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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.
Georg Aichinger; Natálie Živná; Elisabeth Varga; Francesco Crudo; Benedikt Warth; Doris Marko. Microfiltration results in the loss of analytes and affects the in vitro genotoxicity of a complex mixture of Alternaria toxins. Mycotoxin Research 2020, 36, 399 -408.
AMA StyleGeorg Aichinger, Natálie Živná, Elisabeth Varga, Francesco Crudo, Benedikt Warth, Doris Marko. Microfiltration results in the loss of analytes and affects the in vitro genotoxicity of a complex mixture of Alternaria toxins. Mycotoxin Research. 2020; 36 (4):399-408.
Chicago/Turabian StyleGeorg Aichinger; Natálie Živná; Elisabeth Varga; Francesco Crudo; Benedikt Warth; Doris Marko. 2020. "Microfiltration results in the loss of analytes and affects the in vitro genotoxicity of a complex mixture of Alternaria toxins." Mycotoxin Research 36, no. 4: 399-408.
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.
Julia Hohenbichler; Georg Aichinger; Michael Rychlik; Giorgia Del Favero; Doris Marko. Alternaria alternata Toxins Synergistically Activate the Aryl Hydrocarbon Receptor Pathway In Vitro. Biomolecules 2020, 10, 1018 .
AMA StyleJulia Hohenbichler, Georg Aichinger, Michael Rychlik, Giorgia Del Favero, Doris Marko. Alternaria alternata Toxins Synergistically Activate the Aryl Hydrocarbon Receptor Pathway In Vitro. Biomolecules. 2020; 10 (7):1018.
Chicago/Turabian StyleJulia Hohenbichler; Georg Aichinger; Michael Rychlik; Giorgia Del Favero; Doris Marko. 2020. "Alternaria alternata Toxins Synergistically Activate the Aryl Hydrocarbon Receptor Pathway In Vitro." Biomolecules 10, no. 7: 1018.
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.
Francesco Crudo; Georg Aichinger; Jovana Mihajlovic; Luca Dellafiora; Elisabeth Varga; Hannes Puntscher; Benedikt Warth; Chiara Dall'Asta; David Berry; Doris Marko. Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of Alternaria toxins in vitro. Archives of Toxicology 2020, 94, 3541 -3552.
AMA StyleFrancesco Crudo, Georg Aichinger, Jovana Mihajlovic, Luca Dellafiora, Elisabeth Varga, Hannes Puntscher, Benedikt Warth, Chiara Dall'Asta, David Berry, Doris Marko. Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of Alternaria toxins in vitro. Archives of Toxicology. 2020; 94 (10):3541-3552.
Chicago/Turabian StyleFrancesco Crudo; Georg Aichinger; Jovana Mihajlovic; Luca Dellafiora; Elisabeth Varga; Hannes Puntscher; Benedikt Warth; Chiara Dall'Asta; David Berry; Doris Marko. 2020. "Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of Alternaria toxins in vitro." Archives of Toxicology 94, no. 10: 3541-3552.
Gudrun Pahlke; Eva Attakpah; Georg Aichinger; Katarina Ahlberg; Christina Maria Hochkogler; Kerstin Schweiger; Dorothea Schipp; Veronika Somoza; Doris Marko. Corrigendum to “Dark coffee consumption protects human blood cells from spontaneous DNA damage” [J. Funct. Foods 55 (2019) 285–295]. Journal of Functional Foods 2020, 71, 103989 .
AMA StyleGudrun Pahlke, Eva Attakpah, Georg Aichinger, Katarina Ahlberg, Christina Maria Hochkogler, Kerstin Schweiger, Dorothea Schipp, Veronika Somoza, Doris Marko. Corrigendum to “Dark coffee consumption protects human blood cells from spontaneous DNA damage” [J. Funct. Foods 55 (2019) 285–295]. Journal of Functional Foods. 2020; 71 ():103989.
