Document Detail


A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates.
MedLine Citation:
PMID:  8238878     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
This study deals with the development of a sensitive and simple microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes. The method is based on the metabolism by cells cultured in microwells of appropriate substrates at noncytotoxic concentrations (8 microM 7-ethoxyresorufin and 15 microM 7-pentoxyresorufin). After incubation of the probes with the cells, the dealkylated resorufin formed and released into culture medium was quantified. To ensure the hydrolysis of the resorufin conjugates eventually formed, culture supernatants were incubated in the microwells with beta-glucuronidase and arylsulfatase. The fluorescence was then read using a microplate fluorescence reader. A high correlation between the monooxygenase activity measured by this procedure and that measured by conventional procedures in the microsomal fraction of the same cells was found. The major advantages of this method are: (1) the small number of cells required; (2) a drastic reduction in assay time; (3) that the assay is performed in intact cells; and (4) the possibility of performing repeated assays with the same cell monolayer over a period of several days since no injury to cells is detectable during the assay. This method proved to be very convenient for studying cytochrome P450 induction by xenobiotics in primary cultures of human hepatocytes.
Authors:
M T Donato; M J Gómez-Lechón; J V Castell
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical biochemistry     Volume:  213     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  1993 Aug 
Date Detail:
Created Date:  1993-12-03     Completed Date:  1993-12-03     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  29-33     Citation Subset:  IM    
Affiliation:
Unidad de Hepatología, Experimental, Research Center, University Hospital La Fe, Valencia, Spain.
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MeSH Terms
Descriptor/Qualifier:
Adult
Aged
Animals
Cells, Cultured
Cytochrome P-450 CYP2B1
Cytochrome P-450 Enzyme System / metabolism*
Female
Fluorometry
Humans
Liver / cytology,  enzymology*
Male
Microchemistry
Microsomes, Liver / enzymology
Middle Aged
Oxazines / analysis,  metabolism
Oxidoreductases / metabolism*
Rats
Rats, Sprague-Dawley
Reproducibility of Results
Sensitivity and Specificity
Substrate Specificity
Time Factors
Chemical
Reg. No./Substance:
0/Oxazines; 5725-91-7/ethoxyresorufin; 635-78-9/resorufin; 87687-03-4/pentoxyresorufin; 9035-51-2/Cytochrome P-450 Enzyme System; EC 1.-/Oxidoreductases; EC 1.14.14.1/Cytochrome P-450 CYP2B1

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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