marrow (Tang et al. 2022). In this study, AME (1.84, 3.67, and 7.35 µg/kg bw/day) was administered to male Sprague Dawley rats for 28 days by oral gavage. A group receiving the high dose for 28 days followed by a recovery period of 14 days was also included. After the recovery period, the MN frequency in the reticulocytes induced by the high dose of AME was significantly reduced, indicating that the MN induction did not accumulate. The authors also applied the BMD approach to the in vivo MN data suggesting a BMDL<sub>10</sub> and BMDL<sub>50</sub> of 5.33 ng/kg bw/day and 271.42 ng/ kg bw/day, respectively (Tang et al. 2022).

Recent reports on the potential carcinogenicity or cell transformation capacity of *Alternaria* toxins were not found in the literature search. In previous studies, extracts of *A. alternata* containing AME (32, 64, and 128 µg/mL) induced the transformation of NIH/3T3 mouse fibroblasts (Dong et al. 1987). A single exposure of two C3H/10T1 cultures to ATX-I or ATX-III resulted in cell transformation, showing a stronger response to ATX-I (Osborne et al. 1988). Precancerous changes in the esophageal mucosa were discovered in mice (groups of 10 animals) fed with 50–100 mg/kg b.w. per day AME or 25 mg/kg b.w. per day TeA for 10 months in the drinking water, suggesting the possibility of progression to esophageal cancer after prolonged exposure (Yekeler et al. 2001). However, the EFSA Panel on Contaminants was not fully convinced by the results of this study and did not include it in the hazard characterization of these two toxins (EFSA 2011).

## Summary of mutagenicity and genotoxicity studies

AOH and AME are the *Alternaria* toxins most evaluated in terms of genotoxicity. In bacterial systems, they showed weak positive responses in some studies. In mammalian cell systems, both AOH and AME were mutagenic in HPRT/XPRT assays in the V79 cell line. AOH was also positive in the TK gene mutation assay in L51784 tk± cells. Regarding chromosomal damage in vitro, AOH is the only toxin tested with the micronucleus assay, giving positive results in three different cell lines (RAW 264.7, Ishikawa and V79). However, AOH was negative (except for mutations in AOH high-dose recovery group) and AME was positive in vivo in the three genotoxic endpoints evaluated in a 28-day multi-endpoint (comet, micronucleus and mutation in Pig A) study (Miao et al. 2022; Tang et al. 2022). In the in vivo comet assay, AOH was negative in all the assays carried out in rodents except for one study in which positive results were obtained in the parotid gland.

AOH and AME were also tested with in vitro assays not yet contemplated in OECD guidelines such as the γH2AX and comet assay. AOH was positive in all the γH2AX carried out in different cells, while AME was tested only in one study that showed higher levels of γH2AX only at the

highest concentration in the absence of S9. AOH was also positive in almost all the in vitro comet and alkaline unwinding assays except for four assays (3 comet and 1 alkaline unwinding) carried out in HT29 cells. Similarly, AME was positive in all comet and γH2AX assays except one also carried out in HT29 cells.

The perylene quinone toxins ATX-I, ATX-II, ATX-III, and STTX-III are clearly mutagenic in bacterial systems. ATX-II and STTX-III were also positive in the HPRT/XPRT assays carried out in V79 cells. ATX-I and ATX-III were also positive in the in vitro comet and alkaline unwinding assays. ATX-I is the only perylene quinone tested in vivo in a 28-day multi-endpoint (comet, micronucleus, Pig A) study. ATX-I was only positive when using the comet assay in liver and peripheral blood cells.

Finally, TeA, ALT, and TEN produced negative results in the Ames test (bacterial systems), but in experimental designs, in which the criteria for maximum concentration selection were not followed. Therefore, higher test doses might be needed. With respect to mutagenicity assays carried out in mammalian cell systems, TeA was mutagenic in HPRT/XPRT assays carried out in V79 cells but was negative in increasing γH2AX in HepG2 cells and in the comet assay in HT29. TEN was also negative in the in vitro comet assay carried out in HEK 293 T cells. No in vivo studies have been carried out to date for TeA, ALT and TEN.

No genotoxicity or mutagenicity assays, either in vitro or in vivo, have been carried out to date for ALP and ALS; ALT has been only tested in the Ames test. Moreover, no in vitro mutagenicity study in mammalian cells has been found for ATX-I, ATX-III, and TEN. No in vivo studies are available for ALP, ALS, ALT, ATX-III, STTX-III, TeA or TEN. Finally, despite having been more studied, additional experiments with AOH and AME of higher purity, and with higher doses for TeA, ALT and TEN at least in bacterial systems should be performed to generate suitable data for hazard characterization.

## Endocrine disruptive effects

A summary of the studies investigating endocrine disrupting effects of *Alternaria* toxins is presented in Table 6.

## Androgen receptor (AR) transactivation

Possible interactions of AOH with the AR have been investigated in a number of studies, using androgen dependent cell systems. In a study using the TARM-Luc cell line, AR-agonistic effects were not observed with up to 3.87 µM AOH after 48 h of incubation (Frizzell et al. 2013). In line with these results, AOH did not induce an AR-agonistic response in a more recent study in the same cell line at up to 1 µM
marrow (Tang et al. 2022). In this study, AME (1.84, 3.67, and 7.35 µg/kg bw/day) was administered to male Sprague Dawley rats for 28 days by oral gavage. A group receiving the high dose for 28 days followed by a recovery period of 14 days was also included. After the recovery period, the MN frequency in the reticulocytes induced by the high dose of AME was significantly reduced, indicating that the MN induction did not accumulate. The authors also applied the BMD approach to the in vivo MN data suggesting a BMDL<sub>10</sub> and BMDL<sub>50</sub> of 5.33 ng/kg bw/day and 271.42 ng/ kg bw/day, respectively (Tang et al. 2022).

Recent reports on the potential carcinogenicity or cell transformation capacity of *Alternaria* toxins were not found in the literature search. In previous studies, extracts of *A. alternata* containing AME (32, 64, and 128 µg/mL) induced the transformation of NIH/3T3 mouse fibroblasts (Dong et al. 1987). A single exposure of two C3H/10T1 cultures to ATX-I or ATX-III resulted in cell transformation, showing a stronger response to ATX-I (Osborne et al. 1988). Precancerous changes in the esophageal mucosa were discovered in mice (groups of 10 animals) fed with 50–100 mg/kg b.w. per day AME or 25 mg/kg b.w. per day TeA for 10 months in the drinking water, suggesting the possibility of progression to esophageal cancer after prolonged exposure (Yekeler et al. 2001). However, the EFSA Panel on Contaminants was not fully convinced by the results of this study and did not include it in the hazard characterization of these two toxins (EFSA 2011).

## Summary of mutagenicity and genotoxicity studies

AOH and AME are the *Alternaria* toxins most evaluated in terms of genotoxicity. In bacterial systems, they showed weak positive responses in some studies. In mammalian cell systems, both AOH and AME were mutagenic in HPRT/XPRT assays in the V79 cell line. AOH was also positive in the TK gene mutation assay in L51784 tk± cells. Regarding chromosomal damage in vitro, AOH is the only toxin tested with the micronucleus assay, giving positive results in three different cell lines (RAW 264.7, Ishikawa and V79). However, AOH was negative (except for mutations in AOH high-dose recovery group) and AME was positive in vivo in the three genotoxic endpoints evaluated in a 28-day multi-endpoint (comet, micronucleus and mutation in Pig A) study (Miao et al. 2022; Tang et al. 2022). In the in vivo comet assay, AOH was negative in all the assays carried out in rodents except for one study in which positive results were obtained in the parotid gland.

AOH and AME were also tested with in vitro assays not yet contemplated in OECD guidelines such as the γH2AX and comet assay. AOH was positive in all the γH2AX carried out in different cells, while AME was tested only in one study that showed higher levels of γH2AX only at the

highest concentration in the absence of S9. AOH was also positive in almost all the in vitro comet and alkaline unwinding assays except for four assays (3 comet and 1 alkaline unwinding) carried out in HT29 cells. Similarly, AME was positive in all comet and γH2AX assays except one also carried out in HT29 cells.

The perylene quinone toxins ATX-I, ATX-II, ATX-III, and STTX-III are clearly mutagenic in bacterial systems. ATX-II and STTX-III were also positive in the HPRT/XPRT assays carried out in V79 cells. ATX-I and ATX-III were also positive in the in vitro comet and alkaline unwinding assays. ATX-I is the only perylene quinone tested in vivo in a 28-day multi-endpoint (comet, micronucleus, Pig A) study. ATX-I was only positive when using the comet assay in liver and peripheral blood cells.

Finally, TeA, ALT, and TEN produced negative results in the Ames test (bacterial systems), but in experimental designs, in which the criteria for maximum concentration selection were not followed. Therefore, higher test doses might be needed. With respect to mutagenicity assays carried out in mammalian cell systems, TeA was mutagenic in HPRT/XPRT assays carried out in V79 cells but was negative in increasing γH2AX in HepG2 cells and in the comet assay in HT29. TEN was also negative in the in vitro comet assay carried out in HEK 293 T cells. No in vivo studies have been carried out to date for TeA, ALT and TEN.

No genotoxicity or mutagenicity assays, either in vitro or in vivo, have been carried out to date for ALP and ALS; ALT has been only tested in the Ames test. Moreover, no in vitro mutagenicity study in mammalian cells has been found for ATX-I, ATX-III, and TEN. No in vivo studies are available for ALP, ALS, ALT, ATX-III, STTX-III, TeA or TEN. Finally, despite having been more studied, additional experiments with AOH and AME of higher purity, and with higher doses for TeA, ALT and TEN at least in bacterial systems should be performed to generate suitable data for hazard characterization.

## Endocrine disruptive effects

A summary of the studies investigating endocrine disrupting effects of *Alternaria* toxins is presented in Table 6.

### Androgen receptor (AR) transactivation

Possible interactions of AOH with the AR have been investigated in a number of studies, using androgen dependent cell systems. In a study using the TARM-Luc cell line, AR-agonistic effects were not observed with up to 3.87 µM AOH after 48 h of incubation (Frizzell et al. 2013). In line with these results, AOH did not induce an AR-agonistic response in a more recent study in the same cell line at up to 1 µM

<figure><img src="image_11.png" /></figure>
after 48-h exposure (Demaegdt et al. 2016). On the other hand, Stypuła-Trębas et al. (2017) demonstrated an androgenic response at $10^{-2}$ to 400 µM AOH in a yeast bioassay resulting in a remarkably high EC$_{50}$ of 270 µM.

A potent inhibition of testosterone-induced luminescence production was demonstrated for AOH in TARM-Luc cells incubated for 48 h with $9.6 \times 10^{-2}$ to $19.2 \mu M$ (Frizzell et al. 2013). A possible antagonistic activity of more than 50% was observed above 4.8 µM. According to the authors, the reduction in transcriptional activation could be attributed to cytotoxic effects, as a decrease in cell viability of approximately 25% was recorded at the highest concentration used (19,2 µM). Demaegdt et al. (Demaegdt et al. 2016), also suggested a possible antagonistic effect of low potency for AOH at non-cytotoxic doses. An IC$_{50}$ of 3.8 µM was derived for the same experimental system and incubation period. A weak AR antagonistic effect was reported by Stypuła-Trębas et al. (2017) for 5 µM AOH in the yeast reporter bioassay, while an additive effect on the testosterone response was observed at concentrations above 50 µM. Effects on cell viability were not observed in this test system. Interestingly, AOH was successfully docked into the activate pocket of wild-type androgen receptor in a molecular docking study, suggesting possible binding (Agwupuye et al. 2021).

