Elisabeth Varga

Bioactive secondary metabolites produced by the haptophyte microalga Prymnesium parvum are key mediators of ichthyotoxicity during harmful algal bloom events. In the present study, we investigated the effects of a P. parvum extract containing B-type prymnesins, from an isolate obtained during the 2022 Oder River bloom, on mitochondrial health and cellular homeostasis in the rainbow trout gill epithelial cell line RTgill-W1, a widely used model in aquatic toxicology. Mitochondrial function and structure were assessed using multiparameter flow cytometry and high-resolution confocal microscopy (Airyscan). Exposure to prymnesin-containing extract (PRYMCE) induced concentration-dependent mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), changes in mitochondrial mass-associated fluorescence, and reduced cardiolipin-associated 10-N-nonyl acridine orange (NAO) fluorescence. Structural analysis revealed mitochondrial network remodeling across PRYMCE concentrations, including perinuclear clustering and a transition from elongated filamentous structures to condensed spherical forms. Time-resolved imaging further demonstrated that mitochondrial structural alterations preceded overt plasma membrane permeabilization. Single-cell analysis demonstrated pronounced cellular heterogeneity, with distinct injury-associated subpopulations exhibiting increased side scatter and a ΔΨm ^low /mitochondrial mass ^low phenotype. These findings identify mitochondrial impairment and network remodeling as early, quantifiable hallmarks of PRYMCE-induced cytotoxicity and provide mechanistic insight into how P. parvum blooms may damage aquatic organisms.

The occurring food contaminants alternariol monomethyl ether (AME), tenuazonic acid (TeA) and altertoxin II (ATX-II) are recognized as emerging Alternaria mycotoxins, yet data gaps remain regarding their toxicokinetic characteristics. The hepatic metabolism of these three substances was investigated in primary rat (PRH) and human (PHH) hepatocytes by monitoring parent compound depletion and, where applicable, metabolite formation. AME was initially evaluated at 1 µM over 4 h and subsequently investigated in concentration-dependent experiments (0.75, 1.5, 3, and 8 µM). TeA was assessed at 5 µM over 4 h, whereas ATX-II was evaluated at 1.11 µM over 4 h and at 0.22 µM over 30 min. For AME, time-dependent depletion was further investigated in PRHs at two hepatocyte densities (0.25 and 0.5 × 10 ⁶ cells/mL). In PRHs, AME metabolism followed Michaelis–Menten kinetics (V _max = 150.9 pmol·min ^{- ¹} ·10 ^-6 cells, K _m = 1.18 µM), whereas no reliable kinetic model could be established for PHHs. In contrast, TeA exhibited high metabolic stability, with only 9–10% depletion after 4 h, indicating negligible hepatic clearance in both species. ATX-II was also rapidly depleted and became undetectable within 30 min, accompanied by formation of altertoxin I (ATX-I), which was more pronounced in PHHs than in PRHs. Substrate depletion revealed interspecies differences in hepatic clearance capacity and stability. Furthermore, tentative metabolite analysis indicated that AME undergoes phase I and phase II biotransformation. Overall, these findings provide comparative insights into PRH and PHH systems, offering a foundation for future studies on their toxicological relevance and impact on human health.

Freshwater salinization is of increasing concern for integrity and functioning of freshwater habitats worldwide. Experiments so far often have studied drastic salt additions, while gradient designs have been performed less commonly. We tested the effect of freshwater salinization in a mesocosm exposing the plankton community of the oligotrophic Lake Lunz, Austria, to a four-fold salinization gradient (control, 0.2, 1, a 5 ppt salt). Salinity was manipulated in a factorial design with enrichment, with 10 μg L ^-1 and 30 μg L ^-1 phosphorus, resulting in 8 treatments with 3 replicates each. We followed the effects of salinization on diversity, community composition and resource use over 36 days. Community composition was assessed by amplicon sequencing, Diversity loss and community turnover followed upon salt addition. All levels of salinization caused pronounced changes in community composition, with 5 ppt causing the most drastic changes. Salinization caused trophic downgrading by kicking out especially protistan consumers and rotifers, while some green algae and chrysophytes were especially tolerant, resulting in reduced phylogenetic and functional diversity with increasing salinization. In line with reduced top down control, salinization affected temporal variability in chlorophyll- a (chl- a ) and resource use (RUE), with higher salinity causing more extreme fluctuations in chl- a and RUE. Enrichment overall aggravated salinization, enhancing temporal turnover and temporal fluctuations in resource use.

