摘要
随着我国和全球工业化进程的加快，大量重金属以多种方式进入海洋，由此造成海洋重金属污染不断加剧。海洋贝类由于其滤食特性和移动性差而极易富集重金属，重金属超标已成为贝类食品安全的重要威胁，其中以镉（cadmium, Cd）超标尤为严重。已有研究者就如何从已污染贝类中脱除Cd进行了研究，本文则从抑制Cd吸收的角度对解决贝类Cd超标的问题进行了尝试。Cd在双壳贝类中通过Ca~(2+)通道吸收，很多稀土元素具有与Ca~(2+)相近的离子半径，因此是潜在的Cd吸收抑制剂。稀土已在医药、农业等领域获得广泛应用，其中镧（lanthanum,La）和铈（cerium, Ce）已于2008年被农业部纳入了饲料添加剂品种目录用于畜禽及鱼虾养殖。由于La和Ce含量较为丰富且离子半径与Ca~(2+)相近，本文探讨了这两种稀土元素对太平洋牡蛎（Crassostrea gigas）Cd吸收的影响，并研究了这两种稀土元素在太平洋牡蛎中的富集代谢规律及其对太平洋牡蛎部分理化指标的影响，以期为La和Ce在解决海洋贝类Cd超标问题中的应用提供参考。本文结论如下：
1、青岛市售贝类的Cd超标情况较为严重。对青岛城阳水产品批发市场中销售的太平洋牡蛎、紫贻贝（Mytilus edulis）、毛蚶（Scapharca subcrenata）、杂色花蛤（Ruditapes philippinarum）和栉孔扇贝（Chlamys ferrari）体内的Cd和Cu含量进行了为期一年的连续跟踪，每月定期取样，发现除杂色花蛤外，其余四种贝类的Cd含量在所有月份均超标，其中以栉孔扇贝和太平洋牡蛎最为严重，其Cd含量分别在7.1~16.3mg/kg·dw和4~12.7mg/kg·dw之间，而且太平洋牡蛎的Cu含量在部分月份也超标，表明解决贝类Cd超标问题已经迫在眉睫。
2、增加海水的Ca浓度不能降低太平洋牡蛎对Cd的吸收。虽然在海水中添加CaCl_2使Ca浓度达到0.525g/L、0.7g/L和0.875g/L后可使Cd对太平洋牡蛎的96h半致死剂量从17.271mg/L升高至24.988mg/L、43.932mg/L和41.795mg/L，并显著降低由Cd引起的氧化损伤，但并不能抑制太平洋牡蛎对Cd的吸收。
3、La和Ce均显著抑制太平洋牡蛎对Cd的吸收。在第12天，5μmol/L和10μmol/L的La(NO_3)_3对Cd吸收的抑制率分别达到了36%和44%，25μmol/L的Ce(NO_3)_3对Cd吸收的抑制率达到了41%；组织水平研究表明，La和Ce降低了Cd在太平洋牡蛎鳃达到吸附饱和的速度，从而减少了Cd由鳃向消化腺的转运。
4、La和Ce在太平洋牡蛎体内具有较强的蓄积性，鳃和消化腺是La和Ce的主要富集部位。当太平洋牡蛎暴露于含1、5或10μmol/L La(NO_3)_3或Ce(NO_3)_3的海水24天后，其体内的La和Ce含量分别达到了79.75、199.70、425.82mg/kg·dw及42.07、158.23、236.28mg/kg·dw；组织水平研究表明，各组织器官对La和Ce富集能力的强弱顺序均为鳃>消化腺>剩余组织>肌肉；鳃是La和Ce的主要富集部位，24天时其所结合La和Ce的百分比分别为64%和51%，其次是消化腺，分别为13%和25%，肌肉结合的比例最低，仅为1.14%和2.69%；La和Ce的亚细胞水平分布之间无显著差异，均主要与MTLP（metallothionein-like protein）结合，比例分别为38.2%和35.4%，其次是细胞器，分别为25.7%和29.7%，与MRG（metal-rich granules）及HSP（heat-sensitive protein）结合的比例最低。自身脱除实验发现，31天内太平洋牡蛎体内富集的La和Ce含量无明显降低。
5、La和Ce对太平洋牡蛎的急性毒性较低。太平洋牡蛎暴露于75μmol/L的La(NO_3)_3或Ce(NO_3)_396h后均未观察到死亡；La和Ce短时间内不会在鳃和消化腺引起氧化损伤，但是当La和Ce富集到一定程度时会使鳃和消化腺中的MDA含量显著升高；另外，Ce可抑制太平洋牡蛎鳃或消化腺中p-gp（p-glycoprotein）和MT（metallothionein）基因的表达。
6、La和Ce对太平洋牡蛎的肌肉指数和闭壳肌质构无显著影响，但显著影响了蛋白质、脂肪、Zn和Cu含量。1μmol/L的La(NO_3)_3使太平洋牡蛎的总蛋白含量增加，但1、5μmol/L的Ce(NO_3)_3显著降低了总蛋白含量；此外，1μmol/LLa(NO_3)_3组的脂肪含量显著降低，但是10μmol/L Ce(NO_3)_3组的脂肪酸含量与空白组相比显著增加。La和Ce对太平洋牡蛎闭壳肌的质构无显著影响，但是10μmol/L Ce显著降低了壳闭肌的持水力；La和Ce对太平洋牡蛎的金属含量也有一定影响。经1、5、10μmol/L的La(NO_3)_3处理30天后，Zn的含量分别降低了48%、60%和39%，Cu的含量分别降低了63%、32%和39%；而经上述三个剂量的Ce(NO_3)_3处理后，Zn和Cu的含量分别降低了8%、44%、18%和0%、11%、28.6%。
