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CYP4A1 gene transfection studies and the peroxisome proliferator-activated receptor: development of a high-throughput assay to detect peroxisome proliferators

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Summary

An in vitro reporter gene assay has been established to examine cytochrome P4504A1 (CYP4A1) induction. A response element from the upstream region of the rat CYP4A1 gene containing a peroxisome proliferator response element (PPRE) has been linked to the chloramphenicol acetyl-transferase (CAT) gene in a reporter vector (1). This CYP4A1 reporter construct has been co-transfected into human HepG2 cells in the presence and absence of expression vectors encoding the transcription factors PPARα and RXRα. The assay employs calcium phosphate-DNA co-precipitate mediated transfection. Reporter gene products have been quantitated using chemiluminescent based assays. We have shown that, in the presence of PPARα, the above CYP4A1 construct is transcriptionally activated by a range of structurally different peroxisome proliferators including Wy-14,643, ciprofibrate, clofibric acid and nafenopin. Our future efforts will focus on the establishment of a high-throughput assay for the detection of peroxisome proliferators. Such an assay would provide an invaluable in vitro test for the screening of developmental drug candidates prior to in vivo studies.

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Authors and Affiliations

  1. Molecular Toxicology Group, School of Biological Sciences, University of Surrey, GU2 5XH, Guildford, Surrey, UK

    S. J. Giddings & G. G. Gibson

  2. Department of Drug Metabolism, Smith Kline Beecham Pharmaceuticals, Welwyn, UK

    S. E. Clarke

Authors
  1. S. J. Giddings
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  2. S. E. Clarke
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  3. G. G. Gibson
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Giddings, S.J., Clarke, S.E. & Gibson, G.G. CYP4A1 gene transfection studies and the peroxisome proliferator-activated receptor: development of a high-throughput assay to detect peroxisome proliferators. Eur. J. Drug Metab. Pharmacokinet. 22, 315–319 (1997). https://doi.org/10.1007/BF03190963

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