范禮強(qiáng), 鄭關(guān)超, 吳海燕, 郭萌萌, 盧 霞, 李 玉, 譚志軍, 3

貽貝對(duì)麻痹性貝類(lèi)毒素的蓄積代謝研究進(jìn)展

范禮強(qiáng)1, 2, 鄭關(guān)超2, 吳海燕2, 郭萌萌2, 盧 霞1, 李 玉1, 譚志軍2, 3

(1. 江蘇海洋大學(xué)測(cè)繪與海洋信息學(xué)院, 江蘇 連云港 222005; 2. 農(nóng)業(yè)農(nóng)村部水產(chǎn)品質(zhì)量安全檢測(cè)與評(píng)價(jià)重點(diǎn)實(shí)驗(yàn)室, 中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所, 山東 青島 266071; 3. 青島海洋科學(xué)與技術(shù)試點(diǎn)國(guó)家實(shí)驗(yàn)室, 山東 青島 266237)

麻痹性貝類(lèi)毒素(paralytic shellfish toxins, PSTs)能夠?qū)е仑愵?lèi)、魚(yú)類(lèi)等海洋生物染毒或死亡, 危害海洋生態(tài)安全及人類(lèi)健康。其中, 貽貝具有強(qiáng)蓄積性特點(diǎn), 在國(guó)外可作為PSTs的指示性生物。近年來(lái), 我國(guó)因食用貽貝造成多起PSTs中毒事件成為安全關(guān)注重點(diǎn), 因此急需弄清其風(fēng)險(xiǎn)形成機(jī)理。本文通過(guò)綜述我國(guó)近海貽貝等貝類(lèi)中PSTs的風(fēng)險(xiǎn)程度、貽貝對(duì)不同產(chǎn)毒藻來(lái)源的PSTs的蓄積作用, 分析環(huán)境因子對(duì)于蓄積代謝的影響, 深入挖掘貽貝蓄積代謝特異性。進(jìn)而聚焦貽貝代謝階段的表達(dá)調(diào)控過(guò)程、影響因素、轉(zhuǎn)化作用及方式等。未來(lái)應(yīng)側(cè)重于摸清我國(guó)目標(biāo)區(qū)域貽貝等海洋貝類(lèi)中PSTs基礎(chǔ)風(fēng)險(xiǎn)規(guī)律和變化過(guò)程, 并據(jù)此建立區(qū)域性PSTs風(fēng)險(xiǎn)防控技術(shù), 提高我國(guó)貽貝中PSTs風(fēng)險(xiǎn)的主動(dòng)防控能力, 對(duì)于保障消費(fèi)者健康和海洋生態(tài)安全具有積極意義。

貽貝; 蓄積代謝; 免疫; 風(fēng)險(xiǎn)評(píng)價(jià)

近年來(lái), 有害赤潮在世界范圍內(nèi)呈現(xiàn)出頻率增加、區(qū)域擴(kuò)大、危害日益嚴(yán)重的發(fā)展趨勢(shì), 導(dǎo)致貝類(lèi)中貝類(lèi)毒素的安全風(fēng)險(xiǎn)愈加嚴(yán)峻[1]。根據(jù)所引起的中毒癥狀, 可將貝類(lèi)毒素分為: 麻痹性貝類(lèi)毒素(paralytic shellfish toxins, PSTs)、腹瀉性貝類(lèi)毒素(diarrhetic shellfish poisoning, DSP)、神經(jīng)性貝類(lèi)毒素(neurotoxic shellfish poisoning, NSP)、記憶缺失性貝毒(amnesic shellfish poisoning, ASP)和西加魚(yú)毒素(ciguatera fish poisoning, CFP)等[1-2], 其中PSTs是最具代表性的一類(lèi), 分布最廣、毒性最強(qiáng)[2]。PSTs主要由海洋環(huán)境中的亞歷山大藻()、裸甲藻()、盾甲藻()等甲藻及生活在淡水中的藍(lán)綠藻產(chǎn)生[3]。雙殼貝類(lèi)濾食有毒藻類(lèi), 毒素在貝類(lèi)體內(nèi)蓄積或通過(guò)食物鏈作用傳遞至人類(lèi), 引起人類(lèi)食物中毒[1]。全球每年因PSTs導(dǎo)致的中毒事件約2 000起, 占貝類(lèi)毒素中毒事件的64%[4]。其中, 雙殼貝類(lèi)(包括貽貝、扇貝、蛤和牡蠣)是最主要的食物鏈傳播來(lái)源, 也是美國(guó)、加拿大、歐盟等國(guó)家及組織開(kāi)展PSTs管控重點(diǎn)[3], 歐盟限量標(biāo)準(zhǔn)為800 μg STXeq·kg–1[5]。我國(guó)是全球第一貝類(lèi)養(yǎng)殖大國(guó), 同時(shí)也是PSTs有害赤潮高發(fā)國(guó)家之一。