综上所述，La和Ce可显著抑制太平洋牡蛎对Cd的吸收，急性毒性较低，短期内不会对太平洋牡蛎造成氧化损伤，并且对太平洋牡蛎的肌肉指数、部分常规营养组分及闭壳肌的质构无太大影响。但是，La和Ce在太平洋牡蛎体内具有较强的蓄积性，这与其在畜禽中的代谢规律差别很大；此外，长时间接触La和Ce会导致氧化损伤和Zn元素含量降低，并抑制p-gp和MT基因的表达，这可能会对太平洋牡蛎的生理代谢产生不利影响。因此，La和Ce在贝类养殖及解决Cd超标问题中的应用还需要进一步的研究。
With the rapid progression of industrialization in both China and the world, theheavy metal (especially Cd) pollution issue of seawater has turned to be a worlwideconcern. Marine bivalves are filter-feeding molluscs and can accumulate extremelyhigh levels of heavy metals. Heavy metal pollution greatly threatens the safety ofmarine bivalve-derived foods. The selection of unpolluted rearing sea area is thepreferred way for avoding heavy metal pollution in bivalves, but it is still meaningfulto develop alternative solutions. Some researches have attempted to elimiate Cd frompolluted bivalves, the current research aims to solve the problem by inhibiting Cdaccumulation in bivalves. Marine bivalves absorb cadmium through the Ca~(2+)channeland inhibitors of the channel are theoritically able to inhibit Cd accmuluation. Manyrare earth elements have radiuses similar to that of Ca~(2+)and are potential Cdaccumuliation inhibitors. The current research explores the possiblity of applying Laand Ce, which have been approved by the Ministry of Agriculture for addition tolivestock and fish feeds, in inhbiting Cd accumulation in Crassostrea gigas. Theresults are as follows:
1. The Cd pollution issue of marine bivales sold in Qingdao markets is serious.The monthly varition of Cd and Cu contents in five common bivalves sold in aQingdao market, includign Crassostrea gigas, Mytilus edulis, Scapharca subcrenata,Ruditapes philippinarum, and Chlamys ferrari, were followed for one year. It wasfound that the Cd contents of all the bivalves except Ruditapes philippinarumexceeded the permitted value of the national standard. Chlamys ferrari andCrassostrea gigas had the highest Cd contents and were within the ranges7.1~16.3mg/kg·dw and4~12.7mg/kg·dw respectively. Besides, the Cu contents ofCrassostrea gigas also exceeded the permitted values in some months. Crassostreagigas was selected as the model bivale in subsequent researches.
