Abstract
We examined the effects of several agents, including dietary flavonoids, on CYP1A1 expression utilizing a recently developed high-throughput screening system for assessing human cytochrome P450 (CYP) induction. HepG2 cells, stably integrated with regulatory regions of human CYP1A1, were treated with resveratrol, apigenin, curcumin, kaempferol, green tea extract (GTE), (−)-epigallocatechin gallate (EGCG), quercetin, and naringenin. Of these flavonoids, resveratrol produced the greatest increase in CYP1A1-mediated luciferase activity (10-fold), whereas GTE, apigenin, curcumin, and kaempferol produced 2- to 3-fold increases in activity. Compared with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole, or benzanthracene, where increases in luciferase activity ranged from 12- to 35-fold, these flavonoids exhibited weak agonist activity. The remaining compounds, EGCG, quercetin, and naringenin, produced negligible effects. Cotreatment of cells with TCDD and GTE, naringenin, and apigenin resulted in 58, 77, and 74% reductions, respectively, in TCDD-mediated CYP1A1 induction, indicating that these flavonoids exhibit potential antagonist activity toward the aryl hydrocarbon (Ah) receptor. Furthermore, results also suggest that GTE and apigenin possess Ah receptor antagonist and weak agonist activities. Thus, we have shown that a 96-well plate assay allowing high-throughput screening for P450 induction in less than 24 h was efficient in determining the effects of flavonoids on human CYP1A expression. Signal-to-noise ratios were low, and well-to-well and replicate variability was below 10%, allowing induction to be easily detected in this system. These features illustrate the reliability and feasibility of this high-volume screening system for identifying CYP inducers. Furthermore, results produced with the stable cell line were corroborated in HepG2 cells and primary cultures of human hepatocytes, suggesting that stably integrated cell lines harboring enhancer elements of P450 genes may be highly conducive to high-throughput screening.
Footnotes
-
This work was supported by National Institutes of Health Grants GM58287 (S.A.), GM49511 (J.R.), AA08990 (J.R.), and GM54477(L.Q.), by the Liver Transplant, Procurement, and Distribution System (DK62274), and by a grant (99A097) from the American Institute for Cancer Research (to L.Q.). Portions of this work were presented at the International Society for the Study of Xenobiotics (ISSX) meetings in Indianapolis, IN, October, 2000.
-
↵2 U. S. Food and Drug Administration. Guidance for Industry Drug metabolism/drug interaction studies in the drug development process: Studies in vitro. The Drug information Branch, Center for Drug Evaluation and Research. 1998. Ref Type: Unpublished Work.
- Abbreviations used are::
- CYP
- cytochrome P450
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- EGCG
- (−)-epigallocatechin gallate
- GTE
- green tea extract
- HTS
- high-throughput screening
- Ah
- aryl hydrocarbon
- AhR
- Ah receptor
- 3-MC
- 3-methylcholanthrene
- DMSO
- dimethylsulfoxide
- DMEM
- Dulbecco's modified Eagle's medium, FBS, fetal bovine serum
- Received January 26, 2001.
- Accepted April 17, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
