The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes

J.A. Sinclair, C.J. Henderson, I.K. Martin, M.H. Grant, J.N.A. Tettey

Research output: Contribution to journalArticle

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Abstract

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.
LanguageEnglish
Pages256-262
Number of pages7
JournalChemico-Biological Interactions
Volume179
Issue number2-3
DOIs
Publication statusPublished - 15 May 2009

Fingerprint

Rats
Hepatocytes
Glutathione
Enzymes
Metabolism
7-hydroxycoumarin
Trypsin Inhibitors
troglitazone
Cytochrome P-450 Enzyme System
Testosterone
Dinitrochlorobenzene
Oxidation
Scanning electron microscopy
Poisons
Collagenases
Xenobiotics
Glutathione Transferase
Digestion
Cell Survival
Suspensions

Keywords

  • hepatocytes
  • collagenase
  • dispase
  • trypsin inhibitor
  • glutathione
  • reactive intermediates

Cite this

Sinclair, J.A. ; Henderson, C.J. ; Martin, I.K. ; Grant, M.H. ; Tettey, J.N.A. / The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes. In: Chemico-Biological Interactions. 2009 ; Vol. 179, No. 2-3. pp. 256-262.
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The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes. / Sinclair, J.A.; Henderson, C.J.; Martin, I.K.; Grant, M.H.; Tettey, J.N.A.

In: Chemico-Biological Interactions, Vol. 179, No. 2-3, 15.05.2009, p. 256-262.

Research output: Contribution to journalArticle

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T1 - The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes

AU - Sinclair, J.A.

AU - Henderson, C.J.

AU - Martin, I.K.

AU - Grant, M.H.

AU - Tettey, J.N.A.

N1 - PMID: 19330883

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N2 - 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.

AB - 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.

KW - hepatocytes

KW - collagenase

KW - dispase

KW - trypsin inhibitor

KW - glutathione

KW - reactive intermediates

U2 - 10.1016/j.cbi.2009.01.013

DO - 10.1016/j.cbi.2009.01.013

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EP - 262

JO - Chemico-Biological Interactions

T2 - Chemico-Biological Interactions

JF - Chemico-Biological Interactions

SN - 0009-2797

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ER -