Chicago/Turabian StyleGudrun Pahlke; Eva Attakpah; Georg Aichinger; Katarina Ahlberg; Christina Maria Hochkogler; Kerstin Schweiger; Dorothea Schipp; Veronika Somoza; Doris Marko. 2020. "Corrigendum to “Dark coffee consumption protects human blood cells from spontaneous DNA damage” [J. Funct. Foods 55 (2019) 285–295]." Journal of Functional Foods 71, no. : 103989.
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 IC50 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 IC50 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.
Georg Aichinger; Luca Dellafiora; Foteini Pantazi; Giorgia Del Favero; Gianni Galaverna; Chiara Dall'Asta; Doris Marko. Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study. Archives of Toxicology 2020, 94, 2225 -2237.
AMA StyleGeorg Aichinger, Luca Dellafiora, Foteini Pantazi, Giorgia Del Favero, Gianni Galaverna, Chiara Dall'Asta, Doris Marko. Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study. Archives of Toxicology. 2020; 94 (6):2225-2237.
Chicago/Turabian StyleGeorg Aichinger; Luca Dellafiora; Foteini Pantazi; Giorgia Del Favero; Gianni Galaverna; Chiara Dall'Asta; Doris Marko. 2020. "Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study." Archives of Toxicology 94, no. 6: 2225-2237.
The benzo[c]phenanthridine P8-D6 was recently found to suppress the catalytic activity of both human topoisomerase (Topo) I and II. Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo–DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the “in vivo complex of enzyme” (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis (“comet assay”). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells.
Georg Aichinger; Falk-Bach Lichtenberger; Tamara N. Steinhauer; Inken Flörkemeier; Giorgia Del Favero; Bernd Clement; Doris Marko. The Aza-Analogous Benzo[c]phenanthridine P8-D6 Acts as a Dual Topoisomerase I and II Poison, thus Exhibiting Potent Genotoxic Properties. Molecules 2020, 25, 1524 .
AMA StyleGeorg Aichinger, Falk-Bach Lichtenberger, Tamara N. Steinhauer, Inken Flörkemeier, Giorgia Del Favero, Bernd Clement, Doris Marko. The Aza-Analogous Benzo[c]phenanthridine P8-D6 Acts as a Dual Topoisomerase I and II Poison, thus Exhibiting Potent Genotoxic Properties. Molecules. 2020; 25 (7):1524.
Chicago/Turabian StyleGeorg Aichinger; Falk-Bach Lichtenberger; Tamara N. Steinhauer; Inken Flörkemeier; Giorgia Del Favero; Bernd Clement; Doris Marko. 2020. "The Aza-Analogous Benzo[c]phenanthridine P8-D6 Acts as a Dual Topoisomerase I and II Poison, thus Exhibiting Potent Genotoxic Properties." Molecules 25, no. 7: 1524.
Humans are typically exposed to mixtures of substances, whereby their bioactivity can be significantly altered by co-occurring compounds. Thus, over the last years, research on combinatory effects has gained increasing attention. In particular, several xenoestrogens have been recently reported to interact synergistically, among them alternariol (AOH) and zearalenone (ZEN), two toxins produced by molds which contaminate crops or food commodities. Bisphenol A (BPA) is a potential food contaminant arising from its use in plastics and represents a well-known xenoestrogen, acting as an endocrine disruptor. However, little research was yet conducted on its impact on the bioactivity of other xenoestrogens, and vice versa. Thus, in this study, we focused on combinatory estrogenic effects of BPA with AOH and ZEN in Ishikawa cells, which represent a well-established, estrogen-sensitive human cell model. Estrogenic stimuli of the single compounds and binary combinations in constant concentration ratios were measured by assessing the activity of alkaline phosphatase, a natural reporter gene for estrogen receptor activation. In parallel, cytotoxicity was monitored by neutral red assay. For statistical analysis of combinatory effects the "combination index" model was applied. In combination with ZEN, BPA was found to cause additive estrogenic effects. Mixtures of BPA with AOH expressed moderately antagonistic to nearly additive combinatory effects, depending on the concentration ratio. Although no synergistic effects were measured in the applied chemical mixtures, additive estrogenic stimuli were observed, underlining the importance to consider the cumulative impact of endocrine active factors out of different sources and structural classes.
