Somnuek Chawanrat, Boonphakdee Chuta, Cheevaporn Voravit, Tanaka Keiichi
Graduate School of Environmental Science, Faculty of Science, Burapha University, Chonburi, Thailand.
J Environ Biol. 2009 Jan;30(1):83-8.
After serving as an eurotransmitter, acetylcholine is hydrolyzed by acetylcholinesterase (AChE). Inhibition of AChE is considered to be a specific biomarker for exposure to organophosphate and carbamate insecticides. In this study the AChE gene was isolated from hybrid catfish brain by RT-PCR methods using degenerate primers. The amplified fragment of 966 nucleotides generated by PCR was cloned, and sequence analysis showed 82% nucleotide identity with AChE of the Electrophorus electricus. Specific primers of the hybrid catfish AChE gene were then synthesized and used in the examination of AChE gene expression in brain tissue of hybrid catfish exposed to sublethal concentrations of chlorpyrifos (0.43, 4.3 and 43 microM) and carbaryl (1.19, 11.9 and 119 microM) for 24 hr. Real-time PCR was used to compare with the amplified 28S rRNA gene. AChE gene expression was significantly elevated 12.4 times in catfish exposed to 43 microM chlorpyrifos in comparison to the control group (p<0.05). Carbaryl did not produce any significant change. These results indicate thatAChE is more sensitive to administration of chlorpyrifos than carbaryl. The induction of the AChE gene indicates the possibility of using this gene as biomarker for detecting effects of organophosphate insecticides in hybrid catfish.
乙酰胆碱作为神经递质发挥作用后，会被乙酰胆碱酯酶（AChE）水解。抑制乙酰胆碱酯酶被认为是接触有机磷和氨基甲酸酯类杀虫剂的一种特异性生物标志物。在本研究中，使用简并引物通过RT-PCR方法从杂交鲶鱼脑中分离出乙酰胆碱酯酶基因。对PCR扩增产生的966个核苷酸的片段进行克隆，序列分析表明其与电鳗的乙酰胆碱酯酶具有82%的核苷酸同一性。随后合成了杂交鲶鱼乙酰胆碱酯酶基因的特异性引物，并用于检测暴露于亚致死浓度毒死蜱（0.43、4.3和43微摩尔）和西维因（1.19、11.9和119微摩尔）24小时的杂交鲶鱼脑组织中乙酰胆碱酯酶基因的表达。使用实时PCR与扩增的28S rRNA基因进行比较。与对照组相比，暴露于43微摩尔毒死蜱的鲶鱼中乙酰胆碱酯酶基因表达显著升高12.4倍（p<0.05）。西维因未产生任何显著变化。这些结果表明，乙酰胆碱酯酶对毒死蜱的敏感性高于西维因。乙酰胆碱酯酶基因的诱导表明有可能将该基因用作检测杂交鲶鱼中有机磷杀虫剂效应的生物标志物。
