Development of Multi-biomarker Assays Using Mussels for Coastal Monitoring

对单细胞GET电泳法(SCG法)、混合功能加氧酶测定法、吞噬测定法进行了改进，并进行了室内试验和田间试验。用氯化锌处理贻贝血细胞进行SCG测定的最佳条件为：解离时间15分钟，电泳法时间20分钟，表现出最明显的剂量-反应关系。在此条件下的实验室实验中，我们可以观察到DNA损伤的剂量依赖性和时间性，以及DNA损伤和修复的时间模式，表明该方法的实用性。高效液相色谱法测定EROD活性的灵敏度是常规方法的3000倍。以贻贝消化腺制备的微球为试材，采用高效液相色谱(HPL C)方法，可以得到清晰、灵敏的结果。在MMS和B[a]P的实验中观察到EROD活性的显著诱导，但ECOD活性没有被诱导。对吞噬试验的实验条件，如锥虫浓度、荧光大肠埃希菌浓度等进行了考察。随着B[a]P浓度的增加。吞噬作用被激活，在较高浓度时被抑制。在富山湾的6个港口，于2000年8月、10月和12月利用这些已确定的生物标志物进行了初步实地研究。并对生物标志物的检测结果进行了讨论。

The improvement of single cell get electrophoresis assay (SCG assay), mixed function oxygenase assay, and phagocytosis assay, laboratory experiments and field studies had been conducted. As for the improvement of SCG assay using mussel hemocytes exposed to Zn_2Cl, the optimum condition was the combination of unwinding time of 15 minutes and electrophoresis time of 20 minutes, showing the clearest dose-response relationship. In laboratory experiments with this condition, we could observe the dose-dependent and temporal DNA damage, and temporal patter of DNA damage and repair, indicating the usefulness of this assay. HPLC method for EROD activity showed the 3,000 times of sensitivity compared to the conventional method. Using HPLC method with microsome prepared from mussel digestive gland, we could get the clear and sensitive results. Significant induction of EROD activity was observed in the experiment with MMS and B [a] P, but ECOD activity had not been induced. The experimental conditions of phagocytosis assay, such as concentration of trypanbule, concentration of fluorescent E.coli etc., had been examined. With the increase of B [a] P concentration. phagocytosis had been activated, and suppressed at the higher concentration. At 6 harbors in Toyama Bay, the preliminary field study had been conducted using these established biomarkers in August, October and December 2000. The results of biomarkers were discussed.