Georg Aichinger; Foteini Pantazi; Doris Marko. Combinatory estrogenic effects of bisphenol A in mixtures with alternariol and zearalenone in human endometrial cells. Toxicology Letters 2020, 319, 242 -249.
AMA StyleGeorg Aichinger, Foteini Pantazi, Doris Marko. Combinatory estrogenic effects of bisphenol A in mixtures with alternariol and zearalenone in human endometrial cells. Toxicology Letters. 2020; 319 ():242-249.
Chicago/Turabian StyleGeorg Aichinger; Foteini Pantazi; Doris Marko. 2020. "Combinatory estrogenic effects of bisphenol A in mixtures with alternariol and zearalenone in human endometrial cells." Toxicology Letters 319, no. : 242-249.
Mycotoxins are low-molecular weight compounds produced by diverse genera of molds that may contaminate food and feed threatening the health of humans and animals. Recent findings underline the importance of studying the combined occurrence of multiple mycotoxins and the relevance of assessing the toxicity their simultaneous exposure may cause in living organisms. In this context, for the first time, this work has critically reviewed the most relevant data concerning the occurrence and toxicity of mycotoxins produced by Alternaria spp., which are among the most important emerging risks to be assessed in food safety, alone or in combination with other mycotoxins and bioactive food constituents. According to the literature covered, multiple Alternaria mycotoxins may often occur simultaneously in contaminated food, along with several other mycotoxins and food bioactives inherently present in the studied matrices. Although the toxicity of combinations naturally found in food has been rarely assessed experimentally, the data collected so far, clearly point out that chemical mixtures may differ in their toxicity compared to the effect of toxins tested individually. The data presented here may provide a solid foothold to better support the risk assessment of Alternaria mycotoxins highlighting the actual role of chemical mixtures on influencing their toxicity.
Francesco Crudo; Elisabeth Varga; Georg Aichinger; Gianni Galaverna; Doris Marko; Chiara Dall'Asta; Luca Dellafiora. Co-Occurrence and Combinatory Effects of Alternaria Mycotoxins and other Xenobiotics of Food Origin: Current Scenario and Future Perspectives. Toxins 2019, 11, 640 .
AMA StyleFrancesco Crudo, Elisabeth Varga, Georg Aichinger, Gianni Galaverna, Doris Marko, Chiara Dall'Asta, Luca Dellafiora. Co-Occurrence and Combinatory Effects of Alternaria Mycotoxins and other Xenobiotics of Food Origin: Current Scenario and Future Perspectives. Toxins. 2019; 11 (11):640.
Chicago/Turabian StyleFrancesco Crudo; Elisabeth Varga; Georg Aichinger; Gianni Galaverna; Doris Marko; Chiara Dall'Asta; Luca Dellafiora. 2019. "Co-Occurrence and Combinatory Effects of Alternaria Mycotoxins and other Xenobiotics of Food Origin: Current Scenario and Future Perspectives." Toxins 11, no. 11: 640.
Despite the frequent infection of agricultural crops by Alternaria spp., their toxic secondary metabolites and potential food contaminants lack comprehensive metabolic characterization. In this study, we investigated their bioavailability, metabolism, and excretion in vivo. A complex Alternaria culture extract (50 mg/kg body weight) containing 11 known toxins and the isolated lead toxin altertoxin II (0.7 mg/kg body weight) were administered per gavage to groups of 14 Sprague Dawley rats each. After 3 h and 24 h, plasma, urine and feces were collected to determine toxin recoveries. For reliable quantitation, an LC–MS/MS method for the simultaneous detection of 20 Alternaria toxins and metabolites was developed and optimized for either biological matrix. The obtained results demonstrated efficient excretion of alternariol (AOH) and its monomethyl ether (AME) via feces (> 89%) and urine (> 2.6%) after 24 h, while the majority of tenuazonic acid was recovered in urine (20 and 87% after 3 and 24 h, respectively). Moreover, modified forms of AOH and AME were identified in urine and fecal samples confirming both, mammalian phase-I (4-hydroxy-AOH) and phase-II (sulfates) biotransformation in vivo. Despite the comparably high doses, perylene quinones were recovered only at very low levels (altertoxin I, alterperylenol, < 0.06% in urine and plasma, < 5% in feces) or not at all (highly genotoxic, epoxide-holding altertoxin II, stemphyltoxin III). Interestingly, altertoxin I was detected in all matrices of rats receiving altertoxin II and suggests enzymatic de-epoxidation in vivo. In conclusion, the present study contributes valuable information to advance our understanding of the emerging Alternaria mycotoxins and their relevance on food safety.