近年來(lái), 我國(guó)沿海地區(qū)多發(fā)、赤潮, 主要分布在渤海(秦皇島)、東海(浙江、福建及南寧)部分近岸海區(qū)[1, 6]。其中, 秦皇島海域自2016年起春季連續(xù)暴發(fā)赤潮, 貽貝中毒素蓄積量最高為52 072.8 μg STXeq·kg–1, 超過(guò)歐盟限量標(biāo)準(zhǔn)(800 μg STXeq·kg–1)的65倍[7], 可見(jiàn)危害之嚴(yán)重性。同年福建近海暴發(fā)赤潮, 其中貽貝中PSTs蓄積含量最高[8-9]。此外Isabel等[10]的研究也表明, 貽貝較其他貝類(lèi)具有更高的蓄積能力, 它也是造成我國(guó)PSTs食物中毒事件的主要物種[11-12]。因此, 評(píng)估貽貝對(duì)PSTs的蓄積代謝特異性, 對(duì)于我國(guó)質(zhì)量安全監(jiān)管具有重要的現(xiàn)實(shí)意義。

貽貝是我國(guó)四大經(jīng)濟(jì)貝類(lèi)之一, 紫貽貝、翡翠貽貝和厚殼貽貝是我國(guó)沿海常見(jiàn)種類(lèi)[13], 最適生長(zhǎng)溫度為5~22 ℃, 鹽度為15~40, 是環(huán)境適應(yīng)能力較強(qiáng)的廣溫廣鹽類(lèi)生物。貽貝屬于濾食性生物, 并且對(duì)不良環(huán)境具有強(qiáng)耐受力, 因此國(guó)內(nèi)外學(xué)者已經(jīng)將其用于海洋重金屬[14-17]、全氟化合物[16]、農(nóng)藥[18-19]等污染物的監(jiān)測(cè)研究中, 作為生物指示物評(píng)估海洋生態(tài)系統(tǒng)的污染狀況[15]。近十年來(lái), 研究人員在實(shí)驗(yàn)室內(nèi)不斷模擬雙殼貝類(lèi)蓄積和清除PSTs過(guò)程, 發(fā)現(xiàn)貽貝相較其他貝類(lèi), 具有蓄積速率快、蓄積量大、清除快等特點(diǎn)[20-21]。這也使得貽貝成為研究PSTs等海洋污染物初始組分的重要指示生物。因此亟需深入探究貽貝在PSTs污染監(jiān)測(cè)過(guò)程中的蓄積清除及代謝轉(zhuǎn)化機(jī)理, 對(duì)于準(zhǔn)確評(píng)價(jià)其安全風(fēng)險(xiǎn)并構(gòu)建有效防控措施具有重要作用。

1 我國(guó)近海貝類(lèi)中麻痹性貝類(lèi)毒素風(fēng)險(xiǎn)

我國(guó)近海PSTs污染由來(lái)已久, 21世紀(jì)初北黃海、福建沿海、長(zhǎng)江口鄰近海域、廣東沿海PSTs污染問(wèn)題較為突出, 中毒事件時(shí)有發(fā)生[1]。近年來(lái), 渤海和福建沿海PSTs污染較為嚴(yán)重[22]。為準(zhǔn)確探究我國(guó)近海貝類(lèi)中PSTs風(fēng)險(xiǎn)程度, 表1總結(jié)了2005— 2019年我國(guó)近海貝類(lèi)中PSTs調(diào)查結(jié)果。其中, 渤海2016年河北秦皇島貽貝產(chǎn)品PSTs含量超標(biāo), 導(dǎo)致10余人中毒且2人死亡。污染調(diào)查中發(fā)現(xiàn)PSTs最高含量達(dá)到52 072.8 μg STXeq·kg–1, 是歐盟限量標(biāo)準(zhǔn) (800 μg STXeq·kg–1)的65倍, 貽貝污染極為嚴(yán)重。之后2017年、2019 年連續(xù)監(jiān)測(cè), 發(fā)現(xiàn)貽貝等貝類(lèi)中PSTs檢出率和殘留量都有所下降[23-24], 但為防止PSTs污染的突然爆發(fā), 應(yīng)針對(duì)貝類(lèi)中PSTs的風(fēng)險(xiǎn)構(gòu)建有效防控措施, 以便準(zhǔn)確認(rèn)知我國(guó)貝類(lèi)中PSTs的殘留風(fēng)險(xiǎn)。黃海海域2007—2008年貝類(lèi)污染調(diào)查中蝦夷扇貝的PSTs檢出率達(dá)到了79.2%, PSTs殘留量為262.0~8 339.6 μg STXeq·kg–1[23, 25]。2016年牡蠣等貝類(lèi)中PSTs最高含量為562.9 μg STXeq·kg–1, 接近歐盟限量標(biāo)準(zhǔn), 亟待引起關(guān)注。東海海域2016—2017年貝類(lèi)中PSTs檢出率和最高殘留量分別為74.2% 和23 293.0 μg STXeq·kg–1[26], 直接導(dǎo)致2017年6月福建漳州發(fā)生數(shù)十人貽貝PSTs中毒[27], 貝類(lèi)中PSTs風(fēng)險(xiǎn)程度極高。在2017年貽貝污染后續(xù)調(diào)查中, 發(fā)現(xiàn)貝類(lèi)中PSTs殘留量降低至71.0 μg STXeq·kg–1, 存在地域性反復(fù)風(fēng)險(xiǎn)。南海海域2005—2006年貝類(lèi)中PSTs檢出率為14.3%, 最高殘留量達(dá)3 700.0 μg STXeq·kg–1, 是限量標(biāo)準(zhǔn)的4.6倍, 污染嚴(yán)重[28-29]。之后的南海貝類(lèi)中PSTs污染調(diào)查中檢出率逐年增加, 2010—2011年是32.9%, 2015年更是達(dá)到了86.0%[30], 但南海貝類(lèi)中PSTs殘留量卻在逐年下降。綜上可見(jiàn), 隨著我國(guó)環(huán)境污染得到有效控制, 有害赤潮由大面積爆發(fā)轉(zhuǎn)為小范圍板塊化暴發(fā)趨勢(shì)。由于有害赤潮后產(chǎn)生的豐富孢囊沉降至表層沉積物, 受環(huán)境影響成為有害赤潮的“種子庫(kù)”, 顯著提高目標(biāo)海區(qū)的PSTs暴發(fā)風(fēng)險(xiǎn)。