## Effects on steroidogenesis

Reports describing the adverse effects of *Alternaria* toxins on steroidogenesis are scarce and mostly available for AOH. In H295R cells, AOH increased the level of progesterone and estradiol at the highest tested concentration of 3.87 µM, while the testosterone and cortisol levels were not affected (Frizzell et al. 2013). To confirm this effect, qPCR was performed in H295R cells, demonstrating an upregulation of CYP19, encoding for estradiol synthesis, and of HSD3B, a gene involved in the synthesis of progesterone, testosterone, and androstenedione. AOH also inhibited the expression of the nuclear receptor NR0B1, which has an inhibitory effect on CYP1A1, CYP17 and CYP21, which were all upregulated in this study by AOH treatment. AOH did not affect the expression of the steroidogenic acute regulatory protein (StAR) and of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR). Another study using SILAC proteomics identified 22 significantly regulated proteins in H295R cells treated with AOH. Interestingly, seven out of the 22 regulated proteins (SOAT1, NR5A1, NPC1, ACBD5, FDX1, HSD3B, and CYP21A2) are involved in steroidogenesis. For confirmation, the regulatory effects of AOH on the key proteins of steroid biosynthesis, HSD3B and CYP21A2, were investigated by qPCR. Consistent with the proteomic results, AOH upregulated the transcription of HSD3B and CYP21A2. On the contrary, AOH and AME did not alter

the expression of CYP11a1 or HSD3b in pig granulosa cells but decreased the level of progesterone at a concentration of 0.8 µM; however, TEA had no measurable effect (Tiemann et al. 2009).

## Estrogenic response (ER) transactivation

The effects of *Alternaria* toxins on ER transactivation are summarized in Table 6. Most assays have been performed with AOH. Using alkaline phosphatase activity, whose expression is regulated by estrogens through its ERE (estrogen responsive element) regulated promotor as read-out, AOH-stimulated activity was observed in Ishikawa cells with an EC$_{50}$ of 2.7 µM (Aichinger et al. 2020c). The agonistic effect at EC$_{50}$ between 4.7 and 6.2 µM AOH was confirmed in MMV-Luc cell lines bearing a plasmid that contains a luciferase gene under the control of a promoter including the ERE sequences (Demaegdt et al. 2016; Frizzell et al. 2013). Moreover, an EC$_{50}$ of 200 µM AOH was determined in the yeast bioassay using recombinant *Saccharomyces cerevisiae* stably transfected with human estrogen receptor (hER) and the yeast-enhanced green fluorescent protein (yEGFP) under control of the consensus ERE sequences (Stypuła-Trębas et al. 2017). Even though AOH has an agonistic effect on ER, its relative estrogenic potential is low as compared to estradiol (0.005% in Stypuła-Trębas et al. 2017 and 0.004% in Frizzell et al. 2013). In addition, it has been shown in receptor binding assays using isolated recombinant human estrogen receptors α and β that AOH had a tenfold higher affinity for ERβ than ERα (3.1 vs 30 µM, respectively; Lehmann et al. 2006). However, the binding affinities of the natural ligand estrogen were 4 and 1.2 nM, respectively, meaning that the relative binding affinities of AOH were 0.01 and 0.04%. Some studies were also performed on AME showing that this molecule stimulated the activity of alkaline phosphatase in a similar concentration range to AOH (Aichinger et al. 2019; Dellafiora et al. 2018). AME, however, was found to fit better into the binding pocket of ER than AOH (Dellafiora et al. 2018). In addition, the respective hydroxylated phase I metabolites AOH-OH and AME-OH were shown to trigger ER-dependent activity in Ishikawa cells, but less efficiently. Whereas 2.5 µM of the parent compounds was sufficient to significantly induce alkaline phosphatase activity, 5 µM of the metabolites was necessary to achieve a comparable effect (Dellafiora et al. 2018). Receptor binding studies showed that these catecholic AOH and AME-metabolites were unable to bind to the ER. However, in Ishikawa cells, incubation with 4-OH-AOH quickly resulted in the formation of a methoxylated product. In silico modeling indicated a re-occurrence of ER-receptor activity (Dellafiora et al. 2018).

*Alternaria* toxins occur usually in complex mixtures, which can exhibit specific toxicological properties. It
after 48-h exposure (Demaegdt et al. 2016). On the other hand, Stypuła-Trębas et al. (2017) demonstrated an androgenic response at $10^{-2}$ to 400 µM AOH in a yeast bioassay resulting in a remarkably high EC$_{50}$ of 270 µM.

A potent inhibition of testosterone-induced luminescence production was demonstrated for AOH in TARM-Luc cells incubated for 48 h with $9.6 \times 10^{-2}$ to $19.2 \mu M$ (Frizzell et al. 2013). A possible antagonistic activity of more than 50% was observed above 4.8 µM. According to the authors, the reduction in transcriptional activation could be attributed to cytotoxic effects, as a decrease in cell viability of approximately 25% was recorded at the highest concentration used (19,2 µM). Demaegdt et al. (Demaegdt et al. 2016), also suggested a possible antagonistic effect of low potency for AOH at non-cytotoxic doses. An IC$_{50}$ of 3.8 µM was derived for the same experimental system and incubation period. A weak AR antagonistic effect was reported by Stypuła-Trębas et al. (2017) for 5 µM AOH in the yeast reporter bioassay, while an additive effect on the testosterone response was observed at concentrations above 50 µM. Effects on cell viability were not observed in this test system. Interestingly, AOH was successfully docked into the activate pocket of wild-type androgen receptor in a molecular docking study, suggesting possible binding (Agwupuye et al. 2021).

## Effects on steroidogenesis

Reports describing the adverse effects of *Alternaria* toxins on steroidogenesis are scarce and mostly available for AOH. In H295R cells, AOH increased the level of progesterone and estradiol at the highest tested concentration of 3.87 µM, while the testosterone and cortisol levels were not affected (Frizzell et al. 2013). To confirm this effect, qPCR was performed in H295R cells, demonstrating an upregulation of CYP19, encoding for estradiol synthesis, and of HSD3B, a gene involved in the synthesis of progesterone, testosterone, and androstenedione. AOH also inhibited the expression of the nuclear receptor NR0B1, which has an inhibitory effect on CYP1A1, CYP17 and CYP21, which were all upregulated in this study by AOH treatment. AOH did not affect the expression of the steroidogenic acute regulatory protein (StAR) and of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR). Another study using SILAC proteomics identified 22 significantly regulated proteins in H295R cells treated with AOH. Interestingly, seven out of the 22 regulated proteins (SOAT1, NR5A1, NPC1, ACBD5, FDX1, HSD3B, and CYP21A2) are involved in steroidogenesis. For confirmation, the regulatory effects of AOH on the key proteins of steroid biosynthesis, HSD3B and CYP21A2, were investigated by qPCR. Consistent with the proteomic results, AOH upregulated the transcription of HSD3B and CYP21A2. On the contrary, AOH and AME did not alter

the expression of CYP11a1 or HSD3b in pig granulosa cells but decreased the level of progesterone at a concentration of 0.8 µM; however, TEA had no measurable effect (Tiemann et al. 2009).

## Estrogenic response (ER) transactivation

The effects of *Alternaria* toxins on ER transactivation are summarized in Table 6. Most assays have been performed with AOH. Using alkaline phosphatase activity, whose expression is regulated by estrogens through its ERE (estrogen responsive element) regulated promotor as read-out, AOH-stimulated activity was observed in Ishikawa cells with an EC$_{50}$ of 2.7 µM (Aichinger et al. 2020c). The agonistic effect at EC$_{50}$ between 4.7 and 6.2 µM AOH was confirmed in MMV-Luc cell lines bearing a plasmid that contains a luciferase gene under the control of a promoter including the ERE sequences (Demaegdt et al. 2016; Frizzell et al. 2013). Moreover, an EC$_{50}$ of 200 µM AOH was determined in the yeast bioassay using recombinant *Saccharomyces cerevisiae* stably transfected with human estrogen receptor (hER) and the yeast-enhanced green fluorescent protein (yEGFP) under control of the consensus ERE sequences (Stypuła-Trębas et al. 2017). Even though AOH has an agonistic effect on ER, its relative estrogenic potential is low as compared to estradiol (0.005% in Stypuła-Trębas et al. 2017 and 0.004% in Frizzell et al. 2013). In addition, it has been shown in receptor binding assays using isolated recombinant human estrogen receptors α and β that AOH had a tenfold higher affinity for ERβ than ERα (3.1 vs 30 µM, respectively; Lehmann et al. 2006). However, the binding affinities of the natural ligand estrogen were 4 and 1.2 nM, respectively, meaning that the relative binding affinities of AOH were 0.01 and 0.04%. Some studies were also performed on AME showing that this molecule stimulated the activity of alkaline phosphatase in a similar concentration range to AOH (Aichinger et al. 2019; Dellafiora et al. 2018). AME, however, was found to fit better into the binding pocket of ER than AOH (Dellafiora et al. 2018). In addition, the respective hydroxylated phase I metabolites AOH-OH and AME-OH were shown to trigger ER-dependent activity in Ishikawa cells, but less efficiently. Whereas 2.5 µM of the parent compounds was sufficient to significantly induce alkaline phosphatase activity, 5 µM of the metabolites was necessary to achieve a comparable effect (Dellafiora et al. 2018). Receptor binding studies showed that these catecholic AOH and AME-metabolites were unable to bind to the ER. However, in Ishikawa cells, incubation with 4-OH-AOH quickly resulted in the formation of a methoxylated product. In silico modeling indicated a re-occurrence of ER-receptor activity (Dellafiora et al. 2018).

*Alternaria* toxins occur usually in complex mixtures, which can exhibit specific toxicological properties. It
<table><caption>**Table 6** Summary of published studies reporting the endocrine disrupting effects of Alternaria toxins</caption><thead><tr><th>Test item</th><th>Assay</th><th>Cells</th><th>Concentration / exposure time</th><th>Effects</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">AOH</td><td rowspan="4">Alkaline Phosphatase Activity Assay</td><td rowspan="4">Ishikawa</td><td>0.05–10 µM<br>48 h</td><td>EC50 2.7 µM</td><td>Aichinger et al. (2020b)</td></tr><tr><td>0.05–10 µM<br>48 h</td><td>Increased activity at<br>2.5 µM</td><td>Dellafiora et al. (2018)</td></tr><tr><td>0.5–10 µM<br>72 h</td><td>Increased activity at<br>2.5 µM</td><td>Lehmann et al. (2006)</td></tr><tr><td>2.5 nM–2.5 µM<br>48 h</td><td>Increased activity at<br>2.5 µM</td><td>Aichinger et al. (2019)</td></tr><tr><td rowspan="6">Reporter gene Assay</td><td rowspan="3">MMV-Luc estrogen responsive</td><td>0.19–38.7 µM<br>24 h</td><td>Agonistic effect: EC50<br>of 6.2±1.6 µM; No<br>data on antagonistic<br>effect</td><td>Frizzell et al. (2013)</td></tr><tr><td>0–10 µM<br>24 h</td><td>Agonism: EC50 of<br>4.7±2.8 µM<br>Antagonism: IC50 of<br>5.2±2.5 µM</td><td>Demaegdt et al. (2016)</td></tr><tr><td>TARM-Luc androgen and progestagen responsive</td><td>0.19–38.7 µM<br>48 h<br>0–10 µM<br>48 h</td><td>Agonism: negative<br>Antagonism: posi-<br>ti ve ≥ 4.9 µM<br>Agonism: negative<br>Antagonism: IC50 of<br>3.8±0.6 µM</td><td>Frizzell et al. (2013)<br>Demaegdt et al. (2016)</td></tr><tr><td>TM-Luc progestagen responsive</td><td>0.19–38.7 µM<br>48 h</td><td>Agonism: negative<br>Antagonism: posi-<br>ti ve ≥ 4.9 µM</td><td>Frizzell et al. (2013)</td></tr><tr><td>TGRM-Luc glucocorticoid and progestagen responsive</td><td>0.19–38.7 µM<br>48 h</td><td>Agonism: negative<br>Antagonism: posi-<br>ti ve ≥ 4.9 µM</td><td>Frizzell et al. (2013)</td></tr><tr><td>U-2 OS cells stably transfected with human TRβ</td><td>0–10 µM<br>24 h</td><td>Agonism: negative<br>Antagonism: negative</td><td>Demaegdt et al. (2016)</td></tr><tr><td rowspan="2">Receptor binding / localization</td><td>Isolated recombinant human Estrogen receptors α and β</td><td>0.25–10 µM in the presence of 1.04 nM of [3H]E2</td><td>Affinity of 30±20 µM<br>for ERα and<br>3.1±2.9 µM for ERβ</td><td>Lehmann et al. (2006)</td></tr><tr><td>Porcine ER from uterine endometrial cytosol</td><td>0.1–10 µM<br>24 h</td><td>No competing binding</td><td>Wollenhaupt et al. (2008)</td></tr><tr><td rowspan="3">Yeast bioassay</td><td rowspan="3">Yeast cells stably transformed with hER</td><td rowspan="3">Yeast cells stably transformed with hAR</td><td>0.1–10 µM<br>24 h</td><td>Tendency to nuclear<br>translocation at 10 µM</td><td>Aichinger et al. (2020b)</td></tr><tr><td>10 nM–400 µM<br>24 h</td><td>Agonism: EC50 of<br>200 µM<br>Antagonism: negative</td><td>Stypuła-Trębas et al. (2017)</td></tr><tr><td>10 nM–400 µM<br>24 h</td><td>Agonism: EC50 of<br>269.4 µM<br>Antagonism: Weak<br>antagonistic effect at<br>5 µM, additive effect at<br>higher doses</td><td>Stypuła-Trębas et al. (2017)</td></tr></tbody></table>

