Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism

M H Grant, M D Burke, G M Hawksworth, S J Duthie, J Engeset, J C Petrie

Research output: Contribution to journalArticle

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Abstract

The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.
Original languageEnglish
Pages (from-to)2311-6
Number of pages6
JournalBiochemical Pharmacology
Volume36
Issue number14
Publication statusPublished - 1987

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Cytochrome P-450 CYP2B1
Mixed Function Oxygenases
Metabolism
Hepatocytes
Monolayers
Maintenance
Cytochrome P-450 CYP1A1
Phenolphthalein
Pharmaceutical Preparations
Cytochrome P-450 Enzyme System
Cytochrome-B(5) Reductase
Dealkylation
Drug Stability
Epoxide Hydrolases
Sulfotransferases
Glucuronosyltransferase
NADPH-Ferrihemoprotein Reductase
Uridine Diphosphate
Enzyme activity
Phenobarbital

Keywords

  • Adult
  • Biotransformation
  • Cells, Cultured
  • Cytochrome P-450 Enzyme System
  • Female
  • Glucuronosyltransferase
  • Glutathione Transferase
  • Humans
  • Isoenzymes
  • Liver
  • Male
  • Metabolic Detoxication, Drug
  • Mixed Function Oxygenases
  • Phenobarbital

Cite this

Grant, M. H., Burke, M. D., Hawksworth, G. M., Duthie, S. J., Engeset, J., & Petrie, J. C. (1987). Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism. Biochemical Pharmacology, 36(14), 2311-6.

Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism. / Grant, M H; Burke, M D; Hawksworth, G M; Duthie, S J; Engeset, J; Petrie, J C.

In: Biochemical Pharmacology, Vol. 36, No. 14, 1987, p. 2311-6.

Research output: Contribution to journalArticle

Grant, MH, Burke, MD, Hawksworth, GM, Duthie, SJ, Engeset, J & Petrie, JC 1987, 'Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism', Biochemical Pharmacology, vol. 36, no. 14, pp. 2311-6.
Grant, M H ; Burke, M D ; Hawksworth, G M ; Duthie, S J ; Engeset, J ; Petrie, J C. / Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism. In: Biochemical Pharmacology. 1987 ; Vol. 36, No. 14. pp. 2311-6.
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N2 - The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.

AB - The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.

KW - Adult

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KW - Isoenzymes

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KW - Male

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