表1 不同海域貝類(lèi)中麻痹性貝類(lèi)毒素分布情況

注: -表示無(wú)數(shù)據(jù)

從2005—2019年的全國(guó)近海貝類(lèi)養(yǎng)殖區(qū)PSTs污染調(diào)查數(shù)據(jù)發(fā)現(xiàn): 貝類(lèi)毒素污染存在季節(jié)性差異, 與產(chǎn)毒藻的季節(jié)性生長(zhǎng)有較大關(guān)系[31-32], 尤其是受海水溫度影響顯著[33]。我國(guó)海域南北跨度大, 調(diào)查發(fā)現(xiàn)南海貝類(lèi)中PSTs含量多在冬季或早春達(dá)到最高。東海緯度高于南海, 貝類(lèi)中PSTs檢出率和超標(biāo)率也相應(yīng)推遲到夏季6、7、8月份達(dá)到最高[9, 34], 而黃渤海PSTs污染多在4、5月份暴發(fā)[33, 35]。貽貝作為我國(guó)沿海重要養(yǎng)殖經(jīng)濟(jì)貝類(lèi), 四大海域貽貝的PSTs檢出率和殘留量都較高[36], 這也與貽貝具有蓄積速率快、蓄積量大的研究結(jié)果相一致。為保障我國(guó)貝類(lèi)產(chǎn)業(yè)健康發(fā)展和消費(fèi)者生命安全, 亟需深入探究貽貝中PSTs的蓄積清除及代謝轉(zhuǎn)化規(guī)律, 以便更好的對(duì)貝類(lèi)毒素安全風(fēng)險(xiǎn)進(jìn)行有效管控。

2 貽貝對(duì)麻痹性貝類(lèi)毒素的蓄積、清除特性

貽貝能夠快速蓄積大量PSTs, 具有蓄積速率快、蓄積量大、蓄積率高的特點(diǎn)。如表2中所示, 貽貝蓄積速率最高為1 736.0 μg STXeq?kg–1?d–1, 是其他四種主要經(jīng)濟(jì)貝類(lèi)的3~15倍[37-42]。邱江兵等[42]和Katsushi等[40]PSTs暴露實(shí)驗(yàn)中貽貝的整體蓄積率分別達(dá)到13.6%和14.6%, 高于牡蠣的8.3%、菲律賓蛤仔的2.5%和文蛤的12.4%; 貽貝能夠蓄積大量PSTs, 暴露實(shí)驗(yàn)中貽貝體內(nèi)PSTs含量分別達(dá)到了8 680.0 μg STXeq·kg–1和10 960.0 μg STXeq·kg–1, 約是歐盟限量標(biāo)準(zhǔn)(800 μg STXeq·kg–1)10.6倍和13.7倍。Shumway 等[43]研究表明貽貝神經(jīng)元軸突對(duì)PSTs毒素STX不敏感, 這可能是貽貝PSTs蓄積速率快、蓄積量大的主要原因之一。貽貝組織間蓄積速率和蓄積量存在較大差異[44], 內(nèi)臟團(tuán)是貽貝蓄積PSTs的靶器官, 對(duì)PSTs蓄積速率和蓄積量都遠(yuǎn)高于鰓、閉殼肌、性腺等其他組織[42], 內(nèi)臟中毒素含量有時(shí)甚至可以達(dá)到整體的90%以上[45]。