<figure><img src="image_11.png" /></figure>
<table><caption>**Table 6** Summary of published studies reporting the endocrine disrupting effects of Alternaria toxins</caption><thead><tr><th>Test item</th><th>Assay</th><th>Cells</th><th>Concentration / exposure time</th><th>Effects</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">AOH</td><td rowspan="4">Alkaline Phosphatase Activity Assay</td><td rowspan="4">Ishikawa</td><td>0.05–10 µM<br>48 h</td><td>EC50 2.7 µM</td><td>Aichinger et al. (2020b)</td></tr><tr><td>0.05–10 µM<br>48 h</td><td>Increased activity at 2.5 µM</td><td>Dellafiora et al. (2018)</td></tr><tr><td>0.5–10 µM<br>72 h</td><td>Increased activity at 2.5 µM</td><td>Lehmann et al. (2006)</td></tr><tr><td>2.5 nM–2.5 µM<br>48 h</td><td>Increased activity at 2.5 µM</td><td>Aichinger et al. (2019)</td></tr><tr><td rowspan="6">Reporter gene Assay</td><td rowspan="3">MMV-Luc estrogen responsive</td><td>0.19–38.7 µM<br>24 h</td><td>Agonistic effect: EC50 of 6.2±1.6 µM; No data on antagonistic effect</td><td>Frizzell et al. (2013)</td></tr><tr><td>0–10 µM<br>24 h</td><td>Agonism: EC50 of 4.7±2.8 µM<br>Antagonism: IC50 of 5.2±2.5 µM</td><td>Demaegdt et al. (2016)</td></tr><tr><td>TARM-Luc androgen and progestagen responsive</td><td>0.19–38.7 µM<br>48 h<br>0–10 µM<br>48 h</td><td>Agonism: negative<br>Antagonism: positive≥4.9 µM<br>Agonism: negative<br>Antagonism: IC50 of 3.8±0.6 µM</td><td>Frizzell et al. (2013)</td></tr><tr><td>TM-Luc progestagen responsive</td><td>0.19–38.7 µM<br>48 h</td><td>Agonism: negative<br>Antagonism: positive≥4.9 µM</td><td>Frizzell et al. (2013)</td></tr><tr><td>TGRM-Luc glucocorticoid and progestagen responsive</td><td>0.19–38.7 µM<br>48 h</td><td>Agonism: negative<br>Antagonism: positive≥4.9 µM</td><td>Frizzell et al. (2013)</td></tr><tr><td>U-2 OS cells stably transfected with human TRβ</td><td>0–10 µM<br>24 h</td><td>Agonism: negative<br>Antagonism: negative</td><td>Demaegdt et al. (2016)</td></tr><tr><td rowspan="2">Receptor binding / localization</td><td>Isolated recombinant human Estrogen receptors α and β</td><td>0.25–10 µM in the presence of 1.04 nM of [3H]E2</td><td>Affinity of 30±20 µM for ERα and 3.1±2.9 µM for ERβ</td><td>Lehmann et al. (2006)</td></tr><tr><td>Porcine ER from uterine endometrial cytosol</td><td>0.1–10 µM<br>24 h</td><td>No competing binding</td><td>Wollenhaupt et al. (2008)</td></tr><tr><td rowspan="3">Yeast bioassay</td><td rowspan="3">Yeast cells stably transformed with hER</td><td>Ishikawa</td><td>0.1–10 µM<br>24 h</td><td>Tendency to nuclear translocation at 10 µM</td><td>Aichinger et al. (2020b)</td></tr><tr><td>10 nM–400 µM<br>24 h</td><td>Agonism: EC50 of 200 µM<br>Antagonism: negative</td><td>Stypuła-Trębas et al. (2017)</td></tr><tr><td>10 nM–400 µM<br>24 h</td><td>Agonism: EC50 of 269.4 µM<br>Antagonism: Weak antagonistic effect at 5 µM, additive effect at higher doses</td><td>Stypuła-Trębas et al. (2017)</td></tr></tbody></table>
<table><caption>**Table 6** (continued)</caption><thead><tr><th>Test item</th><th>Assay</th><th>Cells</th><th>Concentration / exposure time</th><th>Effects</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Steroidogenesis</td><td rowspan="2">Pig granulosa cells</td><td>0.8–100 µM<br>24 h</td><td>Decreased progesterone secretion at 0.8 µM</td><td>Tiemann et al. (2009)</td></tr><tr><td>0.39 nM–3.87 µM<br>48 h</td><td>Increase in progesterone and estradiol at 1,000 ng/mL (3.87 µM);<br>No effects on testosterone and cortisol secretion</td><td>Frizzell et al. (2013)</td></tr><tr><td rowspan="2">H295R cells</td><td>0.8–100 µM<br>24 h</td><td>No effects on expression of Cyp11a1 and Hsd3b</td><td>Tiemann et al. (2009)</td></tr><tr><td>3.87 µM<br>48 h</td><td>Upregulation of CYP1A1, MC2R, HSD3B2, CYP17, CYP21, CYP11B2, CYP19;<br>Downregulation of NR0B1</td><td>Frizzell et al. (2013)</td></tr><tr><td rowspan="2">H295R cells</td><td>3.87 µM<br>48 h</td><td>Identification of deregulated proteins by using SILAC proteomics;<br>Upregulation of HSD3B, CYP21A2, SOAT1, FDX1;<br>Downregulation of NR5A1, NPC1, ACBD5</td><td>Kalayou et al. (2014)</td></tr><tr><td>4 µM<br>48 h</td><td>Upregulation of HSD3B and CYP21A2</td><td>Kalayou et al. (2014)</td></tr><tr><td rowspan="2">Molecular docking study</td><td rowspan="2">NR</td><td>NR</td><td>Successfully docked into the activate pocket of wild-type AR</td><td>Agwupuye et al. (2021)</td></tr><tr><td>NR</td><td>Favorable interaction with ERα and ERβ ligand pocket</td><td>Aichinger et al. (2020c)</td></tr><tr><td rowspan="4">AME</td><td rowspan="2">Alkaline Phosphatase Activity Assay</td><td>Ishikawa</td><td>0.05–10 µM<br>48 h<br>2.0 nM–2.0 µM<br>48 h</td><td>Increased activity at 2.5 µM<br>Increased activity at 2.0 µM</td><td>Dellafiora et al. (2018)<br>Aichinger et al. (2019)</td></tr><tr><td>Pig granulosa cells</td><td>0.8–100 µM<br>24 h<br>0.8–100 µM<br>24 h</td><td>decreased progesterone secretion at 0.8 µM<br>No effects on expression of Cyp11a1 and Hsd3b</td><td>Tiemann et al. (2009)<br>Tiemann et al. (2009)</td></tr><tr><td rowspan="2">Molecular docking study</td><td rowspan="2">NR</td><td>NR</td><td>Favorable interaction with ERα and ERβ ligand pocket. More favorably than AOH</td><td>Dellafiora et al. (2018)</td></tr><tr><td>NR</td><td>No effects on progesterone secretion by TeA</td><td>Tiemann et al. (2009)</td></tr><tr><td>TeA</td><td>Steroidogenesis</td><td>Pig granulosa cells</td><td>0.8–100 µM<br>24 h</td><td></td><td></td></tr></tbody></table>

was shown that native *Alternaria alternata* extracts did not elicit any estrogenic stimulus (measured as alkaline phosphatase activity) in Ishikawa cells up to 10 µg/mL, but when they were incubated with 1 nM E2, they induced a significant decrease of the expected estrogenic response at concentrations starting at 5 µg/mL (Aichinger et al.

2019). However, the compound(s) responsible for these effects remain(s) to be identified. Similarly, a complex extract obtained from *Alternaria alternata* culture was found to partly quench the estrogenic activity of fecal slurries and fecal water even after 3 h of anaerobic incubation (Crudo et al. 2022). The effect of *Alternaria*
<table><caption>**Table 6** (continued)</caption><thead><tr><th>Test item</th><th>Assay</th><th>Cells</th><th>Concentration / exposure time</th><th>Effects</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Steroidogenesis</td><td rowspan="2">Pig granulosa cells</td><td>0.8–100 µM<br>24 h</td><td>Decreased progesterone secretion at 0.8 µM</td><td>Tiemann et al. (2009)</td></tr><tr><td>0.39 nM–3.87 µM<br>48 h</td><td>Increase in progesterone and estradiol at 1,000 ng/mL (3.87 µM); No effects on testosterone and cortisol secretion</td><td>Frizzell et al. (2013)</td></tr><tr><td rowspan="2">H295R cells</td><td>0.8–100 µM<br>24 h</td><td>No effects on expression of Cyp11a1 and Hsd3b</td><td>Tiemann et al. (2009)</td></tr><tr><td>3.87 µM<br>48 h</td><td>Upregulation of CYP1A1, MC2R, HSD3B2, CYP17, CYP21, CYP11B2, CYP19; Downregulation of NR0B1</td><td>Frizzell et al. (2013)</td></tr><tr><td rowspan="2">H295R cells</td><td>3.87 µM<br>48 h</td><td>Identification of deregulated proteins by using SILAC proteomics; Upregulation of HSD3B, CYP21A2, SOAT1, FDX1; Downregulation of NR5A1, NPC1, ACBD5</td><td>Kalayou et al. (2014)</td></tr><tr><td>4 µM<br>48 h</td><td>Upregulation of HSD3B and CYP21A2</td><td>Kalayou et al. (2014)</td></tr><tr><td rowspan="2">Molecular docking study</td><td rowspan="2">NR</td><td rowspan="2">NR</td><td>Successfully docked into the activate pocket of wild-type AR</td><td>Agwupuye et al. (2021)</td></tr><tr><td>Favorable interaction with ERα and ERβ ligand pocket</td><td>Aichinger et al. (2020c)</td></tr><tr><td rowspan="4">AME</td><td rowspan="2">Alkaline Phosphatase Activity Assay</td><td rowspan="2">Ishikawa</td><td>0.05–10 µM<br>48 h</td><td>Increased activity at 2.5 µM</td><td>Dellafiora et al. (2018)</td></tr><tr><td>2.0 nM–2.0 µM<br>48 h</td><td>Increased activity at 2.0 µM</td><td>Aichinger et al. (2019)</td></tr><tr><td rowspan="2">Steroidogenesis</td><td rowspan="2">Pig granulosa cells</td><td>0.8–100 µM<br>24 h</td><td>decreased progesterone secretion at 0.8 µM</td><td>Tiemann et al. (2009)</td></tr><tr><td>0.8–100 µM<br>24 h</td><td>No effects on expression of Cyp11a1 and Hsd3b</td><td>Tiemann et al. (2009)</td></tr><tr><td>Molecular docking study</td><td>NR</td><td>NR</td><td>Favorable interaction with ERα and ERβ ligand pocket. More favorably than AOH</td><td>Dellafiora et al. (2018)</td></tr><tr><td>TeA</td><td>Steroidogenesis</td><td>Pig granulosa cells</td><td>0.8–100 µM<br>24 h</td><td>No effects on progesterone secretion by TeA</td><td>Tiemann et al. (2009)</td></tr></tbody></table>

was shown that native *Alternaria alternata* extracts did not elicit any estrogenic stimulus (measured as alkaline phosphatase activity) in Ishikawa cells up to 10 µg/mL, but when they were incubated with 1 nM E2, they induced a significant decrease of the expected estrogenic response at concentrations starting at 5 µg/mL (Aichinger et al.

