Simone Baechler

Modulation of the cellular redox status by the Alternaria toxins alternariol and alternariol monomethyl ether.

The mycotoxin alternariol (AOH) has been reported to possess genotoxic properties, inducing enhanced levels of DNA damage after only 1 h of incubation. In the present study we addressed the question whether the induction of oxidative stress might contribute to the genotoxic effects of AOH or its naturally occurring monomethylether (AME). In the dichlorofluorescein (DCF) assay, treatment of HT29 cells for 1 h enhanced the formation of dichlorofluorescein, indicative for ROS formation. The total glutathione (tGSH) was transiently decreased. In accordance with the results of the DCF assay, AOH and AME enhanced the proportion of the transcription factor Nrf2 in the nucleus. Concomitantly, the Nrf2/ARE-dependent genes γ-glutamylcysteine ligase (γ-GCL) and glutathione-S-transferase (GSTA1/2) showed enhanced transcript levels. After 24 h of incubation this effect was also reflected on the protein level by an increase of GST activity. However, in spite of the positive DCF assay and the activation of the redox-sensitive Nrf2/ARE-pathway, the level of oxidative DNA damage, measured in the comet assay by the addition of formamidopyrimidine-DNA-glycosylase (fpg) remained unaffected. Of note, after 3 h of incubation no significant DNA damaging potential of AOH and AME was detectable, indicating either inactivation of the compounds or enhanced DNA repair. In summary, the mycotoxins AOH and AME were found to modulate the redox balance of HT29 cells but without apparent negative effect on DNA integrity.

Anthocyanin-rich extracts suppress the DNA-damaging effects of topoisomerase poisons in human colon cancer cells.

SCOPE The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. METHODS AND RESULTS The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 μg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 μg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 μg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 μg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 μg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 μg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. CONCLUSION These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.

Genotoxicity of dietary, environmental and therapeutic topoisomerase II poisons is uniformly correlated to prolongation of enzyme DNA residence

SCOPE DNA damage by genistein and etoposide is determined by the half-life of topoisomerase II-DNA linkage induced [Bandele O. J. and Osheroff N., Biochemistry 2008, 47, 11900]. Here, we test whether this applies generally to dietary flavonoids and therapeutic compounds enhancing topoisomerase II-DNA cleavage (Topo II poisons). METHODS AND RESULTS We compared the impact of Topo II poisons on DNA residence kinetics of biofluorescent human topoisomerases IIα and IIβ (delineating duration of the DNA-linked enzyme state) with histone 2AX phosphorylation (delineating DNA damage response). Prolongation of topoisomerase II-DNA residence was correlated to DNA damage response, whereas topoisomerase II-DNA linkage was not. Catalytic inhibitors stabilizing topoisomerase II on unbroken DNA also exhibited such a correlation, albeit at a lower level of DNA damage response. Therapeutic Topo II poisons had stronger and more durable effects on enzyme II DNA residence and elicited stronger DNA damage responses than natural or dietary ones. CONCLUSIONS Topoisomerase II-mediated DNA damage appears related to the prolongation of enzyme DNA residence more than to enzyme-DNA cleavage. Due to this reason, genistein and other tested natural and dietary Topo II poisons have a much lower genotoxic potential than therapeutic ones under the conditions of equal topoisomerase II-DNA linkage.

Synthesis, topoisomerase-targeting activity and growth inhibition of lycobetaine analogs

The plant alkaloid lycobetaine has potent topoisomerase-targeting properties and shows anticancer activity. Based on these findings, several lycobetaine analogs were synthesized mainly differing in their substituents at 2, 8 and 9 position and their biological activities were evaluated. The topoisomerase-targeting properties and cytotoxicity of these structural analogs were assessed in the human gastric carcinoma cell line GXF251L. Performing a plasmid relaxation assay, an increased inhibition of topoisomerase I was found with N-methylphenanthridinium chlorides bearing a 8,9-methylenedioxy moiety or a methoxy group in 2-position. Furthermore, quaternized phenanthridinium derivatives bearing either a 2-methoxy or a 8,9-methylenedioxy moiety in conjunction with a 2-hydroxy or 2-methoxy group display potent topoisomerase II inhibition as shown by decatenation of kinetoplast DNA. In general, the N-methylphenanthridinium chlorides possess more potency in inhibiting topoisomerase I than topoisomerase II. All quaternized derivatives also exhibited potent inhibition of tumor cell growth in the low micromolar concentration range. Hence, N-methylphenanthridinium compounds were found to represent a promising class of compounds, potently inhibiting both, topoisomerases I and II, and may be further developed into clinically useful topoisomerase inhibitors.

Anthocyanins suppress the cleavable complex formation by irinotecan and diminish its DNA strand breaking activity in the colon of Wistar rats

In the present study, the question was addressed whether anthocyanins interfere with the topoisomerase I poison irinotecan in vivo. In vivo complexes of enzyme to DNA bioassay was used to detect irinotecan-induced stabilization of topoisomerase I/DNA complexes and single cell gel electrophoresis to determine DNA-strand-break induction in the colon of male Wistar rats. Furthermore, analysis of anthocyanin concentrations in rat plasma and rat colon was included in the testing, demonstrating that anthocyanins reach the colon and the concentrations do not differ between rats that only received anthocyanins and the anthocyanin/irinotecan group. Blackberry extract was found to significantly reduce irinotecan-mediated topoisomerase I/DNA cleavable complex formation. Overall, anthocyanins did not notably increase cleavable complex formation. However, a significant increase of DNA damage was shown after a single dose of irinotecan as well as the single compounds cyanidin (cy) and cyanidin-3-glucoside (cy-3-g). Furthermore, a significant reduction of irinotecan-induced DNA-strand breaks after a pretreatment with cy, cy-3-g and blackberry extract was observed. Thus, the question arises whether anthocyanin-rich preparations might interfere with chemotherapy or whether, due to low systemic bioavailability, the preparations might provide protective potential in the gastrointestinal tract.

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.

Repair of DNA damage induced by the mycotoxin alternariol involves tyrosyl-DNA phosphodiesterase 1

Alternariol (AOH) was reported recently to act as a topoisomerase poison. To underline the relevance of topoisomerase targeting for the genotoxic properties of AOH, we addressed the question whether human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital to the repair of covalent DNA-topoisomerase adducts, affects AOH-mediated genotoxicity. The relevance of TDP1 activity on AOH-induced genotoxicity was investigated by the comet assay in human cells overexpressing GFP chimera of TDP1 or the inactive mutant TDP1H263A as well as in cells subjected to siRNA-mediated knock-down of endogenous TDP1. Cells overexpressing TDP1 exhibited significantly less DNA damage after treatment with AOH in comparison to cells expressing the inactive mutant TDP1H263A. In accordance with these results, levels of AOH inducing DNA strand breaks were increased in TDP1-suppressed cells in comparison to cells transfected with control siRNA. The specific topoisomerase poisons camptothecin and etoposide caused comparable effects, underlining that TDP1 plays an important role in the repair of topoisomerase-mediated DNA damage. In summary, the repair enzyme TDP1 was identified as a factor for the modulation of AOH-mediated DNA damage in human cells.

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.