OBJECTIVE To investigate the feasibility and application of enzyme-linked bridging assay(ELBA)method to the pharmacokinetic evaluation of antisense strand siRNA drug. METHODS Antisense strand RNAs were diluted in LNCap cell lysates from 5 to 50 000 pmol·L-1 to construct the quantification curves. We transfected the intact double-strand siRNA at a final concentration 100 nmol·L-1 targeting Polo-like kinase into the LNCap cells and investigated the specificity of ELBA quantitating the siRNA antisense strand in cell supernatant,cell lysates and RNA-induced silencing complex( RlSC). Quantification curves were constructed and validated in biological matrices such as plasma (5-25 000 pmol·L-1 )and multiple tissues(liver,heart,spleen,and kidneys)(3-6250 pmol·L-1 ). The prostate specific membrane antigen aptamer siRNA delivery system with the intact siRNA concentration of 15 nmol·kg-1 was prepared. The siRNAs were delivered into the LNCap xenogrant tumor model in C57 mice by tail vein injection. The concentration of siRNA antisense strand was determined in plasma and tissues 30 min post administration by ELBA. RESULTS The quantitative range of antisense strand siRNA in cell lysates was 5-50 000 pmol·L-1 ,and ELBA method could quantify the siRNA antisense strand concentration from cell lysates and RlSC in LNCap cells transfected with double-strand siRNA. ln addition,ELBA could specifically reflect the single antisense strand concentration instead of intact siRNA double strands in plasma. The quantification range of siRNA antisense strand using ELBA in plasma was 5-25 000 pmol·L-1 and 3-3125 pmol·L-1 in tissues. About 30 min post administration of PSMA aptamer-siRNA,the antisense strand of siRNA was distributed mainly to the tumor,liver,kidneys,blood and spleen in sequence. The distribution profile might be attributed to the target delivery and siRNA pharma-codynamics. CONCLUSION The ELBA method is successfully applied to the siRNA antisense strand pharmacokinetic evaluation,which provides an alternative for pharmacokinetic studies of siRNA-based drugs.

OBJECTlVE To establish Tg(-6.3CYP3A65∶EGFP) transgenic zebrafish for quick, intuitive detection of heavy metals ( copper, cadmium and zinc) , dioxin-like PCBs ( PCB126) and other environmental pollutants. METHODS Tol2 transposon system was used to generate transgenic zebrafish lines Tg(-6.3CYP3A65∶EGFP) in which CYP3A65 promoter regualated labeled fluorescence. The effect of heavy mentals ( copper, cadmium and zinc ) and PCB126 on the relative amounts of CYP3A65 gene expression was determined by observing the change in fluorescence intensity. RESULTS The relative gene expression of CYP3A65 was significantly increased after 96 h exposure to copper 0.1 and 0.2μmol·L-1 , cadmium 0.35 and 0.7μmol·L-1 , zinc 1.5 and 3μmol·L-1 , and PCB126 2-32μmol·L-1 , respectively ( P<0.01) , but decreased after 96 h exposure to copper 0. 9 μmol·L-1 , cadmium 2. 7 and 5.4 μmol·L-1 , and zinc 24μmol·L-1 , respectively( P<0.01) . CYP3A65 gene expression was significantly increased after 168 h exposure to copper 0.1 and 0.2 μmol·L-1 , cadmium 0.35 and 0.7 μmol·L-1 , zinc 1.5 and 3 μmol·L-1, and PCB126 2-32 μmol·L-1, respectively(P<0.01), but decreased after 168 h exposure to copper 0.9 μmol·L-1, cadmium 2.7 and 5.4 μmol·L-1, and zinc 12 and 24 μmol·L-1( P<0.05) , in a concentration-dependent manner. CONCLUSlON The results suggest that zebrafish CYP3A65 gene expression and the CYP3A65 labeled fluorescence lines can be another candidate biomarker for detecting environmental pollutants.