2019). However, the compound(s) responsible for these effects remain(s) to be identified. Similarly, a complex extract obtained from *Alternaria alternata* culture was found to partly quench the estrogenic activity of fecal slurries and fecal water even after 3 h of anaerobic incubation (Crudo et al. 2022). The effect of *Alternaria*
mycotoxins on estrogen receptor signaling may also be mediated by indirect mechanisms such as the ability of AOH to target casein kinase 2 (Aichinger et al. 2020c). Furthermore, a crosstalk may exist between the effects caused by *Alternaria* extract on airway inflammation and on ERα activity because the pulmonary IL-33 release was decreased in ERα-deficient mice after *Alternaria* extract exposure (Cephus et al. 2021). Finally, using the specific ERβ inhibitor PHTPP, it was observed that oxidative stress induced by 0.1 and 10 µM AOH was partially dependent on ER activation in PNT1A-normal human prostatic cells but that the cells were unprotected against DNA damage due to the lack of activation of this receptor (Kowalska et al. 2021b).

## Summary of endocrine disruption studies

The potential of *Alternaria* toxins to interfere with the endocrine system has been investigated in several studies, with the majority focusing on AOH and AME. Table 6 summarizes the effects of *Alternaria* toxins in different in vitro and in silico endocrine-related systems, assessing possible interactions with ER, AR or steroidogenesis.

AOH did not show AR-agonistic properties in two different studies using the same reporter cell line (Demaegdt et al. 2016; Frizzell et al. 2013). This was not the case for a study using a yeast reporter androgen bioassay, where AOH was reported to elicit a full androgenic response (Stypuła-Trębas et al. 2017). Weak antagonistic effects have been reported for AOH in several transactivation assays using different reporter cell systems (Demaegdt et al. 2016; Frizzell et al. 2013; Stypuła-Trębas et al. 2017). Interpretation of these results suggesting antagonistic activity is challenging, as cytotoxicity was also present in the corresponding cell viability assays. Interestingly, AOH was shown to successfully dock into the pocket of wild-type AR, suggesting possible binding to the receptor (Agwupuye et al. 2021).

Although data on the effects of *Alternaria* toxins on steroidogenesis are scarce, there are some indications for potential interference. In the H295R adrenal cell line, AOH was shown to increase progesterone and estradiol synthesis. This was also confirmed by the upregulation of steroidogenesis-related genes in the same cell system, including CYP19 and HSD3B (Frizzell et al. 2013). Further indications for steroidogenesis disruption came from a proteomic study where a number of steroidogenesis-related proteins were found to be decreased. This study was also coupled with gene expression levels of implicated genes, demonstrating a deregulation of HSD3B among other deregulated genes (Kalayou et al. 2014). On the other hand, AOH and AME decreased the level of progesterone secretion without altering the expression of HSD3B and other steroidogenesis-related genes in pig granulosa cells, while TeA had no effect on progesterone

secretion in the same system (Tiemann et al. 2009). The different effects elicited by AOH in various systems highlighted the different steroidogenesis mechanisms implicated in different tissues as well as the different impacts that *Alternaria* toxins could potentially have on them.

Overall, AOH has shown to exert estrogenic-like effects with low potency in several test systems including reporter gene assays and a yeast estrogen-sensitive bioassay (Frizzel et al. 2013; Demaegdt et al. 2016; Stypuła-Trębas et al. 2017). AOH also induced alkaline phosphatase activity in Ishikawa cells (Aichinger et al. 2020c; Dellafiora et al. 2018). In agreement with these results, AOH was shown to favorably arrange within both ERα and ERβ ligand pockets in a molecular docking study performed by Dellafiora et al. 2018. When the binding affinity of AOH was tested with a recombinant human ER, a tenfold higher affinity for ERβ as compared to ERα was shown (Lehmann et al. 2006). As for ER-antagonistic effects, AOH was inactive in reporter gene assays and in yeast cells (Frizzel et al. 2013, Demaegdt et al. 2016, Stypuła-Trębas et al. 2017). Of note, synergistic estrogenic effects were reported in Ishikawa cells upon co-exposure with AOH and the known mycoestrogen zearalenone (Vejdovszky et al. 2017).

Chemical modification and metabolism appeared to play a crucial role in mediating the estrogenic activity of *Alternaria* toxins. In particular, AME was found to fit better into the binding pocket of ER and to be more potent in the alkaline phosphatase assay, indicating that methylation enhanced estrogenicity. On the other hand, the phase I metabolites AOH-OH and AME-OH were unable to bind to the ER but were able to trigger ER-dependent activity in Ishikawa cells with a low efficiency, which might arise from rapid methoxylation of the catechol structures of AOH-OH and AME-OH (Dellafiora et al. 2018).

Native *Alternata alternata* extracts did not trigger any pro-estrogenic stimulus but showed a potent anti-estrogenic effect in an alkaline phosphatase activity assay (Aichinger et al. 2019). However, the compound(s) responsible for these effects remained to be identified. Some published reports indicated that the effect of *Alternaria* mycotoxins on ER signaling may be complex and could be mediated by indirect mechanisms like the ability of AOH to target casein kinase 2.

In additional reporter gene assays, AOH showed no effects on TRβ activation. Furthermore, AOH displayed antagonistic effects in one progestagen-responsive cell line and one progestagen and glucocorticoid-responsive cell line.

## Immunotoxicology / Immunomodulation

The immunotoxicology studies described in the literature are summarized in Table 7.
mycotoxins on estrogen receptor signaling may also be mediated by indirect mechanisms such as the ability of AOH to target casein kinase 2 (Aichinger et al. 2020c). Furthermore, a crosstalk may exist between the effects caused by *Alternaria* extract on airway inflammation and on ERα activity because the pulmonary IL-33 release was decreased in ERα-deficient mice after *Alternaria* extract exposure (Cephus et al. 2021). Finally, using the specific ERβ inhibitor PHTPP, it was observed that oxidative stress induced by 0.1 and 10 µM AOH was partially dependent on ER activation in PNT1A-normal human prostatic cells but that the cells were unprotected against DNA damage due to the lack of activation of this receptor (Kowalska et al. 2021b).

## Summary of endocrine disruption studies

The potential of *Alternaria* toxins to interfere with the endocrine system has been investigated in several studies, with the majority focusing on AOH and AME. Table 6 summarizes the effects of *Alternaria* toxins in different in vitro and in silico endocrine-related systems, assessing possible interactions with ER, AR or steroidogenesis.

AOH did not show AR-agonistic properties in two different studies using the same reporter cell line (Demaegdt et al. 2016; Frizzell et al. 2013). This was not the case for a study using a yeast reporter androgen bioassay, where AOH was reported to elicit a full androgenic response (Stypuła-Trębas et al. 2017). Weak antagonistic effects have been reported for AOH in several transactivation assays using different reporter cell systems (Demaegdt et al. 2016; Frizzell et al. 2013; Stypuła-Trębas et al. 2017). Interpretation of these results suggesting antagonistic activity is challenging, as cytotoxicity was also present in the corresponding cell viability assays. Interestingly, AOH was shown to successfully dock into the pocket of wild-type AR, suggesting possible binding to the receptor (Agwupuye et al. 2021).

Although data on the effects of *Alternaria* toxins on steroidogenesis are scarce, there are some indications for potential interference. In the H295R adrenal cell line, AOH was shown to increase progesterone and estradiol synthesis. This was also confirmed by the upregulation of steroidogenesis-related genes in the same cell system, including CYP19 and HSD3B (Frizzell et al. 2013). Further indications for steroidogenesis disruption came from a proteomic study where a number of steroidogenesis-related proteins were found to be decreased. This study was also coupled with gene expression levels of implicated genes, demonstrating a deregulation of HSD3B among other deregulated genes (Kalayou et al. 2014). On the other hand, AOH and AME decreased the level of progesterone secretion without altering the expression of HSD3B and other steroidogenesis-related genes in pig granulosa cells, while TeA had no effect on progesterone

secretion in the same system (Tiemann et al. 2009). The different effects elicited by AOH in various systems highlighted the different steroidogenesis mechanisms implicated in different tissues as well as the different impacts that *Alternaria* toxins could potentially have on them.

Overall, AOH has shown to exert estrogenic-like effects with low potency in several test systems including reporter gene assays and a yeast estrogen-sensitive bioassay (Frizzel et al. 2013; Demaegdt et al. 2016; Stypuła-Trębas et al. 2017). AOH also induced alkaline phosphatase activity in Ishikawa cells (Aichinger et al. 2020c; Dellafiora et al. 2018). In agreement with these results, AOH was shown to favorably arrange within both ERα and ERβ ligand pockets in a molecular docking study performed by Dellafiora et al. 2018. When the binding affinity of AOH was tested with a recombinant human ER, a tenfold higher affinity for ERβ as compared to ERα was shown (Lehmann et al. 2006). As for ER-antagonistic effects, AOH was inactive in reporter gene assays and in yeast cells (Frizzel et al. 2013, Demaegdt et al. 2016, Stypuła-Trębas et al. 2017). Of note, synergistic estrogenic effects were reported in Ishikawa cells upon co-exposure with AOH and the known mycoestrogen zearalenone (Vejdovszky et al. 2017).

Chemical modification and metabolism appeared to play a crucial role in mediating the estrogenic activity of *Alternaria* toxins. In particular, AME was found to fit better into the binding pocket of ER and to be more potent in the alkaline phosphatase assay, indicating that methylation enhanced estrogenicity. On the other hand, the phase I metabolites AOH-OH and AME-OH were unable to bind to the ER but were able to trigger ER-dependent activity in Ishikawa cells with a low efficiency, which might arise from rapid methoxylation of the catechol structures of AOH-OH and AME-OH (Dellafiora et al. 2018).

Native *Alternata alternata* extracts did not trigger any pro-estrogenic stimulus but showed a potent anti-estrogenic effect in an alkaline phosphatase activity assay (Aichinger et al. 2019). However, the compound(s) responsible for these effects remained to be identified. Some published reports indicated that the effect of *Alternaria* mycotoxins on ER signaling may be complex and could be mediated by indirect mechanisms like the ability of AOH to target casein kinase 2.

In additional reporter gene assays, AOH showed no effects on TRβ activation. Furthermore, AOH displayed antagonistic effects in one progestagen-responsive cell line and one progestagen and glucocorticoid-responsive cell line.

## Immunotoxicology / Immunomodulation

The immunotoxicology studies described in the literature are summarized in Table 7.

<figure><img src="image_12.png" /></figure>
With immune-related diseases like hypersensitivity and autoimmunity on the rise, it is of important to investigate whether repeated exposure to food contaminants might play a role in their development (Beerweiler et al. 2023). However, only in the last decade studies focused more and more on the immunotoxicity and immunomodulation of mycotoxins. Consequently, knowledge about the effects of *Alternaria* toxins on the immune system and related disorders is still incomplete and will be discussed in this chapter.

Plasticity and functional polarization are hallmarks of macrophages. AOH (30–60 µM, 24–48 h) was found to induce major morphological changes in macrophages of different species. In detail, AOH altered the cellular morphology from round to star-like in murine RAW264.7 macrophages as well as from round to a more needle-like morphology in blood-derived primary human macrophages and primary mouse peritoneal macrophages (Solhaug et al. 2015). Although the morphological findings seemed similar, there were marked differences in the phenotypic changes as measured by immune cell markers (CD receptors). However, the differentiated cells could not be characterized as typical M1/M2 macrophages or as dendritic cells. Furthermore, AOH enhanced the level of TNFα and IL-6 at the mRNA level, but only TNFα showed increased secretion in RAW264.7 cells. In human macrophages, secretion of both TNFα and IL-6 was found in response to AOH, while no changes were found for IL-8, IL-10, or IL-12p70 (Solhaug et al. 2015). In RAW264.7 macrophages, AOH (30 µM, 24–48 h) induced senescence and autophagy, most probably associated with AOH-induced DNA damage (Solhaug et al. 2014).

