Biology subjects

Marko, D.

Publications and source records attributed to Marko, D..

11 recordsLinked to original sources

bioRxiv2026

Species-dependent clearance of alternariol monomethyl ether, tenuazonic acid and altertoxin II in rat and human primary hepatocytes

The ubiquitously occurring food contaminants alternariol monomethyl ether (AME), tenuazonic acid (TeA) and altertoxin II (ATX-II) are recognized as emerging Alternaria mycotoxins, yet substantial 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 5 {micro}M and subsequently investigated over a concentration range of 0.75-8 {micro}M (0.75, 1.5, 3, and 8 {micro}M), whereas TeA was assessed at 5 {micro}M, and ATX-II was assessed at 0.22 and 1.11 {micro}M, with additional short-term experiments conducted over 30 min for AME and ATX-II, and over 10 min for concentration-dependent AME depletion. For AME, time-dependent clearance was further evaluated in PRHs at two hepatocyte densities, 0.25 and 0.5 million cells/mL. In PRHs, AME metabolism followed Michaelis-Menten kinetics (Vmax = 150.9 pmol{middle dot}min-{superscript 1}{middle dot}10- cells, Km = 1.18 {micro}M), 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 transient formation of altertoxin I (ATX-I), which was more pronounced in PHHs than in PRHs. Substrate depletion revealed pronounced interspecies differences in hepatic clearance capacity and stability. Overall, these findings provide comparative insights in primary human and rat hepatocyte systems, offering a foundation for future studies on their toxicological relevance and impact on human health.

pharmacology and toxicology↗

bioRxiv2026

Hazard characterization of Alternaria toxins - filling data gaps on in vitro genotoxicity.

Alternaria toxins are naturally occurring food contaminants with limited and often inconsistent genotoxicity and mutagenicity data. Within the European Partnership for the Assessment of Risks from Chemicals (PARC), an OECD-aligned in vitro testing strategy was applied to fill existing data gaps and to characterize the genotoxic potential of major Alternaria toxins using high-purity test materials. Mutagenicity was assessed using bacterial reverse mutation test (OECD TG 471) and SOS/umu assay, while chromosomal damage was assessed using the in vitro micronucleus (MN) assay (OECD TG 487) in TK6 and HepG2 cells, complemented by fluorescence in situ hybridization (FISH) and {gamma}H2AX assay in HepaRG cells. Alternariol (AOH), alternariol monomethyl ether (AME), and altertoxin-I (ATX-I) showed clear mutagenicity in bacteria, whereas altenuene (ALT), tenuazonic acid (TeA), and tentoxin (TEN) were negative under the tested conditions. In mammalian cells, AOH, AME, and ATX-I induced MN formation in TK6 cells at concentrations [≥]5.5 {micro}M, [≥]2.5 {micro}M, and [≥]0.21 {micro}M, respectively, with FISH analysis supporting a clastogenic mode of action. In HepG2 cells, all tested toxins induced chromosomal damage, with effect threshold ranging from [≥]6.25 {micro}M (AOH) to [≥]50 {micro}M (TeA). {gamma}H2AX induction confirmed DNA damage for AOH and ATX-I, and at higher concentrations for TeA (1000 {micro}M). Overall, the data indicate clear in vitro genotoxic potential for AOH, AME, and ATX-I and provide evidence of chromosomal damage for ALT, TEN, and TeA, thereby reducing critical data gaps for hazard assessment.

pharmacology and toxicology↗

bioRxiv2026

In vitro EAS-mediated activity of Alternaria toxins

Alternaria mycotoxins represent an emerging concern due to their frequent occurrence in food and feed. However, available toxicological data remain limited. Under the current EU regulatory framework, and in line with the EFSA/ECHA/JRC guidance for the identification of endocrine disruptors (EDs), assessment of endocrine activity relies on standardized assays performed according to OECD Test Guidelines (TGs) for the estrogen-, androgen- and steroidogenesis- (EAS) modalities. Within the framework of the European Partnership for the Assessment of Risks from Chemicals (PARC), standardized in vitro methods of regulatory relevance were performed for six chemically characterized Alternaria toxins, aiming to address current regulatory gaps on EAS-mediated activity. Alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altertoxin-I (ATX-I), tentoxin (TEN) and altenuene (ALT) were assessed over a broad concentration range, from 0.001 up to 60 M, depending on cytotoxicity and solubility profile of each compound. Our findings indicate estrogenic activity for AOH (PC50: 3.9 - 4.6 {micro}M) and AME (PC50: 5.2 - 8.5 {micro}M) in the estrogen receptor transactivation assay (OECD TG 455), as well as an anti-estrogenic activity for ATX-I (IC30: 0.27 - 0.37 {micro}M). Minimal positive responses were observed at high concentrations for AOH (from the dose of 3 {micro}M) and for AME (from the dose of 10 {micro}M) in the agonistic part of the androgen receptor transactivation assay (OECD TG 458), which may also reflect glucocorticoid receptor activation. No effects on estradiol or testosterone production were observed for any of the tested Alternaria compounds in the steroidogenesis assay (OECD TG 456).

