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Scope: A range of health benefits are attributed to consuming urolithin A (UA), such as improved muscle health, anti-aging activity, and neuroprotection, whereas few studies raise possible adverse effects at high doses, including genotoxicity and estrogenic effects. Therefore, understanding UA bioactivity and safety depends on its pharmacokinetics. However, there is no physiologically-based pharmacokinetic (PBPK) model available for UA, thus limiting reliable assessment of effects observed from in vitro experimentation., Methods and Results: We characterizes glucuronidation rates of UA by human S9 fractions. Partitioning and other physicochemical parameters are predicted using quantitative structure-activity relationship tools. Solubility and dissolution kinetics are determined experimentally. These parameters are used to construct a PBPK model, and results are compared with data from human intervention studies. We evaluates how different supplementation scenarios may influence UA plasma and tissue concentrations. Concentrations at which either toxic or beneficial effects are previously observed in vitro appear unlikely to be achieved in vivo., Conclusion: A first PBPK model for UA is established. It enables prediction of systemic UA concentrations and is critical for extrapolating in vitro results to in vivo uses. Results support the safety of UA, but also challenge the potential for readily achieving beneficial effects by postbiotic supplementation., (© 2023 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

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)., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: Although associated with anti-oxidative properties, genistein has been reported to induce DNA strand breaks, whereby oxidative stress and topoisomerase poisoning are considered as potential mechanisms. In contrast, delphinidin, a catalytic topoisomerase inhibitor, is known to suppress the DNA-damaging properties of several topoisomerase poisons. Recently, alternariol, a mycotoxin produced by Alternaria spp., was found not only to induce oxidative stress but also to act as a topoisomerase poison. As both, polyphenols and mycotoxins, might occur in our nutrition simultaneously, the question was addressed whether potential combinatory effects on DNA integrity have to be considered., Methods and Results: We determined combinatory effects of either genistein or delphinidin with alternariol in HT-29 cells. Cytotoxicity was assessed by WST-1 and SRB assays, whereby only weak interactions were observed. The comet assay revealed significant antagonistic interactions of both polyphenols with the genotoxicity of AOH. The underlying mechanism comprises the suppression of alternariol-mediated stabilization of DNA/topoisomerase-II-intermediates, as observed in the ICE assay. Furthermore, DEL but not GEN was found to suppress AOH-mediated oxidative stress., Conclusion: Our data indicate that a respective polyphenol-rich diet might aid to protect against genotoxic damages caused by AOH, whereby bioactive concentrations of DEL are predominantly expected locally in the intestines., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthesis of various new structures of a library of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines (BP) and 11-substituted 6-aminobenzo[c]phenanthridines (BP-D) is presented. These structures, further synthetic modifications, and the preparation of follow-up products which delivered about 40 new derivatives are described. Their potential as antiproliferative drug candidates was investigated by comparison of NCI 60 developmental therapeutics program (DTP) human tumor cell line screening data based on the results of in vitro tumor cell growth inhibition, including about 40 hitherto unpublished compound test results with up to 60 cancer cell lines. NCI-COMPARE studies helped to suggest the modes of action of the highly active antiproliferative drugs. These findings are supported by in vitro biological investigations showing either inhibition of tubulin polymerization and depolymerization or topoisomerase inhibition. Together with physicochemical parameters of the drug candidates, structure-activity relationships are critically discussed. Tubulin interaction or inhibition of topoisomerase I and IIα/β activity are two rationales that can explain the antiproliferative activity observed in the NCI 60 DTP human tumor cell line screen. However, it can also be reasonably assumed that these compounds address several targets, thus prohibiting the identification of simple structure-activity relationships. The new structures described herein are thought to act as so-called multitarget drugs, thus being of special interest in the area of multidrug resistance., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been described as an important autoantigen in rheumatoid arthritis (RA) since it is targeted by autoantibodies, autoreactive T cells, and is aberrantly expressed in synovial cells in patients. To identify hnRNP-A2-specific T-cell epitopes possibly associated with pathogenicity, we used an innovative approach. We first scanned 280 overlapping hnRNP-A2 peptides for binding to the RA-associated class II molecules HLA-DR4 and HLA-DR1, leading to a comprehensive selection of binders. The selected peptides were tested in IFN-gamma-specific ELISPOT assay: PBMC from 18% of RA patients showed a significant IFN-gamma response to hnRNP-A2 peptides, 15% to the overlapping sequences 117-133 and/or 120-133, whereas PBMC from healthy individuals tested negative. We measured proliferative responses to these two peptides in another cohort of patients with RA or osteoarthritis: positive responses were found in 28% of RA, but also in 11% of osteoarthritis patients and these responses could be blocked by anti-MHC class II Ab. Remarkably, the presence of 117/120-133-specific T cells was significantly associated with active disease in RA patients, and bone erosion appeared to be more common in T-cell positive patients. These data suggest involvement of hnRNP-A2 specific cellular autoimmune responses in RA pathogenesis.