Another study conducted by Solhaug and co-workers showed that AOH (7.5–30 µM, 48 h) also reduced PMA-induced differentiation of human THP1 monocytes into macrophages (Solhaug et al. 2016b), measured by a lower expression of the surface receptors CD14 and CD11b, as well as higher levels of CD71 compared to fully differentiated macrophages. Also, in accordance with reduced CD14 expression, AOH (15 µM) reduced LPS (0.1 ng/ml)-induced secretion of TNFα, potentially by reducing TNFα gene expression (Solhaug et al. 2016b). Furthermore, at equal effect concentration (EC10), a combination of AOH (2.75 µM) and the estrogenic *Fusarium* toxin zearalenone (4.3 µM), showed a synergistic effect on the reduction of PMA-induced CD14 expression in THP1 cells, analyzed by the isobologram approach (Solhaug et al. 2016b).

In a study performed by Kollarova et al. (2018), exposure of THP1-Lucia™-derived macrophages to AOH (20 h) resulted in a dose-dependent suppression of the LPS-induced NF-κB pathway activation starting at 1 µM. In line with these results, Dong et al. (2021) demonstrated decreased nuclear translocation of NF-κB p65 as well as phosphorylation of STAT1/STAT3 by AOH (2.5–10 µM,

3 h) in TNF-α/IFN-γ-stimulated human keratinocytes (HaCaT) cells. Of note, the immunosuppressive effects exerted by AOH on the NF-κB pathway were in line with the suppression of the pro-inflammatory cytokines IL-8, IL-6, and TNFα and the induction of the anti-inflammatory cytokine IL-10 observed in LPS-stimulated cells at mRNA and protein level (Kollarova et al. 2018). Schmutz et al. (2019) reported that AOH also affected the cytokine expression levels in non-immune cells. In particular, the authors found that AOH inhibited the IL-1β-induced transcription of the pro-inflammatory cytokines IL-8, IL-6, and IL-1β in differentiated Caco-2 cells after 5 h of incubation. Although some of these effects at the mRNA level were no longer found after long-term exposure (20 h), AOH nevertheless was able to suppress the secretion of IL-8 after both 5 and 20 h of exposure. Similar suppressive effects on IL-8, IL-6 and on the MCP-1/CCL2 expression (on protein and mRNA level) after LPS stimulation (10 µg/mL) were observed in human bronchial epithelial cells (BEAS-2B) as well as in RAW264.7 macrophages treated with AOH (10 µM, 24 h) by Grover and Lawrence (2017).

AOH was also found to affect the transcription of two miRNAs known to be involved in the regulation of TLR/NF-κB signaling and NF-κB target genes, namely miR-146a (downregulation) and miR-155 (upregulation) in THP1-derived macrophages (Kollarova et al. 2018). In confirmation, Schmutz et al. (2019) reported that AOH (20–40 µM, 5–21 h) was also able to alter the levels of IL-1β-induced microRNAs, including an upregulation of miR-16, miR-125b, and miR-155 as well as a downregulation of miR-146a in Caco-2 cells (Schmutz et al. 2019).

Noteworthy, Del Favero et al. (2020) suggested that AOH affects the signal transduction of pro-inflammatory stimuli by inducing increased membrane fluidity. In addition, it was shown that AOH (1 µM, 1–3 h) increased the co-localization of TLR4 with caveolin-1 in THP1 cells. This might result in premature non-activated TLR4 internalization and subsequently, reduced signaling and cytokine expression. Thus, the data currently available on the *Alternaria* mycotoxin AOH point to the possible inhibition of immune responses in an inflamed cellular environment.

Besides AOH, also AME demonstrated suppressive effects on IL-8, IL-6 and MCP-1/CCL2 protein secretion in LPS-stimulated (10 µg/mL) BEAS-2B cells at concentrations of 10 µM after 24 h incubation, although to a lesser extent as compared to AOH (Grover and Lawrence 2017).

In a comparative study, the decrease in IL-6 secretion in TNF-α/IFN-γ-stimulated HaCaT cells was measured in the presence of AOH, ALS and ALT (27 h incubation) (Dong et al. 2021). While AOH limited the secretion of IL-6 already at 2.5 µM, ALS decreased the secretion only at 20 µM. No effect was observed with ALT up to 80 µM, indicating the varying immunosuppressive potential of
With immune-related diseases like hypersensitivity and autoimmunity on the rise, it is of important to investigate whether repeated exposure to food contaminants might play a role in their development (Beerweiler et al. 2023). However, only in the last decade studies focused more and more on the immunotoxicity and immunomodulation of mycotoxins. Consequently, knowledge about the effects of *Alternaria* toxins on the immune system and related disorders is still incomplete and will be discussed in this chapter.

Plasticity and functional polarization are hallmarks of macrophages. AOH (30–60 µM, 24–48 h) was found to induce major morphological changes in macrophages of different species. In detail, AOH altered the cellular morphology from round to star-like in murine RAW264.7 macrophages as well as from round to a more needle-like morphology in blood-derived primary human macrophages and primary mouse peritoneal macrophages (Solhaug et al. 2015). Although the morphological findings seemed similar, there were marked differences in the phenotypic changes as measured by immune cell markers (CD receptors). However, the differentiated cells could not be characterized as typical M1/M2 macrophages or as dendritic cells. Furthermore, AOH enhanced the level of TNFα and IL-6 at the mRNA level, but only TNFα showed increased secretion in RAW264.7 cells. In human macrophages, secretion of both TNFα and IL-6 was found in response to AOH, while no changes were found for IL-8, IL-10, or IL-12p70 (Solhaug et al. 2015). In RAW264.7 macrophages, AOH (30 µM, 24–48 h) induced senescence and autophagy, most probably associated with AOH-induced DNA damage (Solhaug et al. 2014).

Another study conducted by Solhaug and co-workers showed that AOH (7.5–30 µM, 48 h) also reduced PMA-induced differentiation of human THP1 monocytes into macrophages (Solhaug et al. 2016b), measured by a lower expression of the surface receptors CD14 and CD11b, as well as higher levels of CD71 compared to fully differentiated macrophages. Also, in accordance with reduced CD14 expression, AOH (15 µM) reduced LPS (0.1 ng/ml)-induced secretion of TNFα, potentially by reducing TNFα gene expression (Solhaug et al. 2016b). Furthermore, at equal effect concentration (EC10), a combination of AOH (2.75 µM) and the estrogenic *Fusarium* toxin zearalenone (4.3 µM), showed a synergistic effect on the reduction of PMA-induced CD14 expression in THP1 cells, analyzed by the isobologram approach (Solhaug et al. 2016b).

In a study performed by Kollarova et al. (2018), exposure of THP1-Lucia™-derived macrophages to AOH (20 h) resulted in a dose-dependent suppression of the LPS-induced NF-κB pathway activation starting at 1 µM. In line with these results, Dong et al. (2021) demonstrated decreased nuclear translocation of NF-κB p65 as well as phosphorylation of STAT1/STAT3 by AOH (2.5–10 µM,

3 h) in TNF-α/IFN-γ-stimulated human keratinocytes (HaCaT) cells. Of note, the immunosuppressive effects exerted by AOH on the NF-κB pathway were in line with the suppression of the pro-inflammatory cytokines IL-8, IL-6, and TNFα and the induction of the anti-inflammatory cytokine IL-10 observed in LPS-stimulated cells at mRNA and protein level (Kollarova et al. 2018). Schmutz et al. (2019) reported that AOH also affected the cytokine expression levels in non-immune cells. In particular, the authors found that AOH inhibited the IL-1β-induced transcription of the pro-inflammatory cytokines IL-8, IL-6, and IL-1β in differentiated Caco-2 cells after 5 h of incubation. Although some of these effects at the mRNA level were no longer found after long-term exposure (20 h), AOH nevertheless was able to suppress the secretion of IL-8 after both 5 and 20 h of exposure. Similar suppressive effects on IL-8, IL-6 and on the MCP-1/CCL2 expression (on protein and mRNA level) after LPS stimulation (10 µg/mL) were observed in human bronchial epithelial cells (BEAS-2B) as well as in RAW264.7 macrophages treated with AOH (10 µM, 24 h) by Grover and Lawrence (2017).

AOH was also found to affect the transcription of two miRNAs known to be involved in the regulation of TLR/NF-κB signaling and NF-κB target genes, namely miR-146a (downregulation) and miR-155 (upregulation) in THP1-derived macrophages (Kollarova et al. 2018). In confirmation, Schmutz et al. (2019) reported that AOH (20–40 µM, 5–21 h) was also able to alter the levels of IL-1β-induced microRNAs, including an upregulation of miR-16, miR-125b, and miR-155 as well as a downregulation of miR-146a in Caco-2 cells (Schmutz et al. 2019).

Noteworthy, Del Favero et al. (2020) suggested that AOH affects the signal transduction of pro-inflammatory stimuli by inducing increased membrane fluidity. In addition, it was shown that AOH (1 µM, 1–3 h) increased the co-localization of TLR4 with caveolin-1 in THP1 cells. This might result in premature non-activated TLR4 internalization and subsequently, reduced signaling and cytokine expression. Thus, the data currently available on the *Alternaria* mycotoxin AOH point to the possible inhibition of immune responses in an inflamed cellular environment.

Besides AOH, also AME demonstrated suppressive effects on IL-8, IL-6 and MCP-1/CCL2 protein secretion in LPS-stimulated (10 µg/mL) BEAS-2B cells at concentrations of 10 µM after 24 h incubation, although to a lesser extent as compared to AOH (Grover and Lawrence 2017).

In a comparative study, the decrease in IL-6 secretion in TNF-α/IFN-γ-stimulated HaCaT cells was measured in the presence of AOH, ALS and ALT (27 h incubation) (Dong et al. 2021). While AOH limited the secretion of IL-6 already at 2.5 µM, ALS decreased the secretion only at 20 µM. No effect was observed with ALT up to 80 µM, indicating the varying immunosuppressive potential of
<figure><img src="image_12.png" /><figcaption>**Table 7** Summary of published studies on the immunological effects of *Alternaria* toxins</figcaption></figure>

<table><thead><tr><th>Cells</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>Cytokines</th><th>Other</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>IN VITRO</strong></td></tr><tr><td>Alternariol (AOH)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RAW264.7</td><td>30</td><td>20, 48</td><td>mRNA: IL-6, TNF-α (up), IL-10, IL-12p40 (no change) protein: TNF-α (up), IL-6 (no change)</td><td>Morphological changes CD86, CD80, CD11b (up), CD83 (no change)</td><td>Solhaug et al. (2015)</td></tr><tr><td>Primary mouse macrophages</td><td>60</td><td>24, 48</td><td></td><td>Morphological changes</td><td>Solhaug et al. (2015)</td></tr><tr><td>Primary human macrophages</td><td>30</td><td>24, 48</td><td>Protein: TNF-α, IL-6 (up), IL-8, IL-10, IL12p70 (no change)</td><td>Morphological changes CD86, CD83 (up), CD68, HLA-DR (down)</td><td>Solhaug et al. (2015)</td></tr><tr><td>THP1</td><td>7.5</td><td>48</td><td>Suppressed LPS-induced TNF-α secretion</td><td>Reduced PMA-induced differentiation into macrophages</td><td>Solhaug et al. (2016b)</td></tr><tr><td>THP1</td><td>1</td><td>1-3</td><td></td><td>Affects signal transduction of pro-inflammatory stimuli by increased membrane fluidity</td><td>Del Favero et al. (2020)</td></tr><tr><td>RAW264.7</td><td>30</td><td>24,48</td><td></td><td>Autophagy and senescence</td><td>Solhaug et al. (2014)</td></tr><tr><td>BEAS-2B</td><td>5-10</td><td>24</td><td>Suppressed LPS-induced IL-6, IL-8, CCL2/MCP-1 upregulation (mRNA and protein)</td><td>Upregulation of Caspase-1, but downregulation of LPS-induced caspase-1 (mRNA)</td><td>Grover and Lawrence (2017)</td></tr><tr><td>RAW264.7</td><td>5-10</td><td>24</td><td>Suppressed LPS-induced IL-6 secretion</td><td></td><td>Grover and Lawrence (2017)</td></tr><tr><td>THP-1dif* (macrophages)</td><td>2-20</td><td>20</td><td></td><td>Suppress LPS-induced NF-kB activation, miR-146a (up), miR-155 (down)</td><td>Kollarova et al. (2018)</td></tr><tr><td>Caco-2dif*</td><td>20-40</td><td>5-21</td><td>AOH + IL-1b: mRNA TNF-α and IL-1b (up), IL-8, IL-6 (down) at 5 h, protein IL-8 down (5 and 20 h)</td><td>AOH + IL-1b: miR-125b, miR-146a (up), miR-155 (down)</td><td>Schmutz et al. (2019)</td></tr><tr><td>HaCaT</td><td>2.5-10</td><td>27</td><td>Suppressed INFγ/TNF-α-induced TNF-a, IL-8, MDC and RANTES expression (protein)</td><td>Suppressed INFγ/TNF-α-induced ICAM (protein). Decreased INFγ/TNF-α-induced nuclear relocation of NF-kB p65, as well as phosphorylation of STAT1 and STAT3 (3 h)</td><td>Dong et al. (2021)</td></tr><tr><td>Alternariol monomethyl ether (AME)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BEAS-2B</td><td>10</td><td>24</td><td>Suppressed LPS-induced IL-6, IL-8, CCL2/MCP-1 upregulation (protein)</td><td></td><td>Grover and Lawrence (2017)</td></tr></tbody></table>
<figure><img src="image_13.png" /><figcaption>**Table 7** Summary of published studies on the immunological effects of *Alternaria* toxins</figcaption></figure>

