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A computational fluid dynamics modell is used for process evaluation and optimazion of reduction shafts within melting and direct reduction processes. The structure of the simulation modell and the optimization procedure are described. Examples are given for evaluation of lab scale experiments, recalculation of real plant operation phases and upscaling studies. [ABSTRACT FROM AUTHOR]

The Alternaria mycotoxins alternariol (AOH) and alternariol 9- O -monomethyl ether (AME) are pervasive food contaminants known to exert adverse effects in vitro, yet their toxicokinetics remain inadequately understood. Thus, this study endeavors to elucidate the qualitative and quantitative aspects of the phase I metabolism of AOH and AME. To pursue this goal, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-fortified porcine, rat, and human liver microsomes were incubated for 0-10 min with AOH or AME within a concentration range of 1-100 and 1-50 μM, respectively. The decline in the parent toxin concentration was monitored via liquid chromatography coupled to tandem mass spectrometry, whereas coupling to high-resolution mass spectrometry provided insights into the composition of the arising metabolic mixture. The collected quantitative data allowed us to calculate the hepatic intrinsic clearance rates of AOH and AME, marking a notable contribution to the field. Moreover, we unveiled interspecies differences in the pattern and rate of the phase I metabolism of the investigated mycotoxins. The presented findings lay the groundwork for physiologically based toxicokinetic modeling aimed at estimating local concentrations of these mycotoxins in specific organs, enhancing our understanding of their mode of action and adverse health effects.

Sphaeropsidins are iso-pimarane diterpenes produced by phytopathogenic fungi that display promising anticancer activities. Sphaeropsidin A, in particular, has been shown to counteract regulatory volume increase, a process used by cancer cells to avoid apoptosis. This study reports the hemi-synthesis of new lipophilic derivatives obtained by modifications of the C15,C16-alkene moiety. Several of these compounds triggered severe ER swelling associated with strong proteasomal inhibition and consequently cell death, a feature that was not observed with respect to mode of action of the natural product. Significantly, an analysis from the National Cancer Institute sixty cell line testing did not reveal any correlations between the most potent derivative and any other compound in the database, except at high concentrations (LC 50 ). This study led to the discovery of a new set of sphaeropsidin derivatives that may be exploited as potential anti-cancer agents, notably due to their maintained activity towards multidrug resistant models., (© 2024. The Author(s).)

There is a clear need to develop new approach methodologies (NAMs) that combine in vitro and in silico testing to reduce and replace animal use in chemical risk assessment. Physiologically based kinetic (PBK) models are gaining popularity as NAMs in toxico/pharmacokinetics, but their coverage of complex metabolic pathways occurring in the gut are incomplete. Chemical modification of xenobiotics by the gut microbiome plays a critical role in the host response, for example, by prolonging exposure to harmful metabolites, but there is not a comprehensive approach to quantify this impact on human health. There are examples of PBK models that have implemented gut microbial biotransformation of xenobiotics with the gut as a dedicated metabolic compartment. However, the integration of microbial metabolism and parameterization of PBK models is not standardized and has only been applied to a few chemical transformations. A challenge in this area is the measurement of microbial metabolic kinetics, for which different fermentation approaches are used. Without a standardized method to measure gut microbial metabolism ex vivo/in vitro, the kinetic constants obtained will lead to conflicting conclusions drawn from model predictions. Nevertheless, there are specific cases where PBK models accurately predict systemic concentrations of gut microbial metabolites, offering potential solutions to the challenges outlined above. This review focuses on models that integrate gut microbial bioconversions and use ex vivo/in vitro methods to quantify metabolic constants that accurately represent in vivo conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Janus kinase (JAK) inhibitors were recently identified as promising drug candidates for repurposing in Alzheimer's disease (AD) due to their capacity to suppress inflammation via modulation of JAK/STAT signaling pathways. Besides interaction with primary therapeutic targets, JAK inhibitor drugs frequently interact with unintended, often unknown, biological off-targets, leading to associated effects. Nevertheless, the relevance of JAK inhibitors' off-target interactions in the context of AD remains unclear., Methods: Putative off-targets of baricitinib and tofacitinib were predicted using a machine learning (ML) approach. After screening scientific literature, off-targets were filtered based on their relevance to AD. Targets that had not been previously identified as off-targets of baricitinib or tofacitinib were subsequently tested using biochemical or cell-based assays. From those, active concentrations were compared to bioavailable concentrations in the brain predicted by physiologically based pharmacokinetic (PBPK) modeling., Results: With the aid of ML and in vitro activity assays, we identified two enzymes previously unknown to be inhibited by baricitinib, namely casein kinase 2 subunit alpha 2 (CK2-α2) and dual leucine zipper kinase (MAP3K12), both with binding constant ( K d ) values of 5.8 μM. Predicted maximum concentrations of baricitinib in brain tissue using PBPK modeling range from 1.3 to 23 nM, which is two to three orders of magnitude below the corresponding binding constant., Conclusion: In this study, we extended the list of baricitinib off-targets that are potentially relevant for AD progression and predicted drug distribution in the brain. The results suggest a low likelihood of successful repurposing in AD due to low brain permeability, even at the maximum recommended daily dose. While additional research is needed to evaluate the potential impact of the off-target interaction on AD, the combined approach of ML-based target prediction, in vitro confirmation, and PBPK modeling may help prioritize drugs with a high likelihood of being effectively repurposed for AD., Highlights: This study explored JAK inhibitors' off-targets in AD using a multidisciplinary approach.We combined machine learning, in vitro tests, and PBPK modelling to predict and validate new off-target interactions of tofacitinib and baricitinib in AD.Previously unknown inhibition of two enzymes (CK2-a2 and MAP3K12) by baricitinib were confirmed using in vitro experiments.Our PBPK model indicates that baricitinib low brain permeability limits AD repurposing.The proposed multidisciplinary approach optimizes drug repurposing efforts in AD research., Competing Interests: P.S. and G.S. declare a potential financial conflict of interest as co‐founders of inSili.com LLC, Zurich, and in their role as scientific consultants to the pharmaceutical industry. M.L.F., L.S., H.B., G.A., S.J.S., and A.M.B. declare no conflicts of interest. Author disclosures are available in the supporting information., (© 2024 The Authors. Alzheimer's & Dementia: Translational Research & Clinical Interventions published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

