Impact of Oxidative Metabolism on the Cytotoxic and Genotoxic Potential of Genistein in Human Colon Cancer Cells.

SCOPE: Genistein (GEN) is known to be genotoxic via targeting topoisomerase-II (TOPII). Oxidative metabolism of GEN is shown to generate hydroxylated metabolites with catecholic structures. The present study focuses on the impact of oxidative metabolism of GEN, exemplified for 3'-hydroxygenistein (3'-OH-GEN) and 6-hydroxygenistein (6-OH-GEN), on topoisomerase interference and the resulting genotoxic potential in HT-29 human colon carcinoma cells. METHODS AND RESULTS: In a cell-free decatenation assay, 3'-OH-GEN slightly exceeds the TOPII-inhibiting potential of GEN. In HT-29 cells, its inhibitory action on TOPII does not differ from GEN, but it has greater activity with respect to causing DNA damage (measured by the comet assay), p53 activation (Western blot), apoptosis induction (ELISA), and cytotoxicity (WST-1 assay). This may to some extent be related to a stronger pro-oxidative potential of 3'-OH-GEN in comparison to GEN, as observed for the highest concentrations (DCF assay). 6-OH-GEN exerts much weaker toxic effects than GEN in cell-based assays, including TOPII poisoning, DNA strand-breaking potential, and ROS generation. This might in part arise from decreased cellular uptake of the metabolite, as measured by HPLC-DAD. CONCLUSION: Oxidative metabolism alters the toxicological potential of GEN. Depending on the site of oxidation, the toxicity of the parent compound is exceeded (3'-OH-GEN) or attenuated (6-OH-GEN).

Methyleugenol and oxidative metabolites induce DNA damage and interact with human topoisomerases.

Methyleugenol is a substituted alkenylbenzene found in several herbs and spices. It is classified by the European Union's Scientific Committee on Food as a genotoxic carcinogen. We addressed the biological mechanism of the genotoxic properties of methyleugenol and its oxidative metabolites. Methyleugenol and the oxidative metabolites significantly enhanced the DNA damage in human colon carcinoma cells (HT29). Methyleugenol did not affect the protein status of γH2AX, a biomarker of DNA double-strand breaks, whereas its metabolites methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol significantly increased the cellular phosphorylated H2AX level. Both of these metabolites also showed a significant induction of micronuclei in HT29 cells. Furthermore, we investigated whether topoisomerase interaction contribute to the observed effect on DNA integrity. Methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol inhibited the activity of recombinant topoisomerase I. In HT29 cells, neither methyleugenol nor the metabolites affected the level of topoisomerase protein bound to DNA, excluding a topoisomerase poisoning mode of action. In addition, 3'-oxomethylisoeugenol potently diminished the level of camptothecin-stabilized topoisomerase I/DNA intermediates and camptothecin-induced DNA strand breaks. In conclusion, it could be suggested that 3'-oxomethylisoeugenol may also interact with classical or food-borne topoisomerase I poisons, diminishing their poisoning effectiveness.

Inhibition of topoisomerase II by phase II metabolites of resveratrol in human colon cancer cells.

SCOPE: The cytotoxic and genotoxic potential of phase II metabolites of resveratrol (RSV) was investigated in human colon cells with special emphasis on human topoisomerase (TOP) II. METHODS AND RESULTS: Cell-free screening of topoisomerase II (TOPII) inhibition by the decatenation assay showed inhibitory potential for RSV (≥200 µM) and for the first time for the three human phase II metabolites RSV-3-sulfate (≥200 µM), RSV-3-glucuronide (≥100 µM) and RSV-disulfate (≥100 µM). Conjugation at the C4'-position (RSV-4'-sulfate and RSV-4'-glucuronide) resulted in loss of the inhibitory potential in this assay. Cell-based experiments with RSV and the most abundant metabolite in humans, RSV-3-Sulf, revealed no TOP poisoning in HT29 and Caco-2 cells up to 250 µM. Further, the phase II metabolite exhibited only minor effects in the comet assay and showed negligible cytotoxic effects and apoptotic potential after 1 and 24 h incubation. Fluorescence microscopy and HPLC-DAD analysis identified cellular uptake of RSV and of RSV-3-Sulf although to a lesser extent when compared to RSV. Furthermore, within the cells fractional deconjugation of RSV-3-Sulf to the parent compound was observed. CONCLUSION: Sulfate- and glucuronide-phase II metabolites might contribute to the genotoxic potential of RSV by inhibition of TOPII activity. By deconjugation at the target site RSV-3-Sulf might serve as a pool of the parent compound.