表2 不同貝類(lèi)對(duì)麻痹性貝類(lèi)毒素的蓄積規(guī)律

貽貝在暴露實(shí)驗(yàn)中能夠快速將PSTs清除到體外, 具有清除速度快、清除率高等特點(diǎn)[17, 42]。根據(jù)Bricelja等[47]研究, 雙殼貝類(lèi)清除毒素速度大致可以分為: 快、中速清除貝類(lèi)(6.0%/d~17.0%/d)和慢速清除貝類(lèi)(0.3%/d~ 4%/d)。貽貝的最高清除速度(1 500.0 μg STXeq?kg–1?d–1)和清除速率(17.4%/d) (見(jiàn)表3)分別是其他四種經(jīng)濟(jì)貝類(lèi)的8~15倍和1.3~5.4倍, 屬于快速清除貝類(lèi)。貽貝大約5 d時(shí)間, 即可將8 680.0 μg STXeq·kg–1迅速排除至限量標(biāo)準(zhǔn)以下[42]。Katsushi等[40]研究發(fā)現(xiàn)貽貝對(duì)PSTs清除速度達(dá)1 326.6 μg STXeq?kg–1?d–1(清除速率為12.1%/d), 遠(yuǎn)高于其他貝類(lèi)。Ana等[48]的貽貝PSTs清除實(shí)驗(yàn)研究發(fā)現(xiàn), 清除實(shí)驗(yàn)中的前24 h貽貝體內(nèi)PSTs毒性便下降了約66.9%。以上研究結(jié)果都證明貽貝在不同環(huán)境條件下都具有較強(qiáng)的毒素清除能力。

2.1 環(huán)境因子對(duì)貽貝蓄積清除PSTs的影響

海洋環(huán)境因子如溫度、鹽度、pH等對(duì)貽貝的過(guò)濾、攝食等生長(zhǎng)生理活動(dòng)有重要影響, 從而直接影響貽貝對(duì)藻毒素的蓄積和清除。溫度是環(huán)境因子中首要影響因素, 高溫同時(shí)降低毒素蓄積量并減緩毒素清除速度。Ana等[48]研究了溫度對(duì)貽貝蓄積、清除能力的影響, 結(jié)果表明19 ℃條件下貽貝5天蓄積了1 493.8 μg STXeq·kg–1毒素, 而當(dāng)溫度升高至24 ℃時(shí)貽貝5天僅蓄積661.9 μg STXeq·kg–1毒素, 蓄積量降低了約49.0%, 且毒素清除效率也顯著降低, 殘留時(shí)間延長(zhǎng)[20]。這是由于高溫環(huán)境下貽貝對(duì)產(chǎn)毒藻的攝食明顯減少[49-51]。研究表明, 24 ℃時(shí), 貽貝的過(guò)濾率降低至19 ℃時(shí)的1/6。此外高溫誘發(fā)貽貝應(yīng)激反應(yīng), 其參與代謝過(guò)程的關(guān)鍵調(diào)節(jié)酶如糖酵酶丙酮酸激酶(PK)等活性受到抑制[49], 從而使得貽貝清除率下降[52-53]。

表3 不同貝類(lèi)對(duì)麻痹性貝類(lèi)毒素的清除規(guī)律

不同鹽度海水中貽貝毒素蓄積速率、蓄積含量存在較大差異。研究發(fā)現(xiàn)鹽度25.7時(shí)貽貝攝食最為活躍, 毒素蓄積含量也相應(yīng)較高[54-56], 但當(dāng)鹽度降低至5.0時(shí), 貽貝過(guò)濾海水速率顯著降低, 毒素蓄積能力減弱[57-59]。這是由于低鹽度刺激貽貝產(chǎn)生應(yīng)激反應(yīng), 貽貝通過(guò)閉合外套膜以降低外界環(huán)境對(duì)生理活動(dòng)的影響[21]。因此, 在低鹽度環(huán)境下, 貽貝過(guò)濾率、攝食率降低, 對(duì)藻毒素的蓄積能力降低。另外, 研究表明pH降低至7.5時(shí)貽貝生長(zhǎng)、代謝等生理活動(dòng)都會(huì)顯著減慢。Ana等[48]研究了氣候變化驅(qū)動(dòng)因素(升溫和酸化)對(duì)貽貝中PSTs蓄積代謝影響。結(jié)果表明上述氣候改變可導(dǎo)致殘留毒素毒性降低但毒素殘留時(shí)間延長(zhǎng)[60]。在pH降低至7.3時(shí)貽貝甚至可能會(huì)直接死亡[61-63]。

除溫度、鹽度、pH會(huì)影響貽貝對(duì)PSTs的蓄積清除外, 貽貝的種內(nèi)/種間競(jìng)爭(zhēng)等也會(huì)對(duì)貽貝的蓄積、清除產(chǎn)生影響, 競(jìng)爭(zhēng)會(huì)增加它們對(duì)食物的需求, 通過(guò)鰓過(guò)濾更多的水, 因此PSTs蓄積量顯著增加[62-64]。

