Document Detail


The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes.
MedLine Citation:
PMID:  19330883     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The preparation of hepatocytes using the two-stage perfusion technique usually involves the use of collagenase (CII) alone or in combination with dispase (C/D) or trypsin inhibitor (CA/TI) as digestion enzymes. The effect of CII, C/D and CA/TI on cell viability, yield, cytochrome P450 mediated oxidation of testosterone, glucuronidation and sulfation of 7-hydroxycoumarin, glutathione content, glutathione-S-transferase activity and glutathione-conjugation capacity of hepatocytes has been assessed. Cytochrome P450 mediated oxidation of testosterone was significantly (p < 0.05) decreased with CII isolated hepatocytes (81.7 +/- 3.3 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (96.6 +/- 1.9 nmol/10(6) cells) or C/D (95.1 +/- 2.1 nmol/10(6) cells). In contrast, glutathione conjugation of the non-specific substrate 1-chloro-2,4-dinitrobenzene was significantly (p < 0.05) increased with CII isolated hepatocytes (56.9 +/- 5.9 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (36.0 +/- 3.7 nmol/10(6) cells) or C/D (31.6 +/- 3.7 nmol/10(6) cells). These findings have significant implications for the interpretation of metabolism data derived from hepatocytes in suspension, particularly in terms of glutathione conjugation of potentially toxic reactive intermediates of xenobiotic metabolism. Indeed, data presented show that the presence of trypsin inhibitor in the preparation of isolated rat hepatocytes significantly affects the formation of glutathione conjugates of reactive intermediate products of troglitazone metabolism.
Authors:
J A Sinclair; C Henderson; I Martin; M H Grant; J N A Tettey
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Chemico-biological interactions     Volume:  179     ISSN:  1872-7786     ISO Abbreviation:  Chem. Biol. Interact.     Publication Date:  2009 May 
Date Detail:
Created Date:  2009-03-27     Completed Date:  2009-05-06     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0227276     Medline TA:  Chem Biol Interact     Country:  Ireland    
Other Details:
Languages:  eng     Pagination:  256-62     Citation Subset:  IM    
Affiliation:
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, United Kingdom.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Separation / methods*
Cell Survival / drug effects
Collagenases / metabolism*
Cytochrome P-450 Enzyme System / metabolism*
Glutathione / metabolism
Glutathione Transferase / metabolism*
Hepatocytes / enzymology*,  metabolism
Male
Perfusion
Rats
Rats, Sprague-Dawley
Testosterone / metabolism
Time Factors
Trypsin Inhibitors / pharmacology
Chemical
Reg. No./Substance:
0/Trypsin Inhibitors; 58-22-0/Testosterone; 70-18-8/Glutathione; 9035-51-2/Cytochrome P-450 Enzyme System; EC 2.5.1.18/Glutathione Transferase; EC 3.4.24.-/Collagenases

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