pharmacology and toxicology↗

bioRxiv2026

Mechanistic characterization of tenuazonic acid-induced cellular stress responses in human esophageal KYSE-510 cells

Tenuazonic acid (TeA) is an emerging Alternaria mycotoxin frequently detected in food and feed commodities, raising concerns about its toxicological relevance. Chronic oral exposure to TeA has been reported to induce dysplastic alterations in the esophageal mucosa of mice, while human biomonitoring data indicate an association between TeA exposure and esophageal cancer, although a causal relationship has not yet been established. At a mechanistic level, the effects of TeA in esophageal cells remain poorly characterized. Therefore, this study investigated the impact of TeA on cytotoxicity, oxidative stress, DNA damage, mitochondrial homeostasis, cell-cycle distribution and transcriptomic stress responses in human esophageal KYSE-510 cells. TeA induced a concentration-dependent reduction in metabolic activity and total protein content after 24 h exposure to 0.1-100 {micro}M. Significant cytotoxicity was measured starting from 20 {micro}M. At sub-cytotoxic concentrations, TeA triggered rapid ROS formation within 5-30 min exposure and induced formamidopyrimidine-DNA glycosylase (FPG) sensitive DNA damage after 1 h exposure (5-7.5 {micro}M), indicating oxidative DNA lesions. In addition, TeA altered mitochondrial morphology after 4 h exposure at 7.5 {micro}M, manifested by shrinkage of the mitochondrial network area and perinuclear redistribution, while mitochondrial respiration showed only a non-significant tendency towards reduced respiratory capacity. RNA sequencing after 6 h exposure to 10 {micro}M TeA revealed oxidative stress-associated transcriptional changes, impaired antioxidant and stress-adaptive responses, and p53-associated stress signaling. Furthermore, TeA induced significant G2/M phase accumulation after 24 h exposure to 1-10 {micro}M. HighlightsO_LITeA triggers rapid oxidative stress in human esophageal KYSE-510 cells C_LIO_LISub-cytotoxic TeA induces FPG-sensitive oxidative DNA lesions C_LIO_LITeA disrupts mitochondrial morphology before overt cytotoxicity occurs C_LIO_LIRNA-seq reveals impaired antioxidant defense and p53-linked stress signaling C_LIO_LITeA promotes G2/M phase accumulation and concentration-dependent cytotoxicity C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/736731v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@a496faorg.highwire.dtl.DTLVardef@1b42072org.highwire.dtl.DTLVardef@be5088org.highwire.dtl.DTLVardef@991b5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Modulation of NF-κB signaling by Alternaria mycotoxins: in vitro and in silico insights into molecular mechanisms of immunosuppression in THP-1 monocytes

Alternaria fungi produce various structurally diverse mycotoxins, several of which exhibit immunomodulatory properties. Among these, alternariol monomethyl ether (AME), alternariol (AOH), alterperylenol (ALTP), altertoxin I (ATX-I), and altersetin (AST) have been reported to suppress lipopolysaccharide (LPS)-induced inflammatory responses. However, the precise molecular mechanisms underlying these effects remain unclear. The present study aimed to elucidate how these selected Alternaria mycotoxins (0.1-50 {micro}M) target the NF-{kappa}B signaling pathway in THP-1 monocytes. Key components of the NF-{kappa}B cascade were analyzed by immunofluorescence microscopy, Western blotting and qRT-PCR. Nuclear translocation of NF-{kappa}B p65 and its phosphorylated form (p- NF-{kappa}B p65) was assessed by Western blot, while cytokine responses were determined at transcript (qRT-PCR) and protein (ELISA) levels. Moreover, in silico docking analyses were performed to investigate potential interactions of the toxins with IKK{beta}, and receptor-mediated crosstalk was studied using the glucocorticoid receptor (GR) antagonist RU486. Co-treatment with RU486 attenuated the immunosuppressive effects of 1 and 5 {micro}M AOH, indicating partial involvement of GR-dependent mechanisms. AME, AOH, ALTP, ATX-I, and AST increased total I{kappa}B levels while reducing its phosphorylated form. Additionally, AST and ALTP decreased the protein levels of Toll-like receptor 4 (TLR4), the I{kappa}B kinase (IKK) complex, NF-{kappa}B p65, and p- NF-{kappa}B p65. While AOH (5 {micro}M) and AST (25 {micro}M) reduced nuclear translocation of p65 and p-p65, ALTP (2 {micro}M) enhanced nuclear localization despite decreasing cytokine expression Together, these findings suggest toxin-specific interference at multiple regulatory levels of NF-{kappa}B signaling and provide novel mechanistic insight into the immunomodulatory effects of Alternaria mycotoxins.

