杀虫单对不同倍性泥鳅鳍细胞系的毒性作用

In Vitro Cytotoxicity Test of Monosultap Using Different Ploidy Loach Fin Cell Lines

以泥鳅鳍二倍体(DIMF)和三倍体(TRMF)细胞系为受试细胞,研究杀虫单对2种细胞系的毒性作用。采用噻唑蓝(MTT)法测得DIMF与TRMF 24 h半致死浓度分别为119.73 mg·L-1、146.26 mg·L^(-1)。DIMF的敏感性明显高于TRMF。经杀虫单处理的活体细胞表现为细胞伸长,空泡化和脱落并游离于培养基表面的现象。2种细胞系酶活力测定的结果显示:杀虫单浓度为0~100 mg·L^(-1)时,SOD和GST活力随着浓度的增加而增加,100~200 mg·L^(-1)浓度组酶活力逐渐降低;0~200 mg·L^(-1)时,ACh E活力与杀虫单浓度呈负相关,并且三倍体3种酶活力均高于二倍体。微核试验结果显示:50 mg·L^(-1)杀虫单就能对细胞核造成损伤并形成微核,微核率随杀虫单浓度增加而增加。当杀虫单浓度达到200 mg·L-1时,微核率出现最大值,DIMF和TRMF分别为3.3‰和3.7‰,2种细胞的测试结果无显著性差异(P>0.05)。电镜观察结果显示:对照组DIMF和TRMF超微结构无明显差异;DIMF和TRMF病理变化情况相似:染色质凝集趋边,细胞核分解成多个,细胞内出现空泡和凋亡小体,表现出凋亡的特征。研究表明杀虫单的细胞毒性机制是通过改变细胞内酶活性从而诱导凋亡,不同倍性细胞系之间的差异主要与多倍体细胞体积大,胞内物质多,分裂快,生长旺盛等有关。

In this study,loach findiploid cell line（ DIMF） and triploid cell line（ TRMF） were used to assess toxicity of monosultap in vitro. The 24 h semi-lethal concentrations of DIMF and TRMF measured by thiazole blue（MTT） method were 119. 73 mg·L^-1and 146. 26 mg·L^-1,respectively. DIMF was more sensitive to monosultap than TRMF. After exposure to monosultap,the cellular morphology showed the phenomenon with elongation,vacu-olization and exfoliation. When the monosultap concentration was 0-100 mg·L^-1,the activities of superoxide dismuta（ SOD） and glutathione S-transferase（ GST） in two cell lines were increased with the increase of concentration,however,while the concentration was 100-200 mg·L^-1,the activities of both enzymes gradually decreased.When the concentration was 0-200 mg·L^-1,the activity of acethlcholine esterase（ACh E） in two cell lines was negatively correlated with the concentration of monosultap. The activties of three enzymes in TRMF were higher than those in DIMF. Micronucleus test showed that 50 mg·L^-1 monosultap could cause the nucleus damage and form micronucleus. The rate of micronucleus increased with the increase of monosultap concentration. When monosultap concentration was 200 mg·L^-1,the maximum micronucleus rates of DIMF and TRMF were 3. 3‰ and 3. 7‰,respectively,and no significant differences were observed between two cell lines（ P 〉0. 05）. The electron microscopic observations showed that DIMF and TRMF presented similar pathological changes,showing chromatin condensation,margination and cell nucleus decomposition. The numbers of mitochondria,ribosomes and endoplasmic reticulum reduced. Vacuoles and apoptotic bodies appeared in cells,which showed the characteristic of apoptosis.The present results showed that the cytotoxic mechanism of monosultap was inducing apoptosis by changing the intracellular enzyme activity. Differences between two ploidy cell lines were mainly related to the large cell volume,much intracellular substance,rapid cell division and vigorous growth of triploid.