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.)

Mycotoxins produced by Alternaria spp. act genotoxic in cell-based studies, but data on their toxicity in vivo is scarce and urgently required for risk assessment. Thus, male Sprague-Dawley rats received single doses of a complex Alternaria toxin extract (CE; 50 mg/kg bw), altertoxin II (ATX-II; 0.21 mg/kg bw) or vehicle by gavage, one of the most genotoxic metabolites in vitro and were sacrificed after 3 or 24 h, respectively. Using SDS-PAGE/Western Blot, a significant increase of histone 2a.X phosphorylation and depletion of the native protein was observed for rats that were exposed to ATX-II for 24 h. Applying RT-PCR array technology we identified genes of interest for qRT-PCR testing, which in turn confirmed an induction of Rnf8 transcription in the colon of rats treated with ATX-II for 3 h and CE for 24 h. A decrease of Cdkn1a transcription was observed in rats exposed to ATX-II for 24 h, possibly indicating tissue repair after chemical injury. In contrast to the observed response in the colon, no markers for genotoxicity were induced in the liver of treated animals. We hereby provide the first report of ATX-II as a genotoxicant in vivo . Deviating results for similar concentrations of ATX-II in a natural Alternaria toxin mixture argue for substantial mixture effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aichinger, Pahlke, Puntscher, Groestlinger, Grabher, Braun, Tillmann, Plasenzotti, Del Favero, Warth, Höger and Marko.)

Several Alternaria mycotoxins are believed to act as endocrine disruptive chemicals (EDCs), since they are reported to bind estrogen receptors in several experimental models. After ingestion of contaminated food commodities, the mycotoxins reach the intestine, where they come into direct contact with food constituents as well as the gut microbiota. Thus, the aim of the present work was to evaluate the modulatory potential of a complex extract of cultured Alternaria fungi (CE; containing eleven chemically characterized compounds) on the estrogenic signaling cascade of mammalian cells before and after anaerobic incubation with fecal slurries, in order to simulate an in vivo-like condition in the gut. Assessing alkaline phosphatase expression in Ishikawa cells as a measure for estrogenicity, we found the CE to partially quench the intrinsic estrogenic properties of fecal slurries and fecal waters, even after 3 h of fecal incubation. Investigation of the mechanisms underlying the effects observed carried out through an in vitro/in silico approach revealed the ability of the extract to decrease the ERα/ERβ nuclear ratio, while a possible action of the mycotoxins as ER-antagonists was excluded. Our results suggest that Alternaria mycotoxins might act as EDCs in vivo, and warrant further investigation in animal models., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Natural dibenzo-α-pyrones (DAPs) can be viewed from two opposite angles. From one angle, the gastrointestinal metabolites urolithins are regarded as beneficial, while from the other, the emerging mycotoxin alternariol and related fungal metabolites are evaluated critically with regards to potential hazardous effects. Thus, the important question is: can the structural characteristics of DAP subgroups be held responsible for distinct bioactivity patterns? If not, certain toxicological and/or pharmacological aspects of natural DAPs might yet await elucidation. Thus, this review focuses on comparing published data on the two groups of natural DAPs regarding both adverse and beneficial effects on human health. Literature on genotoxic, estrogenic, endocrine-disruptive effects, as well as on the induction of the cellular anti-oxidative defense system, anti-inflammatory properties, the inhibition of kinases, the activation of mitophagy and the induction of autophagy, is gathered and critically reviewed. Indeed, comparing published data suggests similar bioactivity profiles of alternariol and urolithin A. Thus, the current stratification into hazardous Alternaria toxins and healthy urolithins seems debatable. An extrapolation of bioactivities to the other DAP sub-class could serve as a promising base for further research. Conclusively, urolithins should be further evaluated toward high-dose toxicity, while alternariol derivatives could be promising chemicals for the development of therapeutics.

