Response of human CYP1-luciferase plasmids to 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons.
The cytochrome P4501 gene family consists of two members, CYP1A1 and CYP1A2, that are induced by halogenated hydrocarbons and polycyclic aromatic hydrocarbons. The human CYP1 promoters and 5'-flanking sequences were cloned into luciferase expression vectors to develop cell lines that stably express luciferase activity in response to CYP1 gene induction. Plasmids were initially tested in transient transfection assays. Transient transfections resulted in high-level expression of luciferase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from both the CYP1 expression vectors, pLUC1A1 and pLUC1A2. In dose-response experiments, 10 nM TCDD caused a maximal induction of pLUC1A2-directed luciferase activity that was 10-fold over control. Maximal pLUC1A1-directed luciferase activity was 65-fold over control in cells treated with 10 nM TCDD. Stable integration of CYP1-luciferase-neo plasmids, pL1A1N and pL1A2N, into the human hepatoma cell line, HepG2, was achieved by selection with G418. G418-resistant colonies were isolated for both pL1A1N and pL1A2N plasmids. The pL1A2N transfectants showed basal-level luciferase activity, but were nonresponsive to treatment with 10 nM TCDD. These results are in contrast to the observed induction of pLUC1A2-mediated luciferase expression in transient transfection experiments. Stable integration of the human CYP1A2 gene sequences appears to silence the transcriptional activation by TCDD. The pL1A1N transfectants showed inducible luciferase activity and one cell line, referred to as 101L, was used to establish dose-response relationships for TCDD and various polycyclic aromatic hydrocarbons. Maximal induction occurred after treatment with 100 nM TCDD, 10 microM 3-methylcholanthrene, 50 microM benz[a]anthracene, and 50 microM benzo[a]pyrene. These studies illustrate the use of the CYP1A1-luciferase cell line for the study of structure-activity relationships.
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Department of Pharmacology, University of California, San Diego, La Jolla 92093-0812.
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D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011083:Polycyclic Compounds / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D014162:Transfection
D000072317:Polychlorinated Dibenzodioxins; D011083:Polycyclic Compounds; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases