In a human pilot intervention study (healthy + ileostomy probands), the questions were addressed whether in vivo consumption of an anthocyanin-rich bilberry (Vaccinium myrtillius L.) pomace extract (BE) affects (i) the transcription of Nrf2-dependent genes in peripheral blood mononuclear cells (PBMC), indicative for systemic effects, and (ii) the level of oxidative DNA damage in these cells. In healthy test subjects transcripts of NAD(P)H quinone oxidoreductase 1 (NQO1) were significantly elevated throughout the observation period (1-8 h), whereas transcription of heme oxygenase 1 (HO-1) and Nrf2 was significantly decreased. NQO1 and HO-1 transcription remained unchanged in the ileostomy probands, whereas Nrf2-transcription was suppressed in both groups. Decrease in oxidative DNA damage was observed 2 h after BE consumption again only in healthy subjects. In vitro studies using a reporter gene approach (CHO) and qPCR (HT29) indicate that not the intact anthocyanins/anthocyanidins are the activating constituents but the intestinal degradation product phloroglucinol aldehyde (PGA). Taken together, consumption of anthocyanin-rich BE was found to modulate Nrf2-dependent gene expression in PBMCs indicative for systemic activity. Limitation of the effect to healthy test subjects suggests a role of colonic processes for bioactivity, supported by the results on Nrf2-activating properties of the intestinal anthocyanin degradation product PGA.

Alternariol (AOH) and altertoxin-II (ALTX-II) have been demonstrated to possess genotoxic properties. However, the underlying mechanisms of action have not been fully elucidated yet. AOH has recently been shown to act as a topoisomerase I and II poison, contributing to its genotoxic properties. The topoisomerase-specific repair factor tyrosyl-DNA-phosphodiesterase-1 (TDP1) is involved in the respective repair processes of damaged DNA induced by topoisomerase II poison. In the present study, we investigated the role of DNA repair pathways for the extent of DNA damage by AOH and addressed the question whether interference with topoisomerase II might play a role in the genotoxicity of ALTX-II. Under cell-free conditions, AOH and ALTX-II suppressed the activity of topoisomerase II at a comparable concentration range. In HT29 cells, AOH enhanced the level of covalent DNA-topoisomerase II complexes, thus acting as a topoisomerase poison in DNA damaging concentrations. In contrast, ALTX-II in genotoxic concentrations did not show any effect on the stability of these complexes, indicating that interference with topoisomerases does not play a relevant role in genotoxicity. The differences in genotoxic mechanisms seem to be reflected in the activation of p53. AOH was found to increase p53 phosphorylation in HT29 cells in DNA damaging concentrations. In contrast, incubation with ALTX-II did not affect p53 phosphorylation despite substantial increase in tail intensity in the comet assay, suggesting that the DNA lesions formed by ALTX-II are not detected by the DNA-repair machinery of HT29 cells. These results are supported by differences in persistence of DNA damage, still maintained after 24 h for ALTX-II but nearly vanished already after 3 h for AOH. Furthermore, microarray and qPCR analysis did not indicate any substantial impact of AOH on the transcription of key elements of DNA repair pathways. However, siRNA-approaches indicate that, in addition to TDP1, the expression of other elements of the DNA repair machinery exemplified by the 70 kDa Ku autoantigen and the proliferating cell nuclear antigen are relevant for AOH-mediated DNA damage.

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 TDP1(H263A) 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 TDP1(H263A). 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.

In cell culture were compared the different release rates of anthocyanins from a bilberry pomace extract encapsulated either in food grade whey protein-based matrix capsules (WPC) or in pectin amid-based hollow spherical capsules (PHS). The impact of the formulations on typical anthocyanin-associated biological end points such as inhibition of the epidermal growth factor receptor (EGFR) and suppression of cell growth in HT29 colon carcinoma cells was assessed. The purpose was to find whether the release rates are sufficient to maintain biological activity and whether encapsulation affected EGFR inhibitory and growth suppressive properties of the extract. Even though anthocyanin release from extract-loaded capsules was proven under cell culture conditions, the inhibitory potential toward the EGFR was diminished. However, nonencapsulated extract as well as both extract-loaded encapsulation systems diminished the growth of HT29 cells to a comparable extent. The loss of EGFR inhibitory properties by encapsulation despite anthocyanin release indicates substantial contribution of other further constituents not monitored so far. Taken together, both applied encapsulation strategies allowed anthocyanin release and maintained biological activity with respect to growth inhibitory properties. However, the loss of EGFR inhibitory effects emphasizes the need for biological profiling to estimate process-induced changes of plant constituent's beneficial potencies.