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Objective@#The effect and mechanism of cadmium telluride quantum dots (CdTe QDs) on cytochrome P450 (CYP450) in the liver of rat were investigated.@*Methods@#CdTe QDs (Ex 350 nm, Em 600 nm) were incubated with microsomes in final concentrations (0.5, 5, 50 μmol/L) using rat liver. And the content of CYP450 was determined by mixed incubation system as time (15, 30, 45 min) went on. Relationship also was detected between particle sizes (Em 620, 580, 540 nm; CdTe QDs-2, CdTe QDs-3, CdTe QDs-4) and expression of CYP450. Twenty male Wistar rats were randomly divided into exposed groups at various concentrations (0.25, 2.5 and 12.5 μmol/kg) of CdTe QDs via tail vein injection, the control group was injected with PBS.@*Results@#In vitro, CdTe QDs(0.5, 5, 50 μmol/L) could significantly increase the content of CYP450 in rat liver microsomes(P<0.05), which increased first and then decreased with the dose adding. Moreover, the trend along with the exposure time (15, 30, 45 min) was the same as that in dosages at certain concentration (P<0.01). For different particle sizes, the smaller CdTe QDs were, the higher content increased, the content of CYP450 in group CdTe QDs-4 was the highest (P<0.05). In vivo, experiment proved that CdTe QDs (0.25, 2.5 and 12.5 μmol/kg) could obviously induce the expression of CYP450 (P<0.01). The content level showed a tendency to rise and then fall.@*Conclusion@#CdTe QDs could promote the content of CYP450 in rat liver microsomes, it indicated that CdTe QDs had dose-effect relationship both in vivo and vitro. There was a certain relationship in time-effect. In addition, the smaller particle size was, the greater impact had.
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Objective To establish in vitro metabolism of fentanyl by human liver microsomes in Chinese population.Methods Thirty patients undergoing elective operation on liver were enrolled in the study.Normal liver specimens were obtained during removal of liver and gall for preparation of liver microsomes (by calcium precipitation) which were used for establishment of the liver microsomal incubation system for fentanyl.Fentanyl served as the metabolic substrate in the incubation reaction.The concentration of fentanyl in the incubation medium was detected at 0,5,10,15,20 and 30 min of incubation using HPLC-UV.Sufentanil served as the interior label element.The n-hexane-ethanol absolute was used to extract the sample.The chromatographic column used in this method was Grace C18 (4.6 mm × 250.0 mm,5 μm).The mobile phase was methyl cyanide-KH2PO4 buffer solution with the flow rate of 1.0 ml/min,detection wavelength of 205 nm and sample size of 20 μl.Linear regression analysis was performed by using the least-squares method.The specimens of the blank incubation system with the final concentration of fentanyl 0.6,2.4 and 10.0 μg/ml were obtained to determine the recovery,precision and stability.The metabolic rate of fentanyl in human hepatic microsomes was calculated.Results Fentanyl and the interior label element sufentanil were separated completely,and the retention time were 5.730 and 9.336 min,respectively.Endogenous matrix of microsomes did not interfere with the analysis.Regression equation was C =0.945 8A-0.140 4,R2 =0.999 2.C was the concentration of fentanyl,and A was the peak area ratio of fentanyl versus sufentanil.The recovery of incubation system with low,medium and high concentrations of fentanyl was 85％-115％,and relative standard deviation (RSD) was less than 10％.The RSD of intra-and inter-day precision and stability was less than 10％.The method was proved to meet the requirements of biological sample analysis.The metabolic rate of fentanyl was (1.6 + 0.8) nmol/min per milligram protein in human hepatic microsomes of 30 cases.Conclusion The in vitro metabolism of fentanyl by human liver microsomes is convenient,and the detectability is high,so it can be used for the research on the in vitro metabolism of fentanyl in Chinese population.
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Cytochrome P450 3A4 (CYP3A4), is the dominant human liver hemoprotein enzyme localized in the endoplasmic reticulum (ER), and is responsible for the metabolism of more than 50% of clinically relevant drugs. While we were studying CYP3A4 expression and activity in human liver, we found that anti-CYP3A4 antibody cross-reacted with a lower band in liver cytoplasmic fraction. We assessed the activities of CYP3A4 and its truncated form in the microsomal and cytoplasmic fraction, respectively. In the cytoplasmic fraction, truncated CYP3A4 showed catalytic activity when reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5. In order to determine which site was deleted in the truncated form in vitro, we transfected cells with N-terminal tagged or C-terminal tagged human CYP3A4 cDNA. The truncated CYP3A4 is the N-terminal deleted form and was present in the soluble cytoplasmic fraction. Our result shows, for the first time, that N-terminal truncated, catalytically active CYP3A4 is present principally in the cytoplasm of human liver cells.