Seta D, Minder I, Belloni V, et al. Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats [J]. Hormones and Behavior, 2006,50(2):301-307.

[20] Bowman R E, Hagedorn J, Madden E, et al. Effects of adolescent Bisphenol-A exposure on memory and spine density in for ovariectomized female rats: Adolescence vs adulthood [J]. Hormones and Behavior, 2019,107:26-34.

[21] Tandon A, Singh S J, Gupta M, et al. Notch pathway up-regulation via curcumin mitigates bisphenol-A (BPA) induced alterations in hippocampal oligodendrogenesis [J]. Journal of Hazardous Materials, 2020,392:122052.

[22] Xu X, Tian D, Hong X, et al. Sex-specific influence of exposure to bisphenol-A between adolescence and young adulthood on mouse behaviors [J]. Neuropharmacology, 2011,61(4):565-573.

[23] Ferguson S A, Law C D, Jr., Abshire J S. Developmental treatment with bisphenol A or ethinyl estradiol causes few alterations on early preweaning measures [J]. Toxicological Sciences, 2011,124(1):149- 160.

[24] Rebuli M E, Camacho L, Adonay M E, et al. Impact of low-dose oral exposure to bisphenol A (BPA) on juvenile and adult rat exploratory and anxiety behavior: A CLARITY-BPA Consortium Study [J]. Toxicological Sciences, 2015,148(2):341-354.

[25] Kimura E, Matsuyoshi C, Miyazaki W, et al. Prenatal exposure to bisphenol A impacts neuronal morphology in the hippocampal CA1region in developing and aged mice [J]. Archives of Toxicology, 2016,90(3):691-700.

[26] Tiwari S K, Agarwal S, Seth B, et al. Inhibitory effects of bisphenol-A on neural stem cells proliferation and differentiation in the rat brain are dependent on Wnt/beta-catenin pathway [J]. Molecular Neurobiology, 2015,52(3):1735-1757.

[27] Tiwari S K, Agarwal S, Tripathi A, et al. Bisphenol-A mediated inhibition of hippocampal neurogenesis attenuated by curcumin via canonical Wnt pathway [J]. Molecular Neurobiology, 2016,53(5): 3010-3029.

[28] Agarwal S, Yadav A, Tiwari S K, et al. Dynamin-related Protein 1Inhibition Mitigates Bisphenol A-mediated Alterations in Mitochondrial Dynamics and Neural Stem Cell Proliferation and Differentiation [J]. Journal of Biological Chemistry, 2016,291(31): 15923-15939.

[29] Nakamura K, Itoh K, Yaoi T, et al. Murine neocortical histogenesis is perturbed by prenatal exposure to low doses of bisphenol A [J]. Journal of Neuroscience Research, 2006,84(6):1197-1205.

[30] Yao J, Wang J, Wu L, et al. Perinatal exposure to bisphenol A causes a disturbance of neurotransmitter metabolic pathways in female mouse offspring: A focus on the tryptophan and dopamine pathways [J]. Chemosphere, 2020,254:126715.

[31] Matsuda S, Matsuzawa D, Ishii D, et al. Perinatal exposure to bisphenol A enhances contextual fear memory and affects the serotoninergic system in juvenile female mice [J]. Hormones and Behavior, 2013,63(5):709-716.

[32] Fernandez M O, Bourguignon N S, Arocena P, et al. Neonatal exposure to bisphenol A alters the hypothalamic-pituitary-thyroid axis in female rats [J]. Toxicology Letters, 2018,285:81-86.

[33] Fernandez M O, Bourguignon N S, Arocena P, et al. Neonatal exposure to bisphenol A alters the hypothalamic-pituitary-thyroid axis in female rats [J]. Toxicology Letters, 2018,285:81-86.

[34] Malloy M A, Kochmanski J J, Jones T R, et al. Perinatal bisphenol A exposure and reprogramming of imprinted gene expression in the adult mouse brain [J]. Frontiers in Genetics, 2019:10.

[35] Cao J, Mickens J A, Mccaffrey K A, et al. Neonatal Bisphenol A exposure alters sexually dimorphic gene expression in the postnatal rat hypothalamus [J]. Neurotoxicology, 2012,33(1):23-36.