Alternaria molds are known to cause the contamination of food with their secondary metabolites, a chemically very heterogeneous group of compounds. Yet, after decades of research on the occurrence and the toxicity of Alternaria toxins in academia, no regulation has been implemented yet, thus leaving these potential food contaminants in the status of so-called "emerging mycotoxins". However, research on this topic has been far from static, leading to the European Food Safety Authority repeatedly calling for more data on the occurrence and toxicity of genotoxic metabolites such as alternariol (AOH) and its monomethyl ether (AME). To give an overview on recent developments in the field, this comprehensive review summarizes published data and addresses current challenges arising from the chemical complexity of Alternaria's metabolome, mixture effects and the emergence of novel biological targets like cell membranes or the interaction with different receptors. Besides toxicodynamics, we review recent research on toxicokinetics, including the first in vivo studies which incorporated the rarely investigated-but highly genotoxic-perylene quinones. Furthermore, a particular focus lies on the advances of liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based analytical tools for determining a broader spectrum of Alternaria toxins including modified/masked forms and assessing exposure via human biomonitoring (HBM)., (© 2021 The Authors. Comprehensive Reviews in Food Science and Food Safety published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

The human gut microbiota plays an important role in the maintenance of human health. Factors able to modify its composition might predispose the host to the development of pathologies. Among the various xenobiotics introduced through the diet, Alternaria mycotoxins are speculated to represent a threat for human health. However, limited data are currently available about the bidirectional relation between gut microbiota and Alternaria mycotoxins. In the present work, we investigated the in vitro effects of different concentrations of a complex extract of Alternaria mycotoxins (CE; containing eleven mycotoxins; e.g. 0.153 µM alternariol and 2.3 µM altersetin, at the maximum CE concentration tested) on human gut bacterial strains, as well as the ability of the latter to metabolize or adsorb these compounds. Results from the minimum inhibitory concentration assay showed the scarce ability of CE to inhibit the growth of the tested strains. However, the growth kinetics of most of the strains were negatively affected by exposure to the various CE concentrations, mainly at the highest dose (50 µg/mL). The CE was also found to antagonize the formation of biofilms, already at concentrations of 0.5 µg/mL. LC-MS/MS data analysis of the mycotoxin concentrations found in bacterial pellets and supernatants after 24 h incubation showed the ability of bacterial strains to adsorb some Alternaria mycotoxins, especially the key toxins alternariol, alternariol monomethyl ether, and altersetin. The tendency of these mycotoxins to accumulate within bacterial pellets, especially in those of Gram-negative strains, was found to be directly related to their lipophilicity.

Some prenylated polyphenols originating from hops, which are thus natural constituents of beer, have been discussed critically for their agonistic potential toward estrogen receptors. So far, little attention has been attributed to the fact that humans are typically not exposed to isolated compounds, but to mixtures which for example might comprise in addition to hop flavonoids further xenoestrogens, e.g., certain pesticides used for plant protection of hops and barley. Thus, we used the alkaline phosphatase assay to assess combinatory estrogenic effects of three signature compounds - xanthohumol, 8-prenylnaringenin and iso-xanthohumol-on Ishikawa cells in a combination that resembled the concentration ratios observable in beer. Moreover, we added this natural flavonoid pattern to a mixture of representative estrogenic pesticides to assess their combined effects. Using state-of-the-art statistical tools, we observed cumulative to slightly synergistic effects between isolated flavonoids as well as the flavonoid and the pesticide mixture. Of potential importance, these effects were found at low nanomolar hop polyphenol concentrations that one can reasonably expect to occur in vivo after the consumption of strongly hopped beer. Taken together, our results imply that cumulative/synergistic estrogenicity should be explored in detail and urgently be incorporated into risk assessment of prenylated chalcones., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aichinger, Bliem and Marko.)