2.2 貽貝對(duì)不同產(chǎn)毒藻的蓄積能力評(píng)估

研究表明, 產(chǎn)毒藻的毒性強(qiáng)弱、密度以及投喂方式都能夠影響貽貝蓄積毒素能力。我國(guó)東部沿海主要優(yōu)勢(shì)產(chǎn)毒藻為、。由于藻毒素來(lái)源不同, 貽貝的蓄積能力具有顯著差異[45]。黃德強(qiáng)等[65]研究發(fā)現(xiàn)貽貝對(duì)于的攝食率顯著低于無(wú)毒餌料藻。且Lee等[66]研究證明貽貝在毒性大于26 pg STXeq·個(gè)–1時(shí)攝食率也降低為原來(lái)的1/2。在我國(guó)近海PSTs調(diào)查中[22, 67], 貽貝在赤潮中的蓄積量比在其他產(chǎn)毒藻中的要高。因此, 貽貝的蓄積能力受產(chǎn)毒藻種類(lèi)、藻密度等因素影響。

此外, PSTs產(chǎn)毒藻的不同密度也會(huì)影響貽貝蓄積PSTs。貽貝的攝食率一般隨浮游植物密度的增加而升高, 這是由于在較高食物密度下, 過(guò)濾同樣多海水能夠獲取更多食物[68]。Negri等[69]對(duì)比密度為2×105個(gè)/mL和2×104個(gè)/mL的藍(lán)綠藻對(duì)淡水貽貝毒素蓄積影響時(shí)發(fā)現(xiàn): 暴露于密度為2×105個(gè)/mL產(chǎn)毒藻中的貽貝體內(nèi)PSTs毒性水平在2~3天就達(dá)到800 μg STXeq·kg–1, 而暴露于2×104個(gè)/mL產(chǎn)毒藻中的貽貝5周后只有少量毒素被檢測(cè)到, 差異顯著。趙俊梅等[68]的暴露實(shí)驗(yàn)也反映了類(lèi)似結(jié)果, 當(dāng)產(chǎn)毒藻密度增加時(shí)貽貝攝食率升高, PSTs蓄積量增加。

PSTs產(chǎn)毒藻的不同投喂方式也會(huì)對(duì)貽貝蓄積PSTs產(chǎn)生影響, 連續(xù)投喂時(shí)貽貝蓄積量要高于定期投喂時(shí)的[42, 70]。造成這種現(xiàn)象的原因可能是貽貝長(zhǎng)時(shí)間處于較低密度的毒藻中, 毒素對(duì)其攝食不產(chǎn)生抑制, 貽貝能夠不斷從水中過(guò)濾藻細(xì)胞, 毒素更容易在體內(nèi)累積。而定期投喂高密度的毒藻會(huì)使貽貝對(duì)毒藻產(chǎn)生生理抑制[69], 并且會(huì)較快速把體內(nèi)PSTs清除進(jìn)水體中。

3 貽貝對(duì)麻痹性貝類(lèi)毒素的代謝轉(zhuǎn)化

3.1 貽貝對(duì)藻毒素代謝轉(zhuǎn)化作用

全球目前已鑒別的PSTs約60余種, 主要包括四大類(lèi): 氨基甲酸脂類(lèi)毒素, 包括石房蛤毒素(STX)、新石房蛤毒素(NEO)和膝溝藻毒素(GTX1-4); N-磺酰氨甲?；?lèi)毒素, 包括GTX5(B1)、GTX6(B2)和C1-C4; 脫甲酰基類(lèi)毒素, 包括dcSTX、dcNEO、dcGTX1-4; 脫氧脫氨甲?；?lèi)毒素, 包括doSTX、doGTX2和doGTX3等[42, 71]。除了常見(jiàn)的赤潮藻種毒素, 更多的組分僅在貝類(lèi)體內(nèi)發(fā)現(xiàn), 為貝類(lèi)的代謝產(chǎn)物[72-73]。如圖1所示, 貝類(lèi)中常見(jiàn)的PSTs組分間轉(zhuǎn)化形式。