Hannes Puntscher; Georg Aichinger; Stephanie Grabher; Eva Attakpah; Franziska Krüger; Katharina Tillmann; Tomas Motschnig; Julia Hohenbichler; Dominik Braun; Roberto Plasenzotti; Gudrun Pahlke; Harald Höger; Doris Marko; Benedikt Warth. Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats. Archives of Toxicology 2019, 93, 3153 -3167.
AMA StyleHannes Puntscher, Georg Aichinger, Stephanie Grabher, Eva Attakpah, Franziska Krüger, Katharina Tillmann, Tomas Motschnig, Julia Hohenbichler, Dominik Braun, Roberto Plasenzotti, Gudrun Pahlke, Harald Höger, Doris Marko, Benedikt Warth. Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats. Archives of Toxicology. 2019; 93 (11):3153-3167.
Chicago/Turabian StyleHannes Puntscher; Georg Aichinger; Stephanie Grabher; Eva Attakpah; Franziska Krüger; Katharina Tillmann; Tomas Motschnig; Julia Hohenbichler; Dominik Braun; Roberto Plasenzotti; Gudrun Pahlke; Harald Höger; Doris Marko; Benedikt Warth. 2019. "Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats." Archives of Toxicology 93, no. 11: 3153-3167.
Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17β-estradiol (E2) at non-cytotoxic concentrations (5–10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC–MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.
Georg Aichinger; Franziska Krüger; Hannes Puntscher; Karin Preindl; Benedikt Warth; Doris Marko. Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro. Archives of Toxicology 2019, 93, 3021 -3031.
AMA StyleGeorg Aichinger, Franziska Krüger, Hannes Puntscher, Karin Preindl, Benedikt Warth, Doris Marko. Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro. Archives of Toxicology. 2019; 93 (10):3021-3031.
Chicago/Turabian StyleGeorg Aichinger; Franziska Krüger; Hannes Puntscher; Karin Preindl; Benedikt Warth; Doris Marko. 2019. "Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro." Archives of Toxicology 93, no. 10: 3021-3031.
We are constantly exposed to a variety of environmental contaminants and hormones, including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are needed but not available to date. Herein, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum, and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 μg/L, 0.03 to 14 μg/L, and 0.03 to 4.6 μg/L in urine, serum, and breast milk, respectively. The applicability of the novel method was demonstrated in proof-of-principle experiments by analyzing urine from Austrian individuals and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods' feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multiclass exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.
Karin Preindl; Dominik Braun; Georg Aichinger; Sabina Sieri; Mingliang Fang; Doris Marko; Benedikt Warth. A Generic Liquid Chromatography−Tandem Mass Spectrometry Exposome Method for the Determination of Xenoestrogens in Biological Matrices. Analytical Chemistry 2019, 91, 11334 -11342.
AMA StyleKarin Preindl, Dominik Braun, Georg Aichinger, Sabina Sieri, Mingliang Fang, Doris Marko, Benedikt Warth. A Generic Liquid Chromatography−Tandem Mass Spectrometry Exposome Method for the Determination of Xenoestrogens in Biological Matrices. Analytical Chemistry. 2019; 91 (17):11334-11342.