Resveratrol modulates the topoisomerase inhibitory potential of doxorubicin in human colon carcinoma cells.

Resveratrol (RSV) is currently being widely discussed as potentially useful for anticancer therapy in combination with classical chemotherapeutics, e.g., the topoisomerase II (TOP II) poison doxorubicin (DOX). However, there is still a lack of knowledge of possible interference at the target enzyme, especially since RSV itself has recently been described to act as a TOP poison. We therefore sought to address the question whether RSV affects DOX-induced genotoxic and cytotoxic effects with special emphasis on TOP II in HT-29 colon carcinoma cells. RSV was found to counteract DOX-induced formation of DNA-TOP-intermediates at ≥100 µM for TOP IIα and at 250 µM for TOP IIß. As a consequence, RSV modulated the DNA-strand breaking potential of DOX by mediating protective effects with an apparent maximum at 100 µM. At higher concentration ranges (≥200 µM) RSV diminished the intracellular concentrations of DOX. Nevertheless, the presence of RSV slightly enhanced the cytotoxic effects of DOX after 1.5 h and 24 h of incubation. Taken together, at least in cell culture RSV was found to affect the TOP-poisoning potential of DOX and to modulate its cytotoxic effectiveness. Thus, further studies are needed to clarify the impact of RSV on the therapeutic effectiveness of DOX under in vivo conditions.

Oxidative metabolism enhances the cytotoxic and genotoxic properties of the soy isoflavone daidzein.

SCOPE: Oxidative metabolism of daidzein (DAI) might result in the formation of hydroxylated metabolites. Here, we address the question whether these metabolites differ in their biological activity from the parent isoflavone, exemplified for the epidermal growth factor receptor and topoisomerase II, potentially resulting in an enhanced toxic profile. METHODS AND RESULTS: In contrast to DAI, 6-hydroxydaidzein (6-HO-DAI) and 8-hydroxydaidzein (8-HO-DAI) were found to inhibit the tyrosine kinase activity of the epidermal growth factor receptor in an ELISA-based test system, but showed no effects within cells. Further, the oxidative metabolites suppressed the catalytic activity of topoisomerase II in the decatenation assay. In the in vivo complexes of enzyme to DNA (ICE) bioassay, 6-HO-DAI and 8-HO-DAI did not affect the level of covalent topoisomerase II-DNA intermediates within HT29 cells, thus arguing for a catalytic inhibition of topoisomerase II rather than poisoning activity. In contrast to DAI, 6-HO-DAI and 8-HO-DAI significantly increased the rate of DNA strand breaks in HT29 cells after 24-h incubation and caused a cell cycle delay in S-phase. Differences were also observed between the oxidative metabolites, with only 6-HO-DAI inducing apoptosis but not 8-HO-DAI. CONCLUSION: These data indicate that oxidative metabolism of DAI generates metabolites with genotoxic properties where interference with topoisomerase II might play a role.

Topoisomerase II-targeting properties of a grapevine-shoot extract and resveratrol oligomers.

Grapevine-shoot extracts (GSE), containing trans-resveratrol and resveratrol oligomers, are commercially available as food supplements. Considering the topoisomerase-targeting properties of trans-resveratrol, the question of whether GSE affect these enzymes, thereby potentially causing DNA damage, was addressed. In a decatenation assay, GSE potently suppressed the catalytic activity of topoisomerase IIα (≥5 µg/mL). The resveratrol oligomers ε-viniferin, r2-viniferin, and hopeaphenol, isolated from GSE, were also identified as topoisomerase IIα inhibitors. In the in vivo complexes of enzyme to DNA (ICE) bioassay, neither GSE, r2-viniferin, nor hopeaphenol affected the level of enzyme-DNA intermediates in A431 cells, thus representing catalytic inhibitors rather than topoisomerase poisons. GSE caused moderate DNA strand breaks (≥25 µg/mL) in the comet assay. Taken together, GSE presumably acts as a catalytic inhibitor of topoisomerase II with r2-viniferin and hopeaphenol as potentially contributing constituents. However, the increase of FPG-sensitive sites points to an additional mechanism that may contribute to the DNA-damaging properties of GSE constituents.