圖1 貝類(lèi)中常見(jiàn)PSTs組分間轉(zhuǎn)化

注: 實(shí)線表示還原反應(yīng); 點(diǎn)虛線表示水解反應(yīng); 橫虛線表示異構(gòu)化

Kim等[74]進(jìn)行了中PSTs毒素組分與貽貝中PSTs組分對(duì)比研究,主要毒素為C2、GTX4, 貽貝暴露初期體內(nèi)PSTs組分與藻細(xì)胞中類(lèi)似, 隨著中毒時(shí)間的增加貽貝中主要毒素變?yōu)镃1和GTX1, 分別是初始C2和GTX4的2.1和2.4倍, Wiese等[75]和Gracia等[39]的研究中也得出相似結(jié)論。但與扇貝、牡蠣等相比, 貽貝體內(nèi)PSTs的代謝轉(zhuǎn)化率相對(duì)較低[76]。Choi等[44]用只含有C2毒素的(ATDP)對(duì)貽貝開(kāi)展暴露實(shí)驗(yàn), 凈化階段在貽貝內(nèi)檢測(cè)到了GTX2/3等毒素, 證實(shí)了貽貝能夠?qū)⒌投拘缘腘-磺酰氨甲?；?lèi)毒素轉(zhuǎn)化成高毒性的氨基甲酸酯類(lèi)毒素。于姬等[25]在體外實(shí)驗(yàn)研究中同樣發(fā)現(xiàn)在酸性加熱條件下N-磺酰氨甲?；?lèi)毒素(C1-4、GTX5和GTX6)易轉(zhuǎn)化為PSTs組分中毒性最強(qiáng)的氨基甲酸酯類(lèi)毒素(GTX1-4、STX和NEO)。Paulo等[77]對(duì)鏈狀裸甲藻()展開(kāi)研究,主要毒素為C1-C4、GTX5、GTX6, 而貽貝體內(nèi)轉(zhuǎn)化成了dcGTX2&3、dcSTX和dcNEO。還原反應(yīng)是貝類(lèi)常見(jiàn)的PSTs轉(zhuǎn)化反應(yīng), 且還原反應(yīng)后貝類(lèi)中PSTs毒性相對(duì)更高, 如GTX2&3還原生成STX、C1&2還原生成GTX5、dcGTX2&3還原生成dcSTX[70]。邱江兵等[42]實(shí)驗(yàn)中表明GTX6的N1位羥基在貽貝消化腺中能夠發(fā)生還原反應(yīng)生成GTX5。

近些年, 在PSTs檢測(cè)中發(fā)現(xiàn)貝類(lèi)中獨(dú)有的PSTs代謝物, 如幾種新的STX變體(M1-M4)[78-80], 在微藻中沒(méi)有發(fā)現(xiàn)過(guò)。根據(jù)這些化合物的結(jié)構(gòu)和活性關(guān)系, 推測(cè)可能是貽貝自體解毒的中間產(chǎn)物[81]。為了解這些新代謝物的來(lái)源和生物轉(zhuǎn)化途徑, Ding等[82]在實(shí)驗(yàn)室條件下用兩種(ATHK株和TIO108) 喂養(yǎng)貽貝, 結(jié)果表明11-羥基-C2毒素(M1)和11-二羥基-C2(M3)由C2轉(zhuǎn)化而來(lái), 11-羥基-C4毒素(M7)和11-二羥基-C4(M9)由C4轉(zhuǎn)化而來(lái)。此外, M2、M4和M6可能是GTX2/3的代謝產(chǎn)物, M8和M10可能是GTX1/4轉(zhuǎn)化而來(lái)。這些研究都表明, 貽貝不僅可以對(duì)藻中已存在的PSTs進(jìn)行相互轉(zhuǎn)化, 還可以產(chǎn)生新的PSTs代謝產(chǎn)物, 且新型代謝物與貝類(lèi)的解毒過(guò)程有關(guān)。

3.2 代謝轉(zhuǎn)化過(guò)程中的影響因素

酶類(lèi)在PSTs的代謝轉(zhuǎn)化過(guò)程中發(fā)揮著重要的催化作用, 如表4所示, 主要包括氨甲酰水解酶、磺基轉(zhuǎn)移酶、氨基甲酰酯酶、N-氧化酶和谷胱甘肽還原酶[42, 78, 83-85]。Lin等[83]從貝類(lèi)消化腺中分離純化了一種氨甲酰水解酶, 實(shí)現(xiàn)R4基團(tuán)中氨甲酰基(或N-磺酰氨甲?；?的水解。Yoshida等[78]研究結(jié)果表明磺基轉(zhuǎn)移酶可將3’-磷酸腺苷-5’-磷酸硫酸(PAPS)中的硫酸基團(tuán)轉(zhuǎn)移到STX和GTX2&3的N-21位氨甲基基團(tuán), 分別生成GTX5和C1&2。卞中園等[84]表明氨基甲酰酯酶能夠催化氨基甲酸酯類(lèi)毒素(STX、NEO、GTX1-4) C11 位上的氨基甲?；l(fā)生水解反應(yīng), 生成脫氨甲酰基類(lèi)毒素(dcSTX、dcNEO、dcGTX1-4)。鄒迎麟等[85]在PSTs生物合成中檢測(cè)到N-氧化酶可以將GTX2&3轉(zhuǎn)化為GTX1&4。邱江兵等[42]進(jìn)行了消化腺體外轉(zhuǎn)化實(shí)驗(yàn), 發(fā)現(xiàn)貽貝消化腺在谷胱甘肽還原酶的催化下, 能夠促使11α-表聚體毒素如GTX1、GTX2、C1向11β-表聚體毒素如GTX3、GTX4、C2轉(zhuǎn)化。

