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Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and
T M Buetler1, E P Gallagher, C Wang
1Department of Environmental Health, University of Washington, Seattle 98195, USA.
Abstract:
Various natural and synthetic compounds are known to protect against cancer by elevating phase II detoxification enzymes. Generally classified as monofunctional, these inducers are believed to trigger cellular signal(s) that activate gene transcription through an antioxidant or electrophile response element (ARE/EpRE) in responsive genes. In contrast, the phase I enzymes of drug metabolism (cytochrome P450s) are not believed to be induced by monofunctional inducers and P450 genes have not been found to contain functional ARE/EpREs. In this study, rats were treated with the monofunctional inducers tert-butylated hydroxyanisole, ethoxyquin, and oltipraz to study the inducibility of individual glutathione S-transferase isozymes, NADP(H):quinone oxidoreductase, gamma-glutamylcysteine synthetase, UDP-glucuronosyl transferase, and cytochrome P450 enzymes. Hepatic mRNAs were analyzed on Northern blots using gene-specific oligonucleotide probes for GST Ya1, Ya2, Yc1, Yc2, Yb1, Yb2, and Yf, for UGT 1*06, and for P450 1A1, 1A2, 2B1, 2C11, 3A2, and 4A1. NADP(H):quinone oxidoreductase and gamma-glutamylcysteine synthetase mRNAs were detected using cDNA probes. All the phase II detoxification enzymes analyzed, except GST Yf, were induced by the three monofunctional inducers, suggesting that these genes may be regulated by a mechanism involving an ARE/EpRE element in their promoter region. Interestingly, it was found that ethoxyquin was a particularly good inducer for both members of the P450 2B family, 2B1 and 2B2, and both ethoxyquin and oltipraz were also capable of modestly inducing P450 1A2 and 3A2. Oltipraz was found to slightly induce P450 2B2, but not 2B1, at the dose and time analyzed. Induction of mRNA generally correlated well with induction of protein levels determined by Western blot and/or enzyme activity measurements for selected enzymes. The results of this study suggest that many phase II enzymes may contain ARE/EpRE elements in addition to those confirmed to be regulated by a mechanism involving ARE/EpRE elements. In addition, it was found that several P450 enzymes were induced by monofunctional inducers, suggesting a possibility that some phase I enzymes may also be regulated by a mechanism involving ARE/EpRE elements.
Insights
Monofunctional inducers, like ethoxyquin and oltipraz, activate phase II detoxification enzymes and surprisingly also some phase I cytochrome P450 enzymes. This suggests a broader role for the antioxidant/electrophile response element (ARE/EpRE) in regulating these crucial metabolic genes.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cancer chemoprevention often involves compounds that boost phase II detoxification enzymes.
- These inducers typically activate genes via antioxidant/electrophile response elements (ARE/EpRE).
- Phase I drug-metabolizing enzymes (cytochrome P450s) were not thought to be induced by these inducers or possess ARE/EpREs.
Purpose of the Study:
- To investigate the induction of various phase II and phase I enzymes by monofunctional inducers.
- To explore the potential role of ARE/EpRE in regulating these enzymes.
Main Methods:
- Rats were treated with tert-butylated hydroxyanisole, ethoxyquin, and oltipraz.
- Hepatic mRNA levels for specific glutathione S-transferase (GST), UDP-glucuronosyl transferase (UGT), and cytochrome P450 (P450) enzymes were analyzed using Northern blots and cDNA probes.
- Protein levels and enzyme activities were measured for selected enzymes.
Main Results:
- All analyzed phase II enzymes, except GST Yf, were induced by the inducers.
- Ethoxyquin strongly induced P450 2B1 and 2B2; ethoxyquin and oltipraz induced P450 1A2 and 3A2.
- Oltipraz showed modest induction of P450 2B2.
- mRNA induction generally correlated with protein and activity levels.
Conclusions:
- Monofunctional inducers activate a broad range of phase II detoxification enzymes, suggesting common ARE/EpRE regulatory elements.
- Several phase I cytochrome P450 enzymes were also induced, indicating that ARE/EpRE elements may regulate some phase I enzymes as well.