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CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

Abstract

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.

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    TNO BIBRA International Ltd, Carshalton, UK.

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    MeSH

    Animals
    Clofenapate
    Coumarins
    Cytochrome P-450 CYP1A1
    Cytochrome P-450 CYP1A2
    Cytochrome P-450 CYP2B1
    Cytochrome P-450 Enzyme System
    Dexamethasone
    Enzyme Induction
    Glucocorticoids
    Glucuronic Acid
    Isoenzymes
    Isoniazid
    Kinetics
    Liver
    Male
    Phenobarbital
    Rats
    Rats, Sprague-Dawley
    Recombinant Proteins
    Substrate Specificity
    Sulfates
    beta-Naphthoflavone

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    11037111

    Citation

    Price, R J., et al. "CYP Isoform Induction Screening in 96-well Plates: Use of 7-benzyloxy-4-trifluoromethylcoumarin as a Substrate for Studies With Rat Hepatocytes." Xenobiotica; the Fate of Foreign Compounds in Biological Systems, vol. 30, no. 8, 2000, pp. 781-95.
    Price RJ, Surry D, Renwick AB, et al. CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes. Xenobiotica. 2000;30(8):781-95.
    Price, R. J., Surry, D., Renwick, A. B., Meneses-Lorente, G., Lake, B. G., & Evans, D. C. (2000). CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes. Xenobiotica; the Fate of Foreign Compounds in Biological Systems, 30(8), pp. 781-95.
    Price RJ, et al. CYP Isoform Induction Screening in 96-well Plates: Use of 7-benzyloxy-4-trifluoromethylcoumarin as a Substrate for Studies With Rat Hepatocytes. Xenobiotica. 2000;30(8):781-95. PubMed PMID: 11037111.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes. AU - Price,R J, AU - Surry,D, AU - Renwick,A B, AU - Meneses-Lorente,G, AU - Lake,B G, AU - Evans,D C, PY - 2000/10/19/pubmed PY - 2001/2/28/medline PY - 2000/10/19/entrez SP - 781 EP - 95 JF - Xenobiotica; the fate of foreign compounds in biological systems JO - Xenobiotica VL - 30 IS - 8 N2 - 1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format. SN - 0049-8254 UR - https://www.unboundmedicine.com/medline/citation/11037111/CYP_isoform_induction_screening_in_96_well_plates:_use_of_7_benzyloxy_4_trifluoromethylcoumarin_as_a_substrate_for_studies_with_rat_hepatocytes_ L2 - http://www.tandfonline.com/doi/full/10.1080/00498250050119844 DB - PRIME DP - Unbound Medicine ER -