The foodborne mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been associated with several adverse effects, including cytotoxicity, genotoxicity, endocrine disruption, and immunomodulation. As these endpoints are typically observed in vitro at micromolar concentrations, the question arises whether such levels are attainable in exposed humans. To address this data gap in chemical risk assessment, a physiologically based kinetic (PBK) model was developed to predict internal exposure doses to AOH and AME in humans. As input parameters, kinetic constants for hepatic glucuronidation were obtained in vitro by incubating Sprague Dawley rat and human liver S9 fractions with 0.5–50 µM AOH and 0.5–20 µM AME, demonstrating rapid biotransformation in both species. Intestinal absorption of AME and physicochemical parameters were estimated using quantitative structure–activity relationship (QSAR) models. Sensitivity analysis identified parameters describing hepatic glucuronidation and gastrointestinal uptake as among the most influential, confirming the importance of their reliable estimation. The PBK model was evaluated against available rodent toxicokinetic data and subsequently extrapolated to humans. Ultimately, the currently available exposure estimates published by EFSA in 2016 were applied to predict target tissue concentrations, which were compared to points of departure (PoDs) for relevant toxicological endpoints. Even in the most susceptible group of male toddlers, predicted internal concentrations (10⁻ ⁴ µM range) were approximately four orders of magnitude below the respective PoDs. Consequently, under the applied exposure assumptions and considering the compounds as isolated chemicals, AOH and AME are not expected to reach systemic or tissue concentrations associated with the investigated effects.

The foodborne mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been associated with several adverse effects, including cytotoxicity, genotoxicity, endocrine disruption, and immunomodulation. As these endpoints are typically observed in vitro at micromolar concentrations, the question arises whether such levels are attainable in exposed humans. To address this data gap in chemical risk assessment, a physiologically based kinetic (PBK) model was developed to predict internal exposure doses to AOH and AME in humans. As input parameters, kinetic constants for hepatic glucuronidation were obtained in vitro by incubating Sprague Dawley rat and human liver S9 fractions with 0.5-50 µM AOH and 0.5-20 µM AME, demonstrating rapid biotransformation in both species. Intestinal absorption of AME and physicochemical parameters were estimated using quantitative structure-activity relationship (QSAR) models. Sensitivity analysis identified parameters describing hepatic glucuronidation and gastrointestinal uptake as among the most influential, confirming the importance of their reliable estimation. The PBK model was evaluated against available rodent toxicokinetic data and subsequently extrapolated to humans. Ultimately, the currently available exposure estimates published by EFSA in 2016 were applied to predict target tissue concentrations, which were compared to points of departure (PoDs) for relevant toxicological endpoints. Even in the most susceptible group of male toddlers, predicted internal concentrations (10□□ µM range) were approximately four orders of magnitude below the respective PoDs. Consequently, under the applied exposure assumptions and considering the compounds as isolated chemicals, AOH and AME are not expected to reach systemic or tissue concentrations associated with the investigated effects.