Chicago/Turabian StyleKarin Preindl; Dominik Braun; Georg Aichinger; Sabina Sieri; Mingliang Fang; Doris Marko; Benedikt Warth. 2019. "A Generic Liquid Chromatography−Tandem Mass Spectrometry Exposome Method for the Determination of Xenoestrogens in Biological Matrices." Analytical Chemistry 91, no. 17: 11334-11342.
We are constantly exposed to a variety of environmental contaminants and hormones including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are urgently needed but still not available. Herein, we describe an LC-MS/MS method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 µg/L, 0.03 to 14 µg/L, and 0.03 to 4.6 µg/L in urine, serum and breast milk, respec-tively. The applicability of the novel method was demonstrated in proof of principle experiments by analyzing urine from Austrian, and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods’ feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multi-class exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.
Karin Preindl; Dominik Braun; Georg Aichinger; Sabina Sieri; Mingliang Fang; Doris Marko; Benedikt Warth. A Generic LC-MS/MS Exposome Method for the Determination of Xenoestrogens in Biological Matrices. 2019, 1 .
AMA StyleKarin Preindl, Dominik Braun, Georg Aichinger, Sabina Sieri, Mingliang Fang, Doris Marko, Benedikt Warth. A Generic LC-MS/MS Exposome Method for the Determination of Xenoestrogens in Biological Matrices. . 2019; ():1.
Chicago/Turabian StyleKarin Preindl; Dominik Braun; Georg Aichinger; Sabina Sieri; Mingliang Fang; Doris Marko; Benedikt Warth. 2019. "A Generic LC-MS/MS Exposome Method for the Determination of Xenoestrogens in Biological Matrices." , no. : 1.
We are constantly exposed to a variety of environmental contaminants and hormones including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are urgently needed but still not available. Herein, we describe an LC-MS/MS method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 µg/L, 0.03 to 14 µg/L, and 0.03 to 4.6 µg/L in urine, serum and breast milk, respec-tively. The applicability of the novel method was demonstrated in proof of principle experiments by analyzing urine from Austrian, and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods’ feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multi-class exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.
Karin Preindl; Dominik Braun; Georg Aichinger; Sabina Sieri; Mingliang Fang; Doris Marko; Benedikt Warth. A Generic LC-MS/MS Exposome Method for the Determination of Xenoestrogens in Biological Matrices. 2019, 1 .
AMA StyleKarin Preindl, Dominik Braun, Georg Aichinger, Sabina Sieri, Mingliang Fang, Doris Marko, Benedikt Warth. A Generic LC-MS/MS Exposome Method for the Determination of Xenoestrogens in Biological Matrices. . 2019; ():1.
Chicago/Turabian StyleKarin Preindl; Dominik Braun; Georg Aichinger; Sabina Sieri; Mingliang Fang; Doris Marko; Benedikt Warth. 2019. "A Generic LC-MS/MS Exposome Method for the Determination of Xenoestrogens in Biological Matrices." , no. : 1.
Gudrun Pahlke; Eva Attakpah; Georg Aichinger; Katarina Ahlberg; Christina Maria Hochkogler; Kerstin Schweiger; Dorothea Schipp; Veronika Somoza; Doris Marko. Dark coffee consumption protects human blood cells from spontaneous DNA damage. Journal of Functional Foods 2019, 55, 285 -295.
AMA StyleGudrun Pahlke, Eva Attakpah, Georg Aichinger, Katarina Ahlberg, Christina Maria Hochkogler, Kerstin Schweiger, Dorothea Schipp, Veronika Somoza, Doris Marko. Dark coffee consumption protects human blood cells from spontaneous DNA damage. Journal of Functional Foods. 2019; 55 ():285-295.
Chicago/Turabian StyleGudrun Pahlke; Eva Attakpah; Georg Aichinger; Katarina Ahlberg; Christina Maria Hochkogler; Kerstin Schweiger; Dorothea Schipp; Veronika Somoza; Doris Marko. 2019. "Dark coffee consumption protects human blood cells from spontaneous DNA damage." Journal of Functional Foods 55, no. : 285-295.
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).