<table><thead><tr><th>Cells</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>Cytokines</th><th>Other</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>IN VITRO</strong></td></tr><tr><td>Alternariol (AOH)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RAW264.7</td><td>30</td><td>20, 48</td><td>mRNA: IL-6, TNF-α (up), IL-10, IL-12p40 (no change) protein: TNF-α (up), IL-6 (no change)</td><td>Morphological changes CD86, CD80, CD11b (up), CD83 (no change)</td><td>Solhaug et al. (2015)</td></tr><tr><td>Primary mouse macrophages</td><td>60</td><td>24, 48</td><td></td><td>Morphological changes</td><td>Solhaug et al. (2015)</td></tr><tr><td>Primary human macrophages</td><td>30</td><td>24, 48</td><td>Protein: TNF-α, IL-6 (up), IL-8, IL-10, IL12p70 (no change)</td><td>Morphological changes CD86, CD83 (up), CD68, HLA-DR (down)</td><td>Solhaug et al. (2015)</td></tr><tr><td>THP1</td><td>7.5</td><td>48</td><td>Suppressed LPS-induced TNF-α secretion</td><td>Reduced PMA-induced differentiation into macrophages</td><td>Solhaug et al. (2016b)</td></tr><tr><td>THP1</td><td>1</td><td>1-3</td><td></td><td>Affects signal transduction of pro-inflammatory stimuli by increased membrane fluidity</td><td>Del Favero et al. (2020)</td></tr><tr><td>RAW264.7</td><td>30</td><td>24,48</td><td></td><td>Autophagy and senescence</td><td>Solhaug et al. (2014)</td></tr><tr><td>BEAS-2B</td><td>5-10</td><td>24</td><td>Suppressed LPS-induced IL-6, IL-8, CCL2/MCP-1 upregulation (mRNA and protein)</td><td>Upregulation of Caspase-1, but downregulation of LPS-induced caspase-1 (mRNA)</td><td>Grover and Lawrence (2017)</td></tr><tr><td>RAW264.7</td><td>5-10</td><td>24</td><td>Suppressed LPS-induced IL-6 secretion</td><td></td><td>Grover and Lawrence (2017)</td></tr><tr><td>THP-1dif* (macrophages)</td><td>2-20</td><td>20</td><td></td><td>Suppress LPS-induced NF-kB activation, miR-146a (up), miR-155 (down)</td><td>Kollarova et al. (2018)</td></tr><tr><td>Caco-2dif*</td><td>20-40</td><td>5-21</td><td>AOH + IL-1b: mRNA TNF-α and IL-1b (up), IL-8, IL-6 (down) at 5 h, protein IL-8 down (5 and 20 h)</td><td>AOH + IL-1b: miR-125b, miR-146a (up), miR-155 (down)</td><td>Schmutz et al. (2019)</td></tr><tr><td>HaCaT</td><td>2.5-10</td><td>27</td><td>Suppressed INFγ/TNF-α-induced TNF-a, IL-8, MDC and RANTES expression (protein)</td><td>Suppressed INFγ/TNF-α-induced ICAM (protein). Decreased INFγ/TNF-α-induced nuclear relocation of NF-kB p65, as well as phosphorylation of STAT1 and STAT3 (3 h)</td><td>Dong et al. (2021)</td></tr><tr><td>Alternariol monomethyl ether (AME)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BEAS-2B</td><td>10</td><td>24</td><td>Suppressed LPS-induced IL-6, IL-8, CCL2/MCP-1 upregulation (protein)</td><td></td><td>Grover and Lawrence (2017)</td></tr></tbody></table>
<table><caption>**Table 7 (continued)**</caption><thead><tr><th>Cells</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>Cytokines</th><th>Other</th><th>References</th></tr></thead><tbody><tr><td>Altenusin (ALS)<br>BV2 microglia</td><td>10–75</td><td>4–24</td><td>Suppressed LPS-induced TNF-α, IL-6, IL-1b, iNOS, CCL-2 (mRNA, 4 h), nitrite and TNF-a (protein, 24 h)</td><td>Used as nSMase inhibitor</td><td>Kumar et al. (2019)</td></tr><tr><td>Primary rat microglia</td><td>10–50</td><td>4–24</td><td>Suppressed LPS induced TNF-α, IL-6, IL-1b, iNOS, CCL-2, IL-10 (mRNA, 4 h), nitrite and TNF-a (protein, 24 h)</td><td>No effect on LPS-induced miR-146 and miR-155</td><td>Kumar et al. (2019)</td></tr><tr><td>HaCaT</td><td>20</td><td>27</td><td>Suppressed INFγ/TNF-α-induced IL-6 secretion</td><td></td><td>Dong et al. (2021)</td></tr><tr><td>Altenuene (ALT)<br>HaCaT</td><td>20–80</td><td>27</td><td>No effect on INFγ/TNF-α-induced IL-6 secretion</td><td></td><td>Dong et al. (2021)</td></tr><tr><td>Altertoxin II (ATX-II)<br>THP1-Lucia™ monocytes</td><td>0.1–1</td><td>20</td><td></td><td>Suppressed (non-induced) NF-κB pathway</td><td>Del Favero et al. (2020)</td></tr><tr><td>THP-1dif*</td><td>1 µM</td><td>1</td><td></td><td>No difference on the immunolocalization of IKBα, NF-κB p65, and NF-κB p65 p-S536 compared to solvent control</td><td>Del Favero et al. (2020)</td></tr><tr><td>In vivo<br>AOH</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mouse blastocytes, liver</td><td>5 mg/kg/day (iv)</td><td>1 day before mating and 4 days after mating</td><td>mRNA: CXCL1, IL-1b, IL-8 (down)</td><td></td><td>Huang et al. (2021)</td></tr><tr><td>Adult rats</td><td>5.51, 10.03, 22.05 µg/kg/day by oral gavage</td><td>28 days</td><td></td><td>Spleen white pulp atrophy, neutrophils decreased and lymphocytes increased (at highest dose)</td><td>Miao et al. (2022)</td></tr><tr><td>Alternariol monomethyl ether (AME)<br>Adult rats</td><td>1.84, 3.67, or 7.35 µg/kg/day by oral gavage</td><td>28 days</td><td>Not measured</td><td>Dose-dependent white pulp atrophy in the spleen</td><td>Tang et al. (2022)</td></tr><tr><td>Altenusin (ALS)<br>Mouse model of traumatic brain injury</td><td>2 and 10 mg/kg (ip)</td><td>once</td><td>24 h post injury 10 mg/kg reduced the expression levels of TNF-α, IL-6, IL-1β, iNOS, and CCL2 in cortex</td><td>Proresolution immune response was unaltered</td><td>Kumar et al. (2019)</td></tr></tbody></table>

THP-1dif*Phorbol 12-myristate 13-acetate (PMA) differentiated into macrophages, Caco-2dif*grown 21 days on transwell insert, ip intraperitoneally, iv intravenously

<figure><img src="image_13.png" /></figure>
<table><caption>**Table 7 (continued)**</caption><thead><tr><th>Cells</th><th>Concentration (µM)</th><th>Exposure (h)</th><th>Cytokines</th><th>Other</th><th>References</th></tr></thead><tbody><tr><td>Altenusin (ALS)<br>BV2 microglia</td><td>10–75</td><td>4–24</td><td>Suppressed LPS-induced TNF-α, IL-6, IL-1b, iNOS, CCL-2 (mRNA, 4 h), nitrite and TNF-a (protein, 24 h)</td><td>Used as nSMase inhibitor</td><td>Kumar et al. (2019)</td></tr><tr><td>Primary rat microglia</td><td>10–50</td><td>4–24</td><td>Suppressed LPS induced TNF-α, IL-6, IL-1b, iNOS, CCL-2, IL-10 (mRNA, 4 h), nitrite and TNF-a (protein, 24 h)</td><td>No effect on LPS-induced miR-146 and miR-155</td><td>Kumar et al. (2019)</td></tr><tr><td>HaCaT</td><td>20</td><td>27</td><td>Suppressed INFγ/TNF-α-induced IL-6 secretion</td><td></td><td>Dong et al. (2021)</td></tr><tr><td>Altenuene (ALT)<br>HaCaT</td><td>20–80</td><td>27</td><td>No effect on INFγ/TNF-α-induced IL-6 secretion</td><td></td><td>Dong et al. (2021)</td></tr><tr><td>Altertoxin II (ATX-II)<br>THP1-Lucia™ monocytes</td><td>0.1–1</td><td>20</td><td></td><td>Suppressed (non-induced) NF-κB pathway</td><td>Del Favero et al. (2020)</td></tr><tr><td>THP-1dif*</td><td>1 µM</td><td>1</td><td></td><td>No difference on the immunolocalization of IKBα, NF-κB p65, and NF-κB p65 p-S536 compared to solvent control</td><td>Del Favero et al. (2020)</td></tr><tr><td>In vivo<br>AOH</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mouse blastocytes, liver</td><td>5 mg/kg/day (iv)</td><td>1 day before mating and 4 days after mating</td><td>mRNA: CXCL1, IL-1b, IL-8 (down)</td><td></td><td>Huang et al. (2021)</td></tr><tr><td>Adult rats</td><td>5.51, 10.03, 22.05 µg/kg/day by oral gavage</td><td>28 days</td><td></td><td>Spleen white pulp atrophy, neutrophils decreased and lymphocytes increased (at highest dose)</td><td>Miao et al. (2022)</td></tr><tr><td>Alternariol monomethyl ether (AME)<br>Adult rats</td><td>1.84, 3.67, or 7.35 µg/kg/day by oral gavage</td><td>28 days</td><td>Not measured</td><td>Dose-dependent white pulp atrophy in the spleen</td><td>Tang et al. (2022)</td></tr><tr><td>Altenusin (ALS)<br>Mouse model of traumatic brain injury</td><td>2 and 10 mg/kg (ip)</td><td>once</td><td>24 h post injury 10 mg/kg reduced the expression levels of TNF-α, IL-6, IL-1β, iNOS, and CCL2 in cortex</td><td>Proresolution immune response was unaltered</td><td>Kumar et al. (2019)</td></tr></tbody></table>

THP-1dif*Phorbol 12-myristate 13-acetate (PMA) differentiated into macrophages, Caco-2dif*grown 21 days on transwell insert, ip intraperitoneally, iv intravenously

<figure><img src="image_14.png" /></figure>
the mycotoxins. Similarly, Kumar et al. (2019) detected a suppression of LPS-induced TNF-α, IL-6, IL-1β, iNOS, CCL-2 mRNA expression (4 h) as well as the release of nitrite and TNF-α (24 h) by increasing concentrations of ALS in BV2 microglia cells and primary rat microglia. However, in contrast to AOH in IL-1β-induced Caco-2 cells (Schmutz et al. 2019), ALS did not alter LPS-induced miR-146 and miR-155 expression in primary microglia (Kumar et al. 2019).

