Toxic Effects Of Butyl Benzyl Phthalate On Zebrafish And Cloning, Expression Of Acetylcholinesterase Gene

As plasticizers, softeners, carriers and additives, BBP are widely used in plastics, automotive, lubricants, cosmetics, clothing, pesticide and other industries. Because its hydrolysis, photolysis and evaporation rate is very slow, BBP has been concerned as a major of global environmental pollutant. At present, the EU, the United States, Japan and other countries has ranked this compound among the priority control pollutants, It was represented "The second global PCB (polychlorinated biphenyls) pollution". It is reported that BBP has obvious testicle toxicity, teratogenicity and embryo toxicity, it can lead serum decrease in testosterone, and promote the increase of follicle-stimulating hormone and hysterauxesis, and has changes in sexual differentiation. In addition, it can cause allergies and asthma, have an impact on the human nervous system. It is significant to study its possible adverse impact on organisms.In this study, fish acute static test was used to explore the toxic effects of BBP on zebrafish. Based on the acute toxicity test,96 hours median lethal concentration (LC50) of BBP effected on zebrafish was determined to be 3.98mg·L-1. Then, zebrafish were exposed to different BBP concentrations (0、0.332、0.665、1.33 mg·L-1). The AChE, SOD and CAT activities were measured in the fish collected at 7 d,14d 21d and 28d post-exposure. It showed that from Od to 28d, AChE activity was significantly inhibited; SOD activity increased firstly and then declined with time extension, and was activated to the highest levels(13.51,14.72,11.81,10.19 U/mg) at 14d for all the treatments. CAT activity, in 7d, except 0.665 mg·L-1 group, was inhibited significantly (p<0.01), the rest groups were of no significant difference compared with the control.Semi-quantitative RT-PCR was used to determine the mRNA transcript levels of AChE, SOD and CYP1A gene in brain and muscle of zebrafish exposed 7d in different concentrations (0、0.332、0.665、1.33 mg·L-1). It showed that the mRNA transcript of AChE gene was down-regulated by BBP exposure both in brain and muscle; this was consistent with the AChE activity measured before. The mRNA transcript of SOD gene in zrbrafish brain were first decreased and rebounded modestly at 1.330 mg·L-1 group. In zrbrafish muscle, the mRNA transcript of SOD gene displayed an inhibition trend, compared with the control, the inhibition rates were 19.33%,17.86%,17.23%. The brain mRNA transcript of CYP1A gene was first increased and then decreased, while it was first decreased and then increased in muscle.In this paper, the cDNA of AChE gene in Zebrafish was cloned by reverse transcription polymerase chain reaction (RT-PCR). The full lengh of the cDNA was 1905 bp. The recombinant plasmid was constructed by subcloning the AChE gene into the prokaryotic expression vector and then transformed into the host bacteria for expression. SDS-PAGE analysis showed that the MW of expressed protein was 69kDa which was the same size with zebrafish AChE protein.