A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates.
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.
Donato
M T
MT
Unidad de Hepatología, Experimental, Research Center, University Hospital La Fe, Valencia, Spain.
Gómez-Lechón
M J
MJ
Castell
J V
JV
D000328:Adult; D000368:Aged; D000818:Animals; D002478:Cells, Cultured; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D005470:Fluorometry; D006801:Humans; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D008832:Microchemistry; D008862:Microsomes, Liver / Q000201:enzymology; D008875:Middle Aged; D010078:Oxazines / Q000032:analysis / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013997:Time Factors
D010078:Oxazines; C007649:ethoxyresorufin; C014180:resorufin; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1