Anika Schroeter; Georg Aichinger; Karin Stornig; Doris Marko. Impact of Oxidative Metabolism on the Cytotoxic and Genotoxic Potential of Genistein in Human Colon Cancer Cells. Molecular Nutrition & Food Research 2018, 63, e1800635 .
AMA StyleAnika Schroeter, Georg Aichinger, Karin Stornig, Doris Marko. Impact of Oxidative Metabolism on the Cytotoxic and Genotoxic Potential of Genistein in Human Colon Cancer Cells. Molecular Nutrition & Food Research. 2018; 63 (2):e1800635.
Chicago/Turabian StyleAnika Schroeter; Georg Aichinger; Karin Stornig; Doris Marko. 2018. "Impact of Oxidative Metabolism on the Cytotoxic and Genotoxic Potential of Genistein in Human Colon Cancer Cells." Molecular Nutrition & Food Research 63, no. 2: e1800635.
Alternaria mycotoxins frequently contaminate agricultural crops and may impact animal and human health. However, data on mammalian metabolism and potential biomarkers of exposure for human biomonitoring (HBM) are scarce. Here, we report the preliminary investigation with respect to metabolism and excretion of Alternaria toxins in Sprague Dawley rats. Four animals were housed in metabolic cages for 24 h after gavage administration of an Alternaria alternata culture extract containing ten known toxins. LC-MS/MS analysis of 17 Alternaria toxins in urine and fecal samples allowed to gain first insights regarding xenobiotic metabolism and excretion rates. Alternariol (6-10%), alternariol monomethyl ether (AME, 6-7%) and tenuazonic acid (up to 55%) were recovered in urine and fecal samples (9%, 87%, 0.3%, respectively), while perylene quinones administered at comparatively high levels, were either determined at very low levels (0.5% altertoxin I in urine and 9% in feces; 0.2% alterperylenol in urine and 3% in feces) or not at all (altertoxin II, stemphyltoxin III). AME-3-sulfate, which was not present in the administered extract, was determined in urine, representing up to 23% of the AME intake. Critical evaluation of the applied sample preparation protocol and LC-MS/MS analysis revealed interesting preliminary results and information crucial for improving follow-up experiments.
Hannes Puntscher; Svenja Hankele; Katharina Tillmann; Eva Attakpah; Dominik Braun; Mary-Liis Kütt; Giorgia Del Favero; Georg Aichinger; Gudrun Pahlke; Harald Höger; Doris Marko; Benedikt Warth. First insights into Alternaria multi-toxin in vivo metabolism. Toxicology Letters 2018, 301, 168 -178.
AMA StyleHannes Puntscher, Svenja Hankele, Katharina Tillmann, Eva Attakpah, Dominik Braun, Mary-Liis Kütt, Giorgia Del Favero, Georg Aichinger, Gudrun Pahlke, Harald Höger, Doris Marko, Benedikt Warth. First insights into Alternaria multi-toxin in vivo metabolism. Toxicology Letters. 2018; 301 ():168-178.
Chicago/Turabian StyleHannes Puntscher; Svenja Hankele; Katharina Tillmann; Eva Attakpah; Dominik Braun; Mary-Liis Kütt; Giorgia Del Favero; Georg Aichinger; Gudrun Pahlke; Harald Höger; Doris Marko; Benedikt Warth. 2018. "First insights into Alternaria multi-toxin in vivo metabolism." Toxicology Letters 301, no. : 168-178.