[36] Cao J, Rebuli M E, Rogers J, et al. Prenatal bisphenol A exposure alters sex-specific estrogen receptor expression in the neonatal rat hypothalamus and amygdala [J]. Toxicological Sciences, 2013,133(1): 157-173.

[37] Miller F D, Gauthier A S. Timing is everything: Making neurons versus glia in the developing cortex [J]. Neuron, 2007,54(3):357-369.

[38] Westberry J M, Trout A L, Wilson M E. Epigenetic regulation of estrogen receptor alpha gene expression in the mouse cortex during early postnatal development [J]. Endocrinology, 2010,151(2):731-740.

[39] Yaoi T, Itoh K, Nakamura K, et al. Genome-wide analysis of epigenomic alterations in fetal mouse forebrain after exposure to low doses of bisphenol A [J]. Biochemical and Biophysical Research Communications, 2008,376(3):563-567.

[40] Kumar D, Thakur M K. Effect of perinatal exposure to Bisphenol-A on DNA methylation and histone acetylation in cerebral cortex and hippocampus of postnatal male mice [J]. Journal of Toxicological Sciences, 2017,42(3):281-289.

[41] Tanila H. Testing cognitive functions in rodent disease models: Present pitfalls and future perspectives [J]. Behavioural Brain Research, 2018, 352:23-27.

[42] Patisaul H B. Achieving CLARITY on bisphenol A, brain and behaviour [J]. Journal of Neuroendocrinology, 2020,32(1):e12730.

[43] Prins G S, Patisaul H B, Belcher S M, et al. CLARITY-BPA academic laboratory studies identify consistent low-dose Bisphenol A effects on multiple organ systems [J]. Basic & Clinical Pharmacology & Toxicology, 2019,125:14-31.

[44] Karl T, Pabst R, Von Horsten S. Behavioral phenotyping of mice in pharmacological and toxicological research [J]. Experimental and Toxicologic Pathology, 2003,55(1):69-83.

Research progress and controversy of low dose bisphenol A on neurodevelopment in mammals.

Lü Lin1,2, DONG Meng-qi1,2, QIN Zhan -fen1,2*

(1.State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China；2.University of Chinese Academy of Sciences, Beijing 100049, China)., 2021,41(10)：4864～4871

Bisphenol A (BPA) is a high-production volume chemical and ubiquitous in both the environment and the human body. Nowadays, a large number of animal studies have shown that BPA can affect brain development, along with epidemiological data supporting the results from animals. However, other studies, especially from regulatory agencies reported no significant developmental neurotoxicity of low doses of BPA. In order to develop a more comprehensive understanding of the effects of low-dose BPA on mammalian neurodevelopment, we obtained research reports on the effects of low-dose BPA on mammalian neurodevelopment from the three databases of CNKI, PubMed and Web of Science, and used the toxR tool to evaluate its credibility, and finally screened 26 relevant studies; obtained 1 latest BPA toxicity report from the U.S. Food and Drug Administration (FDA) work website. The major studies reported that BPA caused abnormalities in neurobehaviors, histological structure and cellular features in certain specific brain regions, neurotransmitters and hormones, brain key gene expression, and epigenetic modifications in mammals. In terms of neurobehavioral alterations, however, some results were inconsistent and even contradictory, which may be due to some differences in experimental design, especial the quality control of neurobehavioral tests and the choose of statistical unit. Overall, further research is needed to clarify the effects of BPA on neurodevelopment in mammals.

bisphenol A；brain development；developmental neurotoxicity；behavioral test；quality control

X503

A

1000-6923(2021)10-4864-08

呂 琳(1998-),女,北京人,中國科學(xué)院生態(tài)環(huán)境研究中心碩士研究生,主要從事環(huán)境毒理學(xué)研究.

2021-02-25

國家重點(diǎn)研發(fā)計(jì)劃(2018YFA0901103);國家自然科學(xué)基金資助項(xiàng)目(21876196)

* 責(zé)任作者, 研究員, qinzhanfen@rcees.ac.cn