pharmacology and toxicology↗

bioRxiv2026

Opposing immunomodulatory effects of the Alternaria mycotoxin tenuazonic acid in immune and intestinal epithelial cells

Tenuazonic acid (TeA) is one of the most frequently detected Alternaria mycotoxins in contaminated food. Despite its frequent occurrence, its immunomodulatory effects remain insufficiently characterized. Therefore, the present study investigated the impact of TeA on inflammatory signaling and cytokine regulation in monocytes and intestinal epithelial cell (IEC) models. NF-{kappa}B activity was assessed using a reporter gene assay in THP1-Lucia monocytes, while cytokine mRNA expression and protein secretion were quantified in Caco-2 and HCEC-1CT cells by qRT-PCR and ELISA, respectively. In THP-1 monocytes, TeA significantly suppressed lipopolysaccharide (LPS)-induced NF-{kappa}B activation in a concentration-dependent manner starting at 25 M, while cytotoxicity occurred only at concentrations [≥]100 M. In HCEC-1CT and differentiated Caco-2 cells, TeA increased IL-6, IL-8, and TNF- mRNA levels at non-cytotoxic concentrations ([≥]10 M). In Caco-2 cells, these transcriptional changes were accompanied by increased cytokine secretion, whereas HCEC-1CT cells showed only partial effects on the protein level after short-term exposure. Following prolonged incubation, TNF- secretion was increased and IL-6 and IL-8 secretion were slightly reduced. IL-10 remained unaffected under all conditions. Overall, TeA exerted cell type-dependent immunomodulatory effects characterized by immunoinhibitory activity in monocytes and pro-inflammatory responses in IECs, highlighting the complex immunotoxic potential of this Alternaria mycotoxin.

pharmacology and toxicology↗

bioRxiv2026

Human internal exposures to alternariol and its monomethyl ether are predicted below thresholds of in vitro toxicity by physiologically based kinetic modeling

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 {micro}M AOH and 0.5-20 {micro}M 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{square}{square} {micro}M 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.

pharmacology and toxicology↗

bioRxiv2026

Comparative metabolism of the Alternaria toxins altenuene and tentoxin in rat and human primary hepatocytes

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 {micro}M 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.

pharmacology and toxicology↗

bioRxiv2025

Sulfoquinovose is differently degraded by the mouse and human gut microbiota and not metabolized by the host

BackgroundSulfoquinovose (SQ) is a green-diet-derived sulfonated glucose and a selective substrate for few human gut bacteria. Complete anaerobic SQ degradation via interspecies metabolite transfer to sulfonate-respiring bacteria produces hydrogen sulfide, which has dose- and context-dependent health effects. Here, we studied potential SQ degradation by the mammalian host and the impact of SQ supplementation on human and murine gut microbiota diversity and metabolism. Results13CO2 breath tests with germ-free C57BL/6 mice gavaged with 13C-SQ were negative. Also, SQ was not degraded by human intestinal cells in vitro, indicating that SQ is not directly metabolized by mice and humans. Addition of increasing SQ concentrations to human fecal microcosms revealed dose-dependent responses of the microbiota and corroborated the relevance of Agathobacter rectalis and Bilophila wadsworthia in cooperative degradation of SQ to hydrogen sulfide via interspecies transfer of 2,3-dihydroxy-1-propanesulfonate (DHPS). Similar to the human gut microbiome, the genetic capacity for SQ or DHPS degradation is sparsely distributed among bacterial species in the mouse gut. Escherichia coli and Enterocloster clostridioformis were identified as primary SQ degraders in the mouse gut. SQ and DHPS supplementation experiments with conventional laboratory mice and their intestinal contents showed that SQ was incompletely catabolized to DHPS. Although some E. clostridioformis genomes encode an extended sulfoglycolytic pathway for both SQ and DHPS fermentation, SQ was only degraded to DHPS by a mouse-derived E. clostridioformis strain. ConclusionsOur findings suggest that SQ is solely a nutrient for the gut microbiota and not for mice and humans, emphasizing its potential as a prebiotic. SQ degradation by the microbiota of conventional laboratory mice differs from the human gut microbiota by absence of DHPS degradation activity. Hence, the microbiota of conventional laboratory mice does not fully represent the SQ metabolism in humans, indicating the need for alternative model systems to assess the impact of SQ on human health. This study advances our understanding of how individual dietary compounds shape the microbial community structure and metabolism in the gut and thereby potentially influence host health.

microbiology↗

bioRxiv2020

Immunosuppressive effect of the Fusarium secondary metabolite butenolide in human colon epithelial cells.