Only one study investigating the immunomodulatory properties of the *Alternaria* mycotoxin ATX-II is currently available. Del Favero and co-workers (2020) demonstrated the ability of subtoxic concentrations of ATX-II (0.1–1 µM, 20 h) to suppress the NF-κB pathway activation in (non-induced) THP1-Lucia™ NF-κB monocytes in a dose-dependent manner. However, as found in immunofluorescence experiments, ATX-II was unable to trigger the nuclear translocation of NF-κB subunit p65 in THP1-derived macrophages, although incubation was only performed for 1 h. Based on the data obtained, the authors suggested lipid peroxidation as a possible mechanism involved in the NF-κB pathway inhibition.

Taken together, the in vitro studies suggested that *Alternaria* toxins, especially AOH, may cause a reduced immune response in case there is an infection and/or a disturbed balance of the adaptive immune system.

With regard to the available in vivo studies, direct evidence for immunotoxicity of *Alternaria* mycotoxins has not been reported so far. However, there are some animal studies, which have shown anatomical changes in secondary lymphoid organs and indicated immunosuppressive effects.

In the study by Puntscher et al. (2019a), a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins was administered to 14 Sprague Dawley rats. Enlarged Peyer's patches were found in the gastrointestinal tract, which indicated an excessive immune response turning toward a pathologic state. Changes observed in the lymphoid organs were also discovered in three other publications describing 28-day multi-endpoint toxicity assessments for ATX-I, AOH, or AME (Zhu et al. 2022; Miao et al. 2022; Tang et al. 2022). The three toxins induced anatomical changes in the spleen such as white pulp atrophy. However, the most prominent effects on the spleen were observed with AOH, already noticeable at the lowest dose (5.51 µg/kg b.w.). The highest AOH dose (22.05 µg/kg b.w.) had an additional immune effect on leukocytes, namely an increase in lymphocytes and a decrease in neutrophils (Miao et al. 2022).

Kumar et al. 2019 investigated the effects of systemic treatment with ALS (2 and 10 mg/kg b.w.) on acute neuroinflammation after brain injury. ALS decreased the gene expression of pro-inflammatory cytokines and chemokines (TNF-α, IL-6, IL-1β, iNOS, and CCL2) in post-traumatic

brain injury at higher doses. ALS, however, did not affect the pro-resolution immune response (IL-10, IL-4ra). In addition, AOH appeared to exert suppressive effects on the innate immune function of newborn mice as indicated by the reduced expression of CXCL1, IL-1β and IL-8 in the liver. This was apparent in the offspring of pregnant mice injected with 5 mg/kg b.w./day for 4 days and suggested to be mediated by apoptotic processes combined with reactive oxygen species generation (Huang et al. 2021).

Taken together, studies on the immunotoxicity of *Alternaria* toxins have mainly focused on a limited selection of immune cells and mainly explored functional markers such as cytokine production or cellular morphological changes. Of note, an overlay of cytotoxic (see 2.2.) and immunotoxic effects cannot be excluded. Furthermore, existing in vitro data derive from submerged cultures. Data from cultures with closer resemblance to the lung, such as air-liquid interface (ALI) cultures with differentiated cells or 3D tissue inserts, are lacking. Such cultures may be more sensitive and reflect other effects or effects at lower mycotoxin concentrations and will be important to use for a more realistic air exposure scenario.

AOH was found to induce morphological changes, suppress NF-κB signaling, and alter immune cell markers, while AME exhibited suppressive effects on the secretion of immunoregulatory proteins. ALS and ATX-II also displayed immunosuppressive properties, although to varying degrees. Evidence from in vivo studies is limited at present and mainly shows atrophy in lymphoid organs such as the spleen by AOH, AME, and ATX-I as well as indicate immunosuppressive effects by AOH and ALS.

## Toxicokinetics including biotransformation

Information on the toxicokinetic properties of a compound is central for hazard identification and characterization. However, data on *Alternaria* toxins are still scarce even if some more studies have been performed since the EFSA had pointed out the limited and inadequate toxicokinetic data as major uncertainty in their risk evaluation in 2011 (EFSA 2011). Some information on the metabolism of *Alternaria* toxins has been obtained from in vitro studies. They showed that at least AOH, AME, and ALT undergo oxidative phase I metabolism and can subsequently form glucuronides and sulfates. In general, one will expect differences in the bioavailability and biotransformation between different exposure routes (oral, inhalational, dermal). By oral uptake, the first pass effect in the liver will apply, whereas absorption through the respiratory epithelium leads directly to the systemic circulation. Intestinal microorganisms represent an additional pathway for the (de-)toxification of ingested mycotoxins that may contribute to explain the bioavailability
the mycotoxins. Similarly, Kumar et al. (2019) detected a suppression of LPS-induced TNF-α, IL-6, IL-1β, iNOS, CCL-2 mRNA expression (4 h) as well as the release of nitrite and TNF-α (24 h) by increasing concentrations of ALS in BV2 microglia cells and primary rat microglia. However, in contrast to AOH in IL-1β-induced Caco-2 cells (Schmutz et al. 2019), ALS did not alter LPS-induced miR-146 and miR-155 expression in primary microglia (Kumar et al. 2019).

Only one study investigating the immunomodulatory properties of the *Alternaria* mycotoxin ATX-II is currently available. Del Favero and co-workers (2020) demonstrated the ability of subtoxic concentrations of ATX-II (0.1–1 µM, 20 h) to suppress the NF-κB pathway activation in (non-induced) THP1-Lucia™ NF-κB monocytes in a dose-dependent manner. However, as found in immunofluorescence experiments, ATX-II was unable to trigger the nuclear translocation of NF-κB subunit p65 in THP1-derived macrophages, although incubation was only performed for 1 h. Based on the data obtained, the authors suggested lipid peroxidation as a possible mechanism involved in the NF-κB pathway inhibition.

Taken together, the in vitro studies suggested that *Alternaria* toxins, especially AOH, may cause a reduced immune response in case there is an infection and/or a disturbed balance of the adaptive immune system.

With regard to the available in vivo studies, direct evidence for immunotoxicity of *Alternaria* mycotoxins has not been reported so far. However, there are some animal studies, which have shown anatomical changes in secondary lymphoid organs and indicated immunosuppressive effects.

In the study by Puntscher et al. (2019a), a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins was administered to 14 Sprague Dawley rats. Enlarged Peyer's patches were found in the gastrointestinal tract, which indicated an excessive immune response turning toward a pathologic state. Changes observed in the lymphoid organs were also discovered in three other publications describing 28-day multi-endpoint toxicity assessments for ATX-I, AOH, or AME (Zhu et al. 2022; Miao et al. 2022; Tang et al. 2022). The three toxins induced anatomical changes in the spleen such as white pulp atrophy. However, the most prominent effects on the spleen were observed with AOH, already noticeable at the lowest dose (5.51 µg/kg b.w.). The highest AOH dose (22.05 µg/kg b.w.) had an additional immune effect on leukocytes, namely an increase in lymphocytes and a decrease in neutrophils (Miao et al. 2022).

Kumar et al. 2019 investigated the effects of systemic treatment with ALS (2 and 10 mg/kg b.w.) on acute neuroinflammation after brain injury. ALS decreased the gene expression of pro-inflammatory cytokines and chemokines (TNF-α, IL-6, IL-1β, iNOS, and CCL2) in post-traumatic

brain injury at higher doses. ALS, however, did not affect the pro-resolution immune response (IL-10, IL-4ra). In addition, AOH appeared to exert suppressive effects on the innate immune function of newborn mice as indicated by the reduced expression of CXCL1, IL-1β and IL-8 in the liver. This was apparent in the offspring of pregnant mice injected with 5 mg/kg b.w./day for 4 days and suggested to be mediated by apoptotic processes combined with reactive oxygen species generation (Huang et al. 2021).

Taken together, studies on the immunotoxicity of *Alternaria* toxins have mainly focused on a limited selection of immune cells and mainly explored functional markers such as cytokine production or cellular morphological changes. Of note, an overlay of cytotoxic (see 2.2.) and immunotoxic effects cannot be excluded. Furthermore, existing in vitro data derive from submerged cultures. Data from cultures with closer resemblance to the lung, such as air-liquid interface (ALI) cultures with differentiated cells or 3D tissue inserts, are lacking. Such cultures may be more sensitive and reflect other effects or effects at lower mycotoxin concentrations and will be important to use for a more realistic air exposure scenario.

AOH was found to induce morphological changes, suppress NF-κB signaling, and alter immune cell markers, while AME exhibited suppressive effects on the secretion of immunoregulatory proteins. ALS and ATX-II also displayed immunosuppressive properties, although to varying degrees. Evidence from in vivo studies is limited at present and mainly shows atrophy in lymphoid organs such as the spleen by AOH, AME, and ATX-I as well as indicate immunosuppressive effects by AOH and ALS.

## Toxicokinetics including biotransformation

Information on the toxicokinetic properties of a compound is central for hazard identification and characterization. However, data on *Alternaria* toxins are still scarce even if some more studies have been performed since the EFSA had pointed out the limited and inadequate toxicokinetic data as major uncertainty in their risk evaluation in 2011 (EFSA 2011). Some information on the metabolism of *Alternaria* toxins has been obtained from in vitro studies. They showed that at least AOH, AME, and ALT undergo oxidative phase I metabolism and can subsequently form glucuronides and sulfates. In general, one will expect differences in the bioavailability and biotransformation between different exposure routes (oral, inhalational, dermal). By oral uptake, the first pass effect in the liver will apply, whereas absorption through the respiratory epithelium leads directly to the systemic circulation. Intestinal microorganisms represent an additional pathway for the (de-)toxification of ingested mycotoxins that may contribute to explain the bioavailability

<figure><img src="image_15.png" /></figure>
difference between the exposure routes, although the lung microbiome has a likely influence on the absorption efficiency of inhaled toxins. However, important information on absorption, distribution, metabolism, and excretion (ADME) is still lacking.

## Alternariol (AOH)

**Absorption** When male NMRI mice were exposed orally with a single dose of 200 or 1000 mg/kg b.w. ¹⁴C-AOH, the oral bioavailability (with hepatic first pass effect) was below 10%, while 90% of the total radioactivity were found in the feces (Schuchardt et al. 2014). The blood levels did not exceed 0.06% of the total dose within 24 h after toxin administration. In a second study, male and female NMRI mice received non-radioactive AOH at 200 mg/kg b.w. and blood samples were taken after 0.5, 1, 2, 3, 4, 5, 6, 24, and 48 h (Schuchardt et al. 2014). Sex differences between females and males in the kinetic profile were observed with, respectively, 0.5 h and 2 h for $T_{max}$, 90.2 and 66.2 ng/mL for $C_{max}$, and 158.6 ng × h/mL and 350 ng × h/mL for the area under the concentration–time curve (AUC). After oral administration of a complex *Alternaria* culture extract (50 mg/kg b.w.) containing eleven known toxins (including AOH at 39 µg/kg b.w.) to male Sprague Dawley rats, AOH was not found in plasma at 3 and 24 h after gavage (Puntscher et al. 2019b). However, low AOH levels were detected in urine at 3 h (10 ± 13.4 ng/mL; 0.1% of dose) and at 24 h (35.7 ± 15.0 ng/mL AOH; 2.8% of dose). The AOH-3-*O*-sulfate (AOH-3-*O*-S) was also detected at 24 h in urine (13.4 ± 7.8 ng/mL; 0.78% of AOH dose), while the hydroxylated metabolite 4-OH-AOH was not detected. In feces, 0.3% of the AOH dose was found unchanged at 3 h, and 89% at 24 h. In addition, 4-OH-AOH corresponding to 1% of the AOH dose was found. Taken together, these data suggested a low bioavailability of AOH after oral administration.

