Development of Multi-biomarker Assays Using Mussels for Coastal Monitoring

对单细胞凝胶电泳法、混合功能加氧酶法和吞噬作用法进行了改进，并进行了室内试验和田间试验。对于Zn_2Cl对贻贝血细胞SCG测定的改进，最佳条件为解链时间15 min和电泳时间20 min，呈现出最明显的剂量-反应关系。在此条件下的实验室实验中，我们可以观察到剂量依赖性和时间DNA损伤，以及DNA损伤和修复的时间模式，表明该测定法的有用性。高效液相色谱法测定EROD活性的灵敏度是常规方法的3，000倍。用贻贝消化腺微粒体进行HPLC测定，结果清晰、灵敏。MMS和B [a] P对EROD活性有明显的诱导作用，但对ECOD活性无诱导作用。吞噬实验的实验条件，如锥虫蓝浓度、荧光大肠杆菌浓度等，已经被检查过了。随着B [a] P浓度的增加。吞噬作用被激活，并在较高浓度下被抑制。在富山湾的6个港口，初步的现场研究已在2000年8月，10月和12月使用这些建立的生物标志物进行。对生物标志物的研究结果进行了讨论。

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.