Alternaria molds produce a variety of chemically diverse secondary metabolites with potentially adverse effects on human health. However, data on occurrence in food and human exposure is inconsistent for some of these mycotoxins. Membrane filtration is a frequent step in many sample preparation procedures for LC-MS-based methods analyzing food contaminants. Yet, little is known about the possibility of adsorptive phenomena that might result in analyte losses. Thus, we treated a complex extract of Alternaria toxins with several types of syringe filters and unraveled the impact on its chemical composition by LC-MS/MS. We observed significant, and in some cases complete, losses of compounds due to filtration. Particularly, two key Alternaria toxins, alternariol (AOH) and its monomethyl ether (AME), were heavily affected. As a comparison with published food surveys indicating a correlation of the type of filtration used with lower incidence reports in food, our results point at a possible underestimation of AME in past exposure assessment. Also, perylene quinones were greatly affected by filtration, underlining the importance to take this into consideration during analytical method development. Furthermore, we applied the comet assay in HT-29 cells to elucidate the impact of filtration on the genotoxicity of the extract. We observed strong coincidences with the loss of epoxide-carrying metabolites and also an intriguing induction of oxidative DNA damage by yet toxicologically uncharacterized Alternaria toxins. In conclusion, we highlight potential issues with sample filtration and call for a critical re-evaluation of previous food occurrence data in the light of the results at hand.

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC-MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC-MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications.

Alternaria molds simultaneously produce a large variety of mycotoxins, of which several were previously reported to induce enzymes of phase I metabolism through aryl hydrocarbon receptor activation. Thus, we investigated the potential of naturally occurring Alternaria toxin mixtures to induce Cytochrome P450 (CYP) 1A1/1A2/1B1 activity. Two variants of an extract from cultured Alternaria alternata, as well as the toxins alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX-I), and altertoxin II (ATX-II), were tested singularly and in binary mixtures applying the 7-ethoxy-resorufin- O -deethylase (EROD) assay in MCF-7 breast cancer cells. Sub-cytotoxic concentrations of the two toxin mixtures, as well as ATX-I, ATX-II and AOH, exhibited dose-dependent enhancements of CYP 1 activity. ATX-I and ATX-II interacted synergistically in this respect, demonstrating the two perylene quinones as major contributors to the extract's potential. Binary mixtures between AOH and the two altertoxins respectively exhibited concentration-dependent antagonistic as well as synergistic combinatory effects. Notably, AME showed no efficacy towards EROD enzyme activity or impact on other toxins' efficacy. Hence, this study provides insights into synergistic and other combinatory effects of Alternaria toxins in natural co-occurrence scenarios in the context of AhR signalling pathway activation in breast cancer cells.

Emerging mycotoxins produced by Alternaria spp. were previously reported to exert cytotoxic, genotoxic, but also estrogenic effects in human cells. The involved mechanisms are very complex and not fully elucidated yet. Thus, we followed an in silico target fishing approach to extend knowledge on the possible biological targets underlying the activity of alternariol, taken as the signature compound of Alternaria toxins. Combining ligand-based screening and structure-based modeling, the ubiquitous casein kinase 2 (CK2) was identified as a potential target for the compound. This result was validated in a cell-free in vitro CK2 activity assay, where alternariol inhibited CK2 with an IC 50 of 707 nM. As CK2 was recently discussed to influence estrogen receptor (ER) transcription and DNA-binding affinity, we assessed a potential impact on the mRNA levels of ERα or ERβ by qRT-PCR and on nuclear localization of the receptors by confocal microscopy, using estrogen-sensitive Ishikawa cells as a model. While AOH did not affect the transcription of ERα or ERβ, an increase in nuclear localization of ERα after incubation with 10 µM AOH was observed. However, this effect might be due to ER binding affinity and therefore estrogenicity of AOH. Furthermore, in silico docking simulation revealed not only AOH, but also a number of other Alternaria toxins as potential inhibitors of CK2, including alternariol monomethyl ether and the perylene quinone derivative altertoxin II (ATX-II). These findings were representatively confirmed in vitro for the perylene quinone derivative altertoxin II, which was found to inhibit the kinase with an IC 50 of 5.1 µM. Taken together, we propose CK2 inhibition as an additional mechanism to consider in future studies for alternariol and several other Alternaria toxins.