The ubiquitously occurring food contaminants altenuene (ALT) and tentoxin (TEN) are recognized as emerging Alternaria mycotoxins, yet substantial data gaps remain when it comes to their toxicological behavior and toxicokinetic characteristics. This study aimed to compare and generate quantitative data on their hepatic metabolism and to obtain semi-quantitative insights into their metabolite profiles. To this end, primary rat and human hepatocytes were incubated with 10 µM ALT or TEN over multiple time points up to 4 h. Both substrate depletion and metabolite identification revealed pronounced interspecies differences. The extent of ALT metabolism was significant, with an 88% and 57% decrease in rat and human hepatocytes after 4 h, respectively. In contrast, TEN showed extensive biotransformation in rats (67%) but only modest turnover in humans (27%) over the same period. Hepatocellular clearances were consistently higher for ALT than TEN, with hepatic extraction ratios indicating intermediate extraction for ALT and low extraction for TEN. High-resolution mass spectrometry combined with targeted analysis of selected metabolites annotated phase II conjugation as the predominant metabolic pathway for ALT and phase I oxidative metabolism for TEN, including mono- and double-metabolized species for the latter. Overall, these results provide a comprehensive characterization of ALT- and TEN-metabolism in hepatocytes, offering a foundation for future studies on their toxicological relevance and impact on human health.

The health benefits of soy and soy derived foods are often attributed to their isoflavone (ISFs) content, known to include phytoestrogens. However, EFSA has concluded that there is insufficient scientific evidence to establish a causal relationship between ISFs consumption and the proposed health claims. Although phase-II metabolites of ISFs are often considered as detoxified products with reduced estrogenic effects, recent studies indicate that ISFs metabolites may contribute and even enhance the biological activity of ISFs. However, current knowledge on phase-II metabolites of ISFs is limited, even though representing the major ISFs metabolites circulating in the bloodstreams in humans. This study investigates the role of phase-II metabolism in the biological activity of ISFs, focusing on estrogenicity and cytotoxicity in estrogen-sensitive Ishikawa cells (expressing estrogen receptors (ER) α and β). ISFs concentrations from 0.001 to 10 µM, with 1:10 dilution steps, were tested. Cleavage to parent compounds during incubation was also evaluated using high-performance liquid chromatography tandem mass spectrometry. The results showed that phase-II metabolites, particularly sulfates, can still exhibit estrogenic activity, challenging the general assumption that phase-II metabolism always leads to the inactivation of ISFs. In particular, at 10 µM daidzein-4'-sulfate exhibited a comparable estrogenic activity to daidzein at the same concentration, and genistein-7-sulfate exhibited estrogenic potential at higher concentrations. Molecular dynamics simulations revealed that genistein and its conjugates can stably interact with ERβ. This study highlights the importance of phase-II metabolism for ISFs activity and suggests that the sulfates formed should be examined more closely in further investigations.

Humans and animals are frequently exposed to dietary compounds that can adversely affect intestinal health. The Fusarium mycotoxin deoxynivalenol (DON) is known to compromise epithelial barrier integrity. DON frequently co-occurs in food and feed with the mycoestrogen zearalenone (ZEN) and soy-derived isoflavones (ISFs) such as genistein (GEN) and daidzein. Therefore, this study investigates the effects of ZEN (10 nM), its metabolites hydrolyzed ZEN (HZEN) and decarboxylated hydrolyzed ZEN (DHZEN) (each 10 nM), and ISFs (GEN and equol (EQ), 10 µM), alone and in combination with DON (10 µM), on intestinal barrier function using the porcine epithelial cell line IPEC-J2. Barrier integrity was assessed via transepithelial electrical resistance (TEER) and paracellular permeability (lucifer yellow). DON (10 µM) significantly reduced barrier integrity, confirming its disruptive effect. Conversely, treatment with ZEN, HZEN, DHZEN, GEN, and EQ did not impair barrier function. Co-exposure of DON with ZEN or HZEN yielded TEER values similar to those observed with DON alone. In contrast, the DHZEN + DON combination resulted in higher TEER values compared to DON alone, suggesting a mitigating effect under the experimental conditions. Quantification of GEN by LC-MS/MS showed consistent levels across treatments, except for reduced basolateral GEN during co-incubation with DHZEN, suggesting altered transport or metabolism. These findings highlight the ability of DHZEN to mitigate the intestinal barrier disruption induced by DON and underscores the complex interaction of maintaining epithelial barrier integrities.