Butenolide (BUT, 4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone) is a secondary metabolite produced by several Fusarium species and is co-produced with the major trichothecene mycotoxin deoxynivalenol (DON) on cereal grains throughout the world. BUT has low acute toxicity and only very limited occurrence and exposure data are available. The intestinal epithelium represents the first physiological barrier against food contaminants. We aimed to elucidate the intestinal inflammatory response of the human, non-cancer epithelial HCEC-1CT cells to BUT and to characterize potential combinatory interactions with co-occurring trichothecenes, such as DON and NX-3. Using a reporter gene approach, BUT ([≥]5 M, 20 h) was found to decrease lipopolysaccharide (LPS; 10 ng/mL) induced nuclear factor kappa B (NF-{kappa}B) activation in a dose-dependent manner, and in combinatory treatments represses trichothecene-induced enhancement of this important inflammatory pathway. Analyzing transcription and secretion levels of NF-{kappa}B-dependent, pro-inflammatory cytokines, revealed a significant down-regulation of IL-1{beta}, IL-6 and TNF- in IL-1{beta}-stimulated (25 ng/mL) HCEC-1CT cells after BUT exposure (10 M). Trichothecene-induced expression of pro-in-flammatory cytokines by the presence of 1 M DON or NX-3 was substantially suppressed in the presence of 10 M BUT. The emerging mycotoxin BUT has the ability to suppress NF-{kappa}B-induced intestinal inflammatory response mechanisms and to modulate substantially the immune responsiveness of HCEC-1CT cells after trichothecene treatment. Our results suggest that BUT, present in naturally occurring mixtures of Fusarium fungal metabolites, should be increasingly monitored, and the mechanism of inhibition of NF-{kappa}B that might affect the pathogenesis or progression of intestinal inflammatory disorders, should be further investigated.

pharmacology and toxicology↗

bioRxiv2020

Longitudinal assessment of mycotoxin co-exposures in exclusively breastfed infants

ABSTRACTEarly-life development of infants may be critically affected by man-made or natural contaminants including mycotoxins. However, data on the occurrence of food contaminants in breast milk is scarce and prohibits a comprehensive exposure and risk assessment for mothers and their infants.Here, we present a longitudinal exposure assessment over the first 211 days of a single newborn girl (study A) by measuring multiple mycotoxins in milk. Eighty-seven consecutive breast milk samples were obtained from the newborn’s mother living in Austria and following a regular mixed diet. Mycotoxins were analyzed by utilizing a highly sensitive LC-MS/MS approach covering 29 mycotoxins and key metabolites. In addition to this longitudinal study, three mothers provided breast milk samples each on five consecutive days, for a preliminary comparison of inter-day and inter-individual variation in exposures (study B). Study A revealed that mycotoxin occurrence in breast milk was limited to the emerging mycotoxins alternariol monomethyl ether (AME), beauvericin (BEA), enniatins (A, A1, B, B1) and to ochratoxin A (OTA), which is regulated in commercial infant food. These mycotoxins were, if present, mostly detected at very low concentrations (<10 ng/L), except AME which exceeded this concentration on two distinct days by a factor of 3x and 5x. Overall, longitudinal results indicated chronic low-dose exposure to the detected mycotoxins. Other regulated mycotoxins including the carcinogenic aflatoxins or the estrogenic zearalenone and their biotransformation products were absent in all tested samples. Study B confirmed the results of study A, with minimal inter-day and inter-individual variation. In addition, a preliminary correlation of OTA levels occurring in breast milk and matched urine samples was found (r=0.64, p=0.034) in study B. Based on the data set obtained in study A, exposure of the infant was estimated. Exposure estimates of individual mycotoxins were on average below 1 ng/kg body weight per day.Our preliminary findings suggest that recommended maximum daily intake levels might not be exceeded in the Austrian population. However, exposure is likely to be higher in populations with lower food safety standards. In the light of co-occurrence of several emerging mycotoxins in breast milk, future studies should address low-dose mixture effects. This also includes other environmental contaminants which may be present in this bio-fluid and should involve an exposome-scale risk assessment. All these efforts must be intended to minimize exposure of mothers and infants in a window of high susceptibility.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