表4 貽貝參與PSTs轉(zhuǎn)化過(guò)程調(diào)節(jié)酶

貝類(lèi)中PSTs的代謝轉(zhuǎn)化還受到pH、溫度和還原劑(谷胱甘肽、半胱氨酸)等因素的影響。如在較高溫度和pH值條件下會(huì)加速PSTs的差向異構(gòu)化[86]。雙殼貝類(lèi)組織中毒素的生物轉(zhuǎn)化除了化學(xué)和酶促作用的結(jié)果, 也可能是由于存在于消化道中細(xì)菌轉(zhuǎn)化的結(jié)果。Kotaki等[87]研究表明弧菌、假單胞菌能夠?qū)TX1/2/3和NEO毒素轉(zhuǎn)化為STX, 而且有氧條件比無(wú)氧條件轉(zhuǎn)化快。

3.3 貽貝對(duì)PSTs的適應(yīng)性機(jī)制

研究表明, 貽貝體內(nèi)的代謝靶器官內(nèi)臟團(tuán)會(huì)啟動(dòng)抗氧化應(yīng)激防御系統(tǒng), 用于減輕PSTs帶來(lái)的損傷[45, 88]。PSTs進(jìn)入貽貝體內(nèi)后, 將產(chǎn)生大量活性氧(ROS), 引起生物體氧化應(yīng)激, 導(dǎo)致脂質(zhì)過(guò)氧化、蛋白質(zhì)變性、DNA 損傷等。內(nèi)臟團(tuán)和鰓組織內(nèi)的抗氧化防御系統(tǒng)中的抗氧化酶, 如谷胱甘肽過(guò)氧化物酶(glutathione peroxidase, GSH-PX)、超氧化物歧化酶(superoxide dismutase, SOD)、酸性磷酸酶(acid phosphatase, ACP)等會(huì)被激活, 以消除ROS, 減少損害[41-42]。Qiu等[89]研究A.tamarense (ATHK)對(duì)貽貝抗氧化系統(tǒng)的影響, 發(fā)現(xiàn)在毒素蓄積和清除期間, ROS在貽貝內(nèi)臟團(tuán)中會(huì)迅速產(chǎn)生并消失, SOD和GSH-PX活性增強(qiáng), 有效清除貽貝體內(nèi)超氧陰離子自由基和過(guò)氧化氫(H2O2)。GSH-PX 還在貽貝體內(nèi)起到脫毒作用, 其能將脂類(lèi)過(guò)氧化物還原為相應(yīng)的醇, 并可以代替過(guò)氧化氫酶(Catalase, CAT)將游離的H2O2原成水, 同時(shí)催化谷胱甘肽(Glutathione, GSH)轉(zhuǎn)變?yōu)檠趸蚚90]。粒細(xì)胞分泌的ACP在抗氧化響應(yīng)過(guò)程中活力增強(qiáng), 魚(yú)煙酰胺腺嘌呤二核甘酸磷酸酶(nicotinamide adenine dinucleotide phosphate, NADPH)是一種還原型輔酶, 含量降低, 部分NADPH可能參與了毒素的生物轉(zhuǎn)化。

血細(xì)胞在貽貝天然免疫過(guò)程中發(fā)揮重要作用, 通過(guò)吞噬或包裹活的病原體來(lái)保護(hù)組織, 并通過(guò)炎癥過(guò)程修復(fù)由損傷、中毒和感染引起的組織損傷。通過(guò)超微結(jié)構(gòu)觀察可以發(fā)現(xiàn)PSTs在暴露初期會(huì)造成貽貝鰓組織柱狀上皮細(xì)胞中線粒體和溶酶體增多且聚集, 后期上皮細(xì)胞腫脹破裂, 黏液細(xì)胞大量釋放粘液顆粒, 細(xì)胞核萎縮變形, 嚴(yán)重時(shí)細(xì)胞壞死裂解[41]。Galimany等[86]研究貽貝的免疫反應(yīng), 發(fā)現(xiàn)血細(xì)胞向腸內(nèi)的滲出, 推測(cè)是為了分離腸內(nèi)有毒藻細(xì)胞, 從而將組織損害降到最低。Pousse等[91]研究發(fā)現(xiàn)血細(xì)胞能將有毒細(xì)胞包裹在消化道內(nèi), 形成吞噬泡, 吞噬泡與初級(jí)溶酶體融合形成次級(jí)溶酶體, 次級(jí)溶酶體內(nèi)的ACP就會(huì)將其降解清除到體外, 從而減少藻細(xì)胞與其他組織的接觸。Bianchi等[92]對(duì)貽貝進(jìn)行免疫功能實(shí)驗(yàn)研究, 發(fā)現(xiàn)PSTs暴露3d后貽貝血細(xì)胞吞噬功能顯著增強(qiáng), 以減輕PSTs對(duì)組織的損害。但PSTs對(duì)貽貝生理活動(dòng)的影響大多是暫時(shí)的, 且在長(zhǎng)期暴露于PSTs產(chǎn)毒藻后, 貽貝表現(xiàn)出了良好的生存適應(yīng)和免疫能力。

