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Hydrogen peroxide is a second messenger in phase 2 enzyme induction by cancer chemopreventive dithiolethiones
Ryan Holland1, Mettachit Navamal, Murugesan Velayutham
1Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA. CA91032
Abstract:
The ability of three dithiolethione cancer chemopreventives, oltipraz 1, anetholedithione (ADT) 2, 1,2-dithiole-3-thione (D3T) 3, and the major metabolite, 4, of 1, to induce the cytoprotective enzyme NQO1 in Hepa 1c1c7 cells and the inhibition of this induction by catalase are demonstrated. The ability of 1, 3, and 4 to form O(2)(*) has been reported, and it is here demonstrated that 2 decomposes in the presence of GSH to form, upon addition of the nitrone spin trap DMPO, the DMPO-OH adduct that is detectable by EPR. Decomposition of 2 in the presence of GSH elicits, upon the addition of hydroethidine and excitation at 510 nm, fluorescence at 580 nm that is diminished by the addition of superoxide dismutase. The compound 4, is a product of the reduction of 1, and it is demonstrated that 2 and 3 decompose in the presence of reductants such as thiolates and NaBH(4), followed by addition of CH(3)I, to form the dimethylated products of reductive cleavage of the S(1)-S(2) bond. The same products are isolated subsequent to lysis in buffer containing CH(3)I of Hepa 1c1c7 cells treated with 2 or 3. Reductive cleavage of 2 and 3 in aqueous ethanol by NaBH(4) in an argon atmosphere, followed by acidic destruction of remaining borohydride and neutralization and introduction of O(2) results in the reformation of 2 and 3 to the extent of 80 and 33%, respectively. The data in toto are consistent with a model in which dithiolethiones, generally, undergo reductive cleavage in Hepa 1c1c7 cells, thereby resulting in the generation of O(2)(*) that dismutates to H(2)O(2), that subsequently, by direct or indirect means, effects the nuclear translocation of transcription factor Nrf2, that upregulates phase 2 enzyme expression.
Insights
Three dithiolethione cancer chemopreventives, including oltipraz, anetholedithione, and 1,2-dithiole-3-thione, induce the enzyme NQO1. This induction involves reductive cleavage and superoxide generation, ultimately upregulating protective phase 2 enzyme expression.
Area of Science:
- Biochemistry
- Cancer Chemoprevention
- Cellular Signaling
Background:
- Dithiolethiones are a class of compounds investigated for cancer chemopreventive properties.
- Induction of cytoprotective enzymes like NQO1 is a key mechanism in chemoprevention.
- The role of reactive oxygen species (ROS) in cellular signaling pathways is complex and context-dependent.
Purpose of the Study:
- To investigate the ability of dithiolethiones (oltipraz, anetholedithione, 1,2-dithiole-3-thione) and a metabolite to induce the enzyme NQO1 in Hepa 1c1c7 cells.
- To elucidate the mechanism of NQO1 induction, focusing on the role of reductive cleavage and reactive oxygen species generation.
- To explore the involvement of catalase and superoxide dismutase in modulating these cellular responses.
Main Methods:
- Cell culture experiments using Hepa 1c1c7 cells.
- Enzyme induction assays for NQO1.
- Electron paramagnetic resonance (EPR) spectroscopy to detect radical adducts (DMPO-OH).
- Fluorescence spectroscopy to detect superoxide generation using hydroethidine.
- Chemical analysis to identify reductive cleavage products using thiolates, NaBH(4), and CH(3)I.
- In vitro studies on reductive cleavage and reformation of dithiolethiones.
Main Results:
- Oltipraz, anetholedithione, 1,2-dithiole-3-thione, and a metabolite induced NQO1 in Hepa 1c1c7 cells, an effect inhibited by catalase.
- Anetholedithione decomposed in the presence of glutathione (GSH) to generate superoxide radicals (O(2)(*)), confirmed by EPR and fluorescence assays.
- Dithiolethiones underwent reductive cleavage of the S-S bond in the presence of reductants, forming methylated products, and could reform under oxidative conditions.
Conclusions:
- Dithiolethiones undergo reductive cleavage within Hepa 1c1c7 cells, leading to the generation of superoxide radicals.
- Superoxide dismutates to hydrogen peroxide (H(2)O(2)), which, directly or indirectly, promotes Nrf2 nuclear translocation.
- This pathway culminates in the upregulation of phase 2 enzymes, contributing to the chemopreventive effects of dithiolethiones.
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