The Fusarium toxin zearalenone (ZEN) and its reductive metabolite α-zearalenol (α-ZEL) are well-documented endocrine disruptors that are frequently found to contaminate cereal products, including beer. But also hop is known to represent a source for endocrine active compounds, containing amongst others xanthohumol (XAN), which might be converted to the potent phytoestrogen 8-prenylnaringenin (8-PN). In the present study, we investigated the interaction of these xenoestrogens in mixtures which might occur in beer. Estrogenicity was measured as induction of alkaline phosphatase (AlP) expression in estrogen-sensitive Ishikawa cells. In binary combinations, XAN was found to act as a potent antagonist of mycotoxin-induced estrogenicity, significantly suppressing the AlP-inducing impact of both ZEN and α-ZEL at nanomolar concentrations. Also 8-PN antagonized the estrogenic stimulus of the two fungal metabolites, although less pronounced. These effects also manifested in combinations of three or four test compounds, and at the level of cell proliferation, that was assessed via an E-screen-like approach in Ishikawa cells. Of note, co-exposure to the investigated myco- and phyto-estrogens did not result in additive or overadditive/synergistic estrogenic effects in the applied test system. Being aware that the actual study is still limited to the in vitro situation, our results even suggest that prenylated chalkones from hops might protect against Fusarium toxin–induced endocrine disruptive activities at concentrations that can be reached by moderate beer consumption.
Georg Aichinger; Julia Beisl; Doris Marko. The Hop Polyphenols Xanthohumol and 8-Prenyl-Naringenin Antagonize the Estrogenic Effects of Fusarium Mycotoxins in Human Endometrial Cancer Cells. Frontiers in Nutrition 2018, 5, 85 .
AMA StyleGeorg Aichinger, Julia Beisl, Doris Marko. The Hop Polyphenols Xanthohumol and 8-Prenyl-Naringenin Antagonize the Estrogenic Effects of Fusarium Mycotoxins in Human Endometrial Cancer Cells. Frontiers in Nutrition. 2018; 5 ():85.
Chicago/Turabian StyleGeorg Aichinger; Julia Beisl; Doris Marko. 2018. "The Hop Polyphenols Xanthohumol and 8-Prenyl-Naringenin Antagonize the Estrogenic Effects of Fusarium Mycotoxins in Human Endometrial Cancer Cells." Frontiers in Nutrition 5, no. : 85.
Many small molecules of food origin may effect human health but lack an adequate description of their biological activity. To fill this knowledge gap, a first-line workflow is needed to assign putative functions, rank the endpoints for testing and guide wet-lab experiments. In this framework, the identification of potential biological targets can be used to probe the activity of orphan compounds using a so-called “target fishing” approach. Here, we present a proof of concept study using an in silico/in vitro target fishing approach on the fungal secondary metabolite atromentin. The procedure relies on a computational screening for activity identification coupled with experimental trials for dose-response characterization. Computational results identified estrogen receptors and 17-β-hydroxysteroid dehydrogenase as potential targets. Experiments confirmed a weak estrogenic activity, supporting the reliability of the procedure. Despite limited estrogenicity of atromentin, the proposed inhibition of 17-β-hydroxysteroid dehydrogenase should be considered as a source for endocrine disruptive effects.
Luca Dellafiora; Georg Aichinger; Elena Geib; Leyre Sánchez-Barrionuevo; Matthias Brock; David Cánovas; Chiara Dall'Asta; Doris Marko. Hybrid in silico/in vitro target fishing to assign function to “orphan” compounds of food origin – The case of the fungal metabolite atromentin. Food Chemistry 2018, 270, 61 -69.
AMA StyleLuca Dellafiora, Georg Aichinger, Elena Geib, Leyre Sánchez-Barrionuevo, Matthias Brock, David Cánovas, Chiara Dall'Asta, Doris Marko. Hybrid in silico/in vitro target fishing to assign function to “orphan” compounds of food origin – The case of the fungal metabolite atromentin. Food Chemistry. 2018; 270 ():61-69.
Chicago/Turabian StyleLuca Dellafiora; Georg Aichinger; Elena Geib; Leyre Sánchez-Barrionuevo; Matthias Brock; David Cánovas; Chiara Dall'Asta; Doris Marko. 2018. "Hybrid in silico/in vitro target fishing to assign function to “orphan” compounds of food origin – The case of the fungal metabolite atromentin." Food Chemistry 270, no. : 61-69.