4 結(jié)論與展望

現(xiàn)有結(jié)果表明, PSTs已成為危及我國(guó)近海生態(tài)及貝類(lèi)質(zhì)量安全的關(guān)鍵因子, 其中尤以貽貝中PSTs風(fēng)險(xiǎn)最為嚴(yán)峻, 我國(guó)近海中不斷檢出且多次誘發(fā)中毒事件, 亟需進(jìn)行重點(diǎn)防控。國(guó)內(nèi)外研究表明, PSTs可以被貽貝快速蓄積并極易超過(guò)現(xiàn)有安全限量標(biāo)準(zhǔn), 隨后在貽貝體內(nèi)發(fā)生分布、代謝及轉(zhuǎn)化等過(guò)程, 而環(huán)境因素如溫度、pH、鹽度、種群密度及產(chǎn)毒藻種類(lèi)等能夠影響貽貝的過(guò)濾率、攝食率, 從而改變貽貝對(duì)PSTs的蓄積率、蓄積量以及清除率。目前已經(jīng)從藻和貝中分離鑒定了60多種PSTs及其代謝產(chǎn)物, 它們的殘留能力、毒性大小和靶器官等決定了貝類(lèi)中PSTs的風(fēng)險(xiǎn)表征及危害程度。因此, 后續(xù)應(yīng)側(cè)重于我國(guó)近海重點(diǎn)貝類(lèi)增殖區(qū), 摸清目標(biāo)區(qū)域貽貝等海洋貝類(lèi)中PSTs基礎(chǔ)風(fēng)險(xiǎn)規(guī)律和變化過(guò)程, 闡明貽貝體內(nèi)PSTs風(fēng)險(xiǎn)形成的內(nèi)源過(guò)程及調(diào)控機(jī)理, 比較并評(píng)估不同區(qū)域貽貝中PSTs風(fēng)險(xiǎn)大小, 并據(jù)此建立區(qū)域性PSTs風(fēng)險(xiǎn)防控技術(shù), 提高我國(guó)貽貝中PSTs風(fēng)險(xiǎn)的主動(dòng)防控能力, 保障消費(fèi)者健康安全和產(chǎn)業(yè)可持續(xù)發(fā)展。

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Research progress on the accumulation and metabolism of paralytic shellfish toxin in mussels

FAN Li-qiang1, 2, ZHENG Guan-chao2, WU Hai-yan2, GUO Meng-meng2, LU Xia1, LI Yu1, TAN Zhi-jun2, 3

(1. School of Geomatics and Marine Information, Jiangsu Ocean University, Lianyungang 222005, China; 2. Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture and Rural Affairs, P. R. China, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 3. Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China)

Poisoning with paralysis shellfish toxins (PSTs) can cause death in shellfish, other fish, and other marine organisms as well as hamper the marine ecology and human health. Mussels are highly accumulative and serve as indicators of PSTs in foreign countries. In recent years, several cases of PST poisoning by mussel consumption in China have become the focus of safety attention. Therefore, understanding the mechanism of risk formation is essential and urgent. This study evaluates the risk degree of PSTs in offshore mussels and other shellfishes in China and the accumulation effect of mussels on PSTs from different toxigenic alga sources, analyzes the influence of environmental factors on the accumulation and metabolism, and explores in depth the specificity of mussel accumulation and metabolism. Furthermore, focusing on the expression regulation process of the mussel metabolic stage, our study clarifies the influencing factors, transformation functions, and modes. In future studies, we aim to focus on understanding the basic risk rule and change process of PSTs in mussels as well as other marine shellfish in certain target areas of China and establish regional risk prevention and control technology for active prevention and control of PST risk in mussels of China. This will contribute positively and significantly toward protecting consumer health and the marine ecology.

mussels; accumulation and metabolism; immune; risk assessment

Nov. 2, 2020

S917

A

1000-3096(2021)04-0201-12

10.11759/hykx20201102003

2020-11-02;

2020-12-25

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2017YFC1600701); 國(guó)家自然科學(xué)基金面上項(xiàng)目(31772075, 32072329); 2019江蘇省研究生科研創(chuàng)新項(xiàng)目(KYCX19_2282)

[National Key R&D Program of China, No.2017YFC1600701; The National Natural Science Foundation of China, No.31772075, 32072329; 2019 Jiangsu Graduate Scientific Research Innovation Program, No.KYCX19_ 2282]

范禮強(qiáng)(1996—), 男, 在讀研究生, 研究方向?yàn)樗a(chǎn)品質(zhì)量與安全, E-mail: liqiangfan0@gmail.com; 譚志軍(1978—),通信作者, 研究員, 主要從事貝類(lèi)毒素安全檢測(cè)及評(píng)價(jià)研究, E-mail: tanzj@ysfri.ac.cn

(本文編輯: 趙衛(wèi)紅)