2. Increased Ca concentration in seawater cannot inhibit the absorption of Cd byCrassostrea gigas. Though elevated seawater Ca concentration of0.525g/L,0.7g/L,and0.875g/L increased the96h LD50of Cd from17.271mg/L to24.988mg/L,43.932mg/L, and41.795mg/L respectively and decreased the oxidative damageinduced by Cd in oyster gill, it could not significantly inhibit the accumulation of Cdin Crassostrea gigas.
3. Both La and Ce significantly inhibit the accumulation of Cd in Crassostrea gigas. On day12, the inhibition rates of Cd accumulation by5μmol/L and10μmol/LLa(NO_3)_3reached36%and44%respectively and that of25μmol/L Ce(NO_3)_3reached41%. Organ-level experiments revealed that the two rare earth metals elongated thetime for saturated absorption of Cd in the gill and thus reduced the transfer of Cd fromgill to digestive gland.
4. La and Ce are accumulated in high levels in Crassostrea gigas. After exposureto1,5, and10μmol/L La(NO_3)_3or Ce(NO_3)_3for24days, the contents of La reached79.75,199.70, and425.82mg/kg·dw respectively and that of Ce reached42.07,158.23, and236.28mg/kg·dw respectively. Distribution in organs revealed that gillhad the highest La and Ce contents, followed by digestive gland, remainder, andmuscle. Gill was the main organ for La and Ce storage and the percentages of the twometals were64%and51%on day24, followed by digestive gland with percentages of13%and25%respectively. Subcellular research indicated that both La and Ce boundmainly with MTLP (metallothionein-like protein) and their percentages were38.2%and35.4%respectively. Organelles were the second most important fractions for Laand Ce sequestration and the percentages were25.7%and29.7%respectively.Depuration experiments showed that no significant reduction of La and Ce contentswas observed after one month, indicating slow elimination of the two elements fromCrassostrea gigas.
5. The acute toxicity of La or Ce to Crassostrea gigas is low. After exposure to75μmol/L (the highest concentration available in seawater) La(NO_3)_3or Ce(NO_3)_3for96h, no mortality was observed. La and Ce did not induce oxidative damage aftershort-term exposure, but long-term exposure resulted in elevated MDA levels in boththe gill and digestive gland. Besides, Ce suppressed the expression of p-gp and MTgenes, indicating difference behavior from heavy metals.
6. Exposure to La or Ce does not cause changes in muscle index or muscletexture, but influences the contents of protein, lipid, Zn, and Cu. La(NO_3)_3in1μmol/Lincreased the protein contents in Crassostrea gigas, but1and5μmol/L Ce(NO_3)_3reduced the index. Besides,1μmol/L La(NO_3)_3reduced the lipid content, but10μmol/L Ce(NO_3)_3increased the lipid content. Exposure to La and Ce did not affectthe texture of adductor muscle, but10μmol/L Ce(NO_3)_3significantly reduced thewater-holding capacity of the muscle. Exposure to La(NO_3)_3and Ce(NO_3)_3significantly reduced the contents of Ca, Cu, and Zn. After exposure to1,5, and10μmol/L La for30days, the Zn contents decreased by48%,60%,39%and the Cu contents decreased by63%,32%,39%. After exposure to the three concentrations ofCe(NO_3)_3, the contents of Zn and Cu decreased by8%,44%,18%and0%,11%, and28.6%respectively.
It was concluded that both La and Ce significantly inhibited the accumulation ofCd in Crassostrea gigas and did not induce oxidative damage or cause markedchanges in texture and nutrient contents. Because rare earth elements have gainedwide practical applcations freshwater aquaculture, La and Ce have potentialapplications in reducing Cd accumulation in marine bivalves. However, La and Cewere accumulated in very high levels in oyster soft tissues and cannot be eliminatedwithin a short time, which differed significantly from the metabolism of rare earthelements in livestocks; besides, long-term exposure to rare earth element led tooxidatve damage and reduced Zn contents, which possibly cause adverse effect on thephysiological activities of bivalves. Further researches are necessary for promotingthe application of La and Ce in marine bivalve rearing and Cd bioaccumulationihnibition.
引文
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