Document Detail


Primary culture of rat hepatocytes in 96-well plates: effects of extracellular matrix configuration on cytochrome P450 enzyme activity and inducibility, and its application in in vitro cytotoxicity screening.
MedLine Citation:
PMID:  17141466     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Basal level enzyme activities and enzyme inducibility were compared for rat hepatocytes that were cultured in 96-well plates with three different extracellular matrix configurations: single layer (SL) collagen type I, SL Matrigel, and collagen/Matrigel (C/M) sandwich. Overall, C/M sandwich and SL Matrigel plates were both superior to SL collagen type I plates in maintaining enzyme activities and inducibility and C/M sandwich plates had higher induced activity for CYP3A enzymes than SL Matrigel plates did. Cytotoxicity of nine reference compounds to rat hepatocytes (C/M sandwich configuration), rat hepatoma H4IIE and mouse fibroblast Balb/c 3T3 (3T3) cells was evaluated in 96-well plates using neutral red uptake (for 3T3) and tetrazolium salt MTS assays (for H4IIE and rat hepatocytes). For compounds chlorpromazine, quinidine, trichlorfon, thiopental, and antipyrine, the absolute differences in cytotoxicity LogIC(50) values obtained from different cell types were relatively small and without an obvious trend. The DeltaLogIC(50) values between cultured hepatocytes and the cell lines were much larger for acetaminophen and cyclophosphamide (1.35 < or =/DeltaLogIC(50)/ < or = 3.40), and for clofibrate and thioacetamide (not cytotoxic in hepatocytes at their highest dose levels). These large differences were likely the result of metabolism of these compounds in rat hepatocytes. The relationship between in vitro cytotoxicity LogIC(50) values and in vivo mouse or rat oral acute LogLD(50) values showed that compared to the cell lines, cultured rat hepatocytes improved correlation for acetaminophen and cyclophosphamide. The potential benefit of conducting in vitro cytotoxicity screening using a combination of permanent cell lines and cultured hepatocytes would allow us to obtain mechanistic insight on bioactivation, as well as improve the predictability of metabolism-mediated toxicity.
Authors:
Robert T Mingoia; Diane L Nabb; Ching-Hui Yang; Xing Han
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Publication Detail:
Type:  Journal Article     Date:  2006-10-28
Journal Detail:
Title:  Toxicology in vitro : an international journal published in association with BIBRA     Volume:  21     ISSN:  0887-2333     ISO Abbreviation:  Toxicol In Vitro     Publication Date:  2007 Feb 
Date Detail:
Created Date:  2006-12-12     Completed Date:  2007-04-09     Revised Date:  2009-04-10    
Medline Journal Info:
Nlm Unique ID:  8712158     Medline TA:  Toxicol In Vitro     Country:  England    
Other Details:
Languages:  eng     Pagination:  165-73     Citation Subset:  IM    
Affiliation:
DuPont Haskell Laboratory for Health and Environmental Sciences, P.O. Box 50, 1090 Elkton Road, Newark, Delaware 19714, USA.
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MeSH Terms
Descriptor/Qualifier:
3T3 Cells
Animals
Cell Line, Tumor
Cell Separation
Cells, Cultured
Cytochrome P-450 Enzyme System / biosynthesis*,  metabolism*
Drug Screening Assays, Antitumor / methods*
Enzyme Induction / drug effects
Extracellular Matrix / enzymology*,  ultrastructure*
Fibroblasts / drug effects
Hepatocytes / enzymology*,  ultrastructure*
Liver Neoplasms, Experimental / pathology
Mice
Mice, Inbred BALB C
Rats
Rats, Sprague-Dawley
Reproducibility of Results
Chemical
Reg. No./Substance:
9035-51-2/Cytochrome P-450 Enzyme System

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


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