Alternaria spp. are ubiquitous molds that are able to produce toxic secondary metabolites which may contaminate food globally. One of those is the mycotoxin altertoxin II (ATX-II), a genotoxic and mutagenic compound. In recent years, different flavonoids that may co-occur with mycotoxins in food were demonstrated to temper toxic effects of molds, mostly through their anti-oxidant properties. Thus, in this study, we assessed the influence of the berry anthocyanidin delphinidin on the toxicity of ATX-II in HT-29 colon carcinoma cells. We performed coupled SRB/WST-1 cytotoxicity assays which revealed only weak antagonistic interactions, and single-cell gel electrophoresis ("comet") assays, where we observed a potent protective effect of delphinidin on the DNA-damaging properties of ATX-II. Furthermore, we investigated the mechanism for this interaction. In the DCF assay delphinidin was found to reduce intracellular oxidative stress levels, which might contribute partly to the latter protection. However, LC-MS experiments showed that co-incubation of the mycotoxin with either delphinidin or its potential degradation product phloroglucinol aldehyde significantly decreased ATX-II concentrations in aqueous solutions, indicating that a direct chemical reaction of ATX-II with these components is likely responsible for the observed loss of toxicity. Our results indicate that delphinidin - and possibly other anthocyanins as well - might play a role in the protection of the gut from Alternaria-induced genotoxicity.
Georg Aichinger; Hannes Puntscher; Julia Beisl; Mary-Liis Kütt; Benedikt Warth; Doris Marko. Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II. Toxicology Letters 2018, 284, 136 -142.
AMA StyleGeorg Aichinger, Hannes Puntscher, Julia Beisl, Mary-Liis Kütt, Benedikt Warth, Doris Marko. Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II. Toxicology Letters. 2018; 284 ():136-142.
Chicago/Turabian StyleGeorg Aichinger; Hannes Puntscher; Julia Beisl; Mary-Liis Kütt; Benedikt Warth; Doris Marko. 2018. "Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II." Toxicology Letters 284, no. : 136-142.
Alternariol (AOH) and altertoxin II (ATX II) are mycotoxins formed by Alternaria spp. Since they are expected to co-occur in Alternaria-infested food and feed, we addressed the question of combinatory effects. In addition, potentially involved regulatory microRNAs were surveyed in an exploratory approach. Cytotoxicity measurements in constant ratio combinations of 1:10 or 1:1 (ATX II: AOH) mainly revealed additive effects in HepG2, HT29 and HCEC-1CT cells. Yet, in specific high doses antagonism was found. Microarray analysis of miRNA expression profiles in HepG2 cells indicated different patterns of miRNA regulation by AOH and ATX II, including several miRNA species for which no distinct functions are currently known. Among others, miR-4654, miR-4715_3p and miR-6720_3p were up-regulated by AOH and miR-5583_5p was down-regulated by ATX II. Additionally, miR-1323, involved in hindering DNA repair mechanisms, was decreased by ATX II. Digital droplet PCR (ddPCR) analysis of selected miRNAs indicated regulation of miR-29a by AOH, which might play a role in AOH-induced apoptosis. miR-192 and miR-224 regulation was associated with antagonistic cytotoxic effects of AOH and ATX II combinations. Our study represents the first evaluation on combinatory effects of AOH and ATX II.
Katharina Vejdovszky; Matej Sack; Katharina Jarolim; Georg Aichinger; Mark M. Somoza; Doris Marko. In vitro combinatory effects of the Alternaria mycotoxins alternariol and altertoxin II and potentially involved miRNAs. Toxicology Letters 2017, 267, 45 -52.
AMA StyleKatharina Vejdovszky, Matej Sack, Katharina Jarolim, Georg Aichinger, Mark M. Somoza, Doris Marko. In vitro combinatory effects of the Alternaria mycotoxins alternariol and altertoxin II and potentially involved miRNAs. Toxicology Letters. 2017; 267 ():45-52.
Chicago/Turabian StyleKatharina Vejdovszky; Matej Sack; Katharina Jarolim; Georg Aichinger; Mark M. Somoza; Doris Marko. 2017. "In vitro combinatory effects of the Alternaria mycotoxins alternariol and altertoxin II and potentially involved miRNAs." Toxicology Letters 267, no. : 45-52.