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Cafestol and kahweol are diterpene compounds present in unfiltered coffees. Cafestol is known as the most potent cholesterol-raising agent that may be present in the human diet. Remarkably, the mechanisms behind this effect have only been partly resolved so far. Even less is known about the metabolic fate of cafestol and kahweol. From the structure of cafestol, carrying a furan moiety, we hypothesized that epoxidation may not only be an important biotransformation route but that this also plays a role in its effects found. In bile duct-cannulated mice, dosed with cafestol, we were able to demonstrate the presence of epoxy-glutathione (GSH) conjugates, GSH conjugates and glucuronide conjugates. In addition, it was shown that cafestol was able to induce an electrophile-responsive element (EpRE). Using a murine hepatoma cell line with a luciferase reporter gene under control of an EpRE from the human NQO1 regulatory region, we also found that metabolic activation by CYP450 enzymes is needed for EpRE induction. Furthermore, raising intracellular GSH resulted in a decrease in EpRE-mediated gene induction, whereas lowering intracellular GSH levels increased EpRE-mediated gene induction. In conclusion, evidence suggests that cafestol induces EpRE, apparently via a bioactivation process that possibly involves epoxidation of the furan ring. The epoxides themselves appear subject to conjugation with GSH. The effects on EpRE can also explain the induction of GSH which seems to be involved in the reported beneficial effects of cafestol, for example, when administered with aflatoxin B1 or other toxic or carcinogenic compounds., (Copyright 2010 Elsevier Inc. All rights reserved.)

Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)-LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC(50) (0.8-3.2 microM) and a comparable induction factor (17-22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC(50) 3.9-6.5 microM, induction factor 17.5-23). After 24h of exposure, on average (+/-SD) 23+/-5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression.

In the present study, the ability of green tea catechins to induce electrophile-responsive element (EpRE)-mediated gene expression and the role of their quinones in the mechanism of this induction were investigated. To this end, Hepa1c1c7 mouse hepatoma cells were used, stably transfected with a luciferase reporter gene under the expression regulation of an EpRE from the human NAD(P)H:quinone oxidoreductase 1 (NQO1) gene. The results obtained show that several, but not all, catechins tested are able to induce EpRE-mediated gene transcription, with epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG), both containing a pyrogallol and a galloyl moiety, being the most powerful inducers. Moreover, it was demonstrated that the EpRE-mediated response to catechins was increased in cells with reduced cellular glutathione (GSH) levels and decreased in cells with increased levels of GSH, corroborating a role for catechin quinones. The intrinsic capacity of catechins to form quinone type metabolites upon their oxidation was demonstrated using incubations of epigallocatechin (EGC) and EGCG with tyrosinase and the GSH-trapping method. Glutathione conjugates formed in these incubations were identified as 2'-glutathionyl-EGC, 2',6'-diglutathionyl-EGC, 2'-glutathionyl-EGCG, and 2',6'-diglutathionyl-EGCG, supporting the formation of quinone type metabolites involving especially the pyrogallol moiety of these catechins. Formation of the EGCG-quinone-glutathionyl adducts was also observed in the EpRE-LUX cellular system. This further supports the importance of the pyrogallol moiety for the quinone chemistry of the catechins. Finally, the presence of the pyrogallol moiety in the catechins also results in a relatively lower half-wave oxidation potential (E1/2) and calculated heat of formation (DHF) for conversion of the catechins to their corresponding quinones, pointing at an increased ability to become oxidized. Altogether, our studies reveal that catechins, especially those containing a pyrogallol moiety, induce EpRE-mediated detoxifying gene expression and that this induction is likely to be the result of their quinone chemistry.

Binding and activation of the aryl hydrocarbon receptor (AhR) is thought to be an essential step in the toxicity of the environmental pollutants dioxins and dioxin-like PCBs. However, also a number of natural compounds, referred to as NAhRAs (natural Ah-receptor agonists), which are present in, for example, fruits and vegetables, can bind and activate this receptor. To study their potential effects in humans, we first investigated the effect of the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gene expression in ex vivo exposed freshly isolated human lymphocytes, and compared the resulting gene expression profile with those caused by the well-known NAhRA indolo[3,2-b]carbazole (ICZ), originating from cruciferous vegetables, and by a hexane extract of NAhRA-containing grapefruit juice (GJE). Only ICZ induced a gene expression profile similar to TCDD in the lymphocytes, and both significantly up-regulated CYP1B1 and TIPARP (TCDD-inducible poly (ADP-ribose) polymerase) mRNA. Next, we performed a human intervention study with NAhRA-containing cruciferous vegetables and grapefruit juice. The expression of the prototypical AhR-responsive genes CYP1A1, CYP1B1 and NQO1 in whole blood cells and in freshly isolated lymphocytes was not significantly affected. Also enzyme activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO), as judged by caffeine metabolites in urine, were unaffected, except for a small down-regulation of NAT2 activity by grapefruit juice. Examination of blood plasma with DR CALUX showed a 12% increased AhR agonist activity 3 and 24 h after consumption of cruciferous vegetables, but did not show a significant effect of grapefruit juice consumption. We conclude that intake of NAhRAs from food may result in minor AhR-related effects measurable in human blood and urine.

Flavonoids are important bioactive compounds, omnipresent in the human diet, and are reported to be bifunctional inducers. These phytochemicals are able to induce xenobiotic-responsive element (XRE)- and electrophile-responsive element (EpRE)-mediated gene expression, resulting in the induction of biotransformation enzymes. To test whether flavonoid-induced EpRE-mediated gene expression could be the result of upstream XRE-mediated gene expression, several flavonoids were tested for their ability to induce XRE- and EpRE-mediated gene expression using two stably transfected reporter gene cell lines constructed in the same mouse Hepa-1c1c7 hepatoma background. Although classified as bifunctional inducers, all flavonoids were found to induce EpRE- and XRE-mediated gene expression in a different concentration range, which presents an issue not considered by the current definition of a bifunctional inducer. At physiological relevant concentrations, the induction of gene expression via the EpRE transcriptional enhancer element is dominant, leading in particular to elevated levels of EpRE-regulated detoxifying enzymes. Furthermore, these results strongly suggest that EpRE-mediated gene expression induced by flavonoids is not a downstream reaction of XRE-mediated gene expression.

Flavonoids are important bioactive dietary compounds. They induce electrophile-responsive element (EpRE)-mediated expression of enzymes, such as NAD(P)H-quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs), which are major defense enzymes against electrophilic toxicants and oxidative stress. The induction of EpRE-mediated gene transcription involves the release of the transcription factor Nrf2 from a complex with Keap1, either by a direct interaction of the inducer with Keap1 or by protein kinase C (PKC)-mediated phosphorylation of Nrf2. The inhibition of PKC in Hepa1c1c7 cells, stably transfected with human NQO1-EpRE-controlled luciferase revealed that PKC is not involved in flavonoid-induced EpRE-mediated gene transcription. However, the ability of flavonoids to activate an EpRE-mediated response correlates with their redox properties characterized by quantum mechanical calculations. Flavonoids with a higher intrinsic potential to generate oxidative stress and redox cycling are the most potent inducers of EpRE-mediated gene expression. Modulation of the intracellular glutathione (GSH) level showed that the EpRE-activation by flavonoids increased with decreasing GSH and vice versa, supporting an oxidative mechanism. In conclusion, the pro-oxidant activity of flavonoids can contribute to their health-promoting activity by inducing important detoxifying enzymes, pointing to a beneficial effect of a supposed toxic chemical reaction.

In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.

Background: Many different mechanisms are involved in nutrient-related prevention of colon cancer. In this study, a comprehensive assessment of the spectrum of possible biological actions of the bioactive compound quercetin is made using multiple gene expression analysis. Quercetin is a flavonoid that can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci, although increase of number of colon tumors was also reported., Aim of the Study: In order to elucidate possible mechanisms involved in its mode of action the effect of quercetin on expression of 4000 human genes in Caco-2 cells was studied and related to functional effects., Methods: Caco-2 cells were exposed to 5 or 50 microM quercetin for 48 hours, differential expression of 4000 human genes was studied using microarrays and related to functional effects. Differentially expressed genes were categorized in seven functional groups: cell cycle and differentiation, apoptosis, tumor suppressor genes and oncogenes, cell adhesion and cell-cell interaction, transcription, signal transduction and energy metabolism. Also, cell proliferation and cell cycle distribution were measured., Results: Quercetin (5 microM) downregulated expression of cell cycle genes (for example CDC6, CDK4 and cyclin D1), downregulated cell proliferation and induced cell cycle arrest in Caco-2 cells. After exposure to 50 microM quercetin cell proliferation decreased to 51.3% of control, and further decrease of the percentage of cells in the G1 phase coincided with an increase of the percentage of cells in the sub-G1 phase. Quercetin upregulated expression of several tumor suppressor genes. In addition, genes involved in signal transduction pathways like beta catenin/TCF signalling and MAPK signal transduction were influenced by quercetin., Conclusions: This study shows that large-scale gene expression analysis in combination with functional assays yields a considerable amount of information on (anti-)carcinogenic potential of food components like quercetin.