In in vitro experiments performed on Caco-2 monolayers, around 25% of AOH was absorbed reaching the basolateral compartment 3 h after apical exposure, either as the parent compound or the conjugated metabolites AOH-3-*O*-glucuronide, AOH-9-*O*-glucuronide, and AOH-3-*O*-sulfate (Burkhardt et al. 2009; Nübler et al. 2023). The apparent permeability coefficient ($P_{app}$) was determined as 8.1 ± 2.6 10⁻⁶ cm/s for unconjugated AOH and as 34.9 ± 5.6 10⁻⁶ cm/s for total AOH after 1-h incubation with 20 µM AOH. The rate of AOH transfer in the Caco-2 system can, however, be affected during exposure to chemical mixtures. The presence of urolithin C, a structurally related gut ellagitannin-derived metabolite, in the apical compartment reduced AOH transport and conjugation to glucuronides and sulfates considerably (Crudo et al. 2021b).

**Distribution** The tissue distribution of radiolabeled AOH was measured after a single oral application in NRMI mice

(Schuchardt et al. 2014). The highest radioactivity levels were detected in the gastrointestinal tract after 24 h. However, the total radioactivity in all organs and tissues including the gut was less than 1% of the dose after 24 h, and this fraction decreased further to below 0.01% after 7 days (Schuchardt et al. 2014). AOH binds with higher affinity to rat serum albumin (RSA) than to human, bovine and porcine serum albumins, and the stability of the AOH-RSA complex is eight-fold higher than that of the other species (Fliszár-Nyúl et al. 2019).

**Metabolism** AOH has been found to undergo both oxidoreductive phase I metabolism and conjugative phase II metabolism, in in vivo and in vitro studies (Dall'Asta et al. 2014). At 2 h after oral administration of 2000 mg/kg b.w. unlabeled AOH to NMRI mice, the three hydroxylated metabolites 2-OH-AOH, 4-OH-AOH and 10-OH-AOH were detected in the blood. These metabolites as well as 8-OH-AOH were also detected in the urine during the 72 h-collocation period (Schuchardt et al. 2014). In Sprague Dawley rats receiving an *Alternaria* culture extract (50 mg/kg b.w.) containing 35 µg/kg b.w. AOH, the phase II metabolite AOH-3-*O*-S was found in plasma after 3 h and in urine after 3 and 24 h (Puntscher et al. 2019b). Moreover, 4-OH-AOH was detected in feces after 24 h, accounting for 1% of the AOH intake. Glucuronides were not investigated in this study.

The in vitro metabolism of AOH has been studied using different liver fractions of several species. After incubation of 50 µM AOH for 40 min with microsomes from rat, human and pig, 2-OH-AOH, 4-OH-AOH, 8-OH-AOH and 10-OH-AOH, as well as methyl-OH-AOH were identified (Pfeiffer et al. 2009a). Monohydroxylation in any of the four possible positions in the AOH molecule leads to a metabolite with a catechol-like (1, 2-dihydroxybenzene) structure, possibly resulting in a toxication as compared to the parent compound. Catechols are suspected to form reactive intermediates such as quinones and semiquinones, resulting in DNA adducts and the production of reactive oxygen species (EFSA 2011). The formation rates of the hydroxylated metabolites depend on the species with 228, 491, and 128 pmol/min/nmol cytochrome P450 (CYP) enzymes in rat, human and pig microsomes, respectively (Pfeiffer et al. 2008). Moreover, the individual metabolites were produced with different efficiencies. In human microsomes, 2-OH-AOH was the major metabolite formed (75%), but it was less important in pig (45%) and rat (20%) microsomes. Whereas 10-OH-AOH was predominant in rat microsomes (relative amount of 70%), it occurred only in trace amounts in both human and pig liver microsomes. In contrast, 4-OH-AOH was the major oxidative metabolite in pig microsomes (45%) but was less relevant in rat (7%) and human (19%) microsomes. Finally, 8-OH-AOH was only formed in small amounts (< 10%) in all three species, and methyl-OH-AOH was even less present. When AOH
difference between the exposure routes, although the lung microbiome has a likely influence on the absorption efficiency of inhaled toxins. However, important information on absorption, distribution, metabolism, and excretion (ADME) is still lacking.

## Alternariol (AOH)

**Absorption** When male NMRI mice were exposed orally with a single dose of 200 or 1000 mg/kg b.w. ¹⁴C-AOH, the oral bioavailability (with hepatic first pass effect) was below 10%, while 90% of the total radioactivity were found in the feces (Schuchardt et al. 2014). The blood levels did not exceed 0.06% of the total dose within 24 h after toxin administration. In a second study, male and female NMRI mice received non-radioactive AOH at 200 mg/kg b.w. and blood samples were taken after 0.5, 1, 2, 3, 4, 5, 6, 24, and 48 h (Schuchardt et al. 2014). Sex differences between females and males in the kinetic profile were observed with, respectively, 0.5 h and 2 h for $T_{max}$, 90.2 and 66.2 ng/mL for $C_{max}$, and 158.6 ng × h/mL and 350 ng × h/mL for the area under the concentration–time curve (AUC). After oral administration of a complex *Alternaria* culture extract (50 mg/kg b.w.) containing eleven known toxins (including AOH at 39 µg/kg b.w.) to male Sprague Dawley rats, AOH was not found in plasma at 3 and 24 h after gavage (Puntscher et al. 2019b). However, low AOH levels were detected in urine at 3 h (10 ± 13.4 ng/mL; 0.1% of dose) and at 24 h (35.7 ± 15.0 ng/mL AOH; 2.8% of dose). The AOH-3-*O*-sulfate (AOH-3-*O*-S) was also detected at 24 h in urine (13.4 ± 7.8 ng/mL; 0.78% of AOH dose), while the hydroxylated metabolite 4-OH-AOH was not detected. In feces, 0.3% of the AOH dose was found unchanged at 3 h, and 89% at 24 h. In addition, 4-OH-AOH corresponding to 1% of the AOH dose was found. Taken together, these data suggested a low bioavailability of AOH after oral administration.

In in vitro experiments performed on Caco-2 monolayers, around 25% of AOH was absorbed reaching the basolateral compartment 3 h after apical exposure, either as the parent compound or the conjugated metabolites AOH-3-*O*-glucuronide, AOH-9-*O*-glucuronide, and AOH-3-*O*-sulfate (Burkhardt et al. 2009; Nübler et al. 2023). The apparent permeability coefficient ($P_{app}$) was determined as 8.1 ± 2.6 10⁻⁶ cm/s for unconjugated AOH and as 34.9 ± 5.6 10⁻⁶ cm/s for total AOH after 1-h incubation with 20 µM AOH. The rate of AOH transfer in the Caco-2 system can, however, be affected during exposure to chemical mixtures. The presence of urolithin C, a structurally related gut ellagitannin-derived metabolite, in the apical compartment reduced AOH transport and conjugation to glucuronides and sulfates considerably (Crudo et al. 2021b).

**Distribution** The tissue distribution of radiolabeled AOH was measured after a single oral application in NRMI mice

(Schuchardt et al. 2014). The highest radioactivity levels were detected in the gastrointestinal tract after 24 h. However, the total radioactivity in all organs and tissues including the gut was less than 1% of the dose after 24 h, and this fraction decreased further to below 0.01% after 7 days (Schuchardt et al. 2014). AOH binds with higher affinity to rat serum albumin (RSA) than to human, bovine and porcine serum albumins, and the stability of the AOH-RSA complex is eight-fold higher than that of the other species (Fliszár-Nyúl et al. 2019).

**Metabolism** AOH has been found to undergo both oxidoreductive phase I metabolism and conjugative phase II metabolism, in in vivo and in vitro studies (Dall'Asta et al. 2014). At 2 h after oral administration of 2000 mg/kg b.w. unlabeled AOH to NMRI mice, the three hydroxylated metabolites 2-OH-AOH, 4-OH-AOH and 10-OH-AOH were detected in the blood. These metabolites as well as 8-OH-AOH were also detected in the urine during the 72 h-collocation period (Schuchardt et al. 2014). In Sprague Dawley rats receiving an *Alternaria* culture extract (50 mg/kg b.w.) containing 35 µg/kg b.w. AOH, the phase II metabolite AOH-3-*O*-S was found in plasma after 3 h and in urine after 3 and 24 h (Puntscher et al. 2019b). Moreover, 4-OH-AOH was detected in feces after 24 h, accounting for 1% of the AOH intake. Glucuronides were not investigated in this study.

The in vitro metabolism of AOH has been studied using different liver fractions of several species. After incubation of 50 µM AOH for 40 min with microsomes from rat, human and pig, 2-OH-AOH, 4-OH-AOH, 8-OH-AOH and 10-OH-AOH, as well as methyl-OH-AOH were identified (Pfeiffer et al. 2009a). Monohydroxylation in any of the four possible positions in the AOH molecule leads to a metabolite with a catechol-like (1, 2-dihydroxybenzene) structure, possibly resulting in a toxication as compared to the parent compound. Catechols are suspected to form reactive intermediates such as quinones and semiquinones, resulting in DNA adducts and the production of reactive oxygen species (EFSA 2011). The formation rates of the hydroxylated metabolites depend on the species with 228, 491, and 128 pmol/min/nmol cytochrome P450 (CYP) enzymes in rat, human and pig microsomes, respectively (Pfeiffer et al. 2008). Moreover, the individual metabolites were produced with different efficiencies. In human microsomes, 2-OH-AOH was the major metabolite formed (75%), but it was less important in pig (45%) and rat (20%) microsomes. Whereas 10-OH-AOH was predominant in rat microsomes (relative amount of 70%), it occurred only in trace amounts in both human and pig liver microsomes. In contrast, 4-OH-AOH was the major oxidative metabolite in pig microsomes (45%) but was less relevant in rat (7%) and human (19%) microsomes. Finally, 8-OH-AOH was only formed in small amounts (< 10%) in all three species, and methyl-OH-AOH was even less present. When AOH
metabolism experiments were carried out with specific human recombinant CYP isoforms, it could be shown that CYP1A1 had the greatest activity, followed by CYP1A2, CYP2C19 and CYP3A4 (Pfeiffer et al. 2008). From this profile, it was concluded that significant extrahepatic hydroxylation, e.g., in the lungs and esophagus, could be expected.

The incubation of AOH with hepatic and intestinal microsomes from rats, pigs, and humans in the presence of uridine diphosphate glucuronic acid (UDPGA) resulted in the production of two glucuronide conjugates, tentatively identified as AOH-3-*O*-GlcA and AOH-9-*O*-GlcA (Pfeiffer et al. 2009a). The formation rates differed among species, sexes, and tissues. While AOH-3-*O*-GlcA was the prevalent glucuronide in liver microsomes of female Sprague Dawley rats and sows, and in intestinal microsomes of sows and men, AOH-9-*O*-GlcA predominated in the liver and intestinal microsomes of male Sprague Dawley rats. In liver microsomes of male Wistar rats and men, both glucuronides occurred with about 50%. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) showed that AOH was a substrate for UGT1A9, 1A1, 1A8, 1A10, 1A7, 2B15, 2B7, 1A3, and 1A6 in decreasing order, but not for UGT1A4 (Pfeiffer et al. 2009a). Metabolism studies of 50 µM AOH in precision-cut rat liver slices for 24 h gave rise to the four OH-metabolites of AOH with 2-OH-AOH as the preferred isomer, in contrast to the result obtained in the rat liver microsome assay (Burkhardt et al. 2011). Further incubation of the hydroxylated metabolites in rat liver cytosol fortified with S-adenosyl-L-methionine (SAM) produced *O*-methylated metabolites, possibly formed by catalysis of catechol-O-methyltransferase. From hydrolysis experiments, it was concluded that the majority of the phase I metabolites were subsequently conjugated with glucuronate or sulfate (Burkhardt et al. 2011). In a preliminary study with bile duct-cannulated male Sprague Dawley rats dosed with about 6.5 mg/kg b.w. AOH by gavage, the presence of the four OH-AOH metabolites and several *O*-methyl ethers could be demonstrated (Burkhardt et al. 2011). Moreover, glucuronides and sulfates were indirectly determined by hydrolysis. However, the formation of the phase II metabolites AOH-3-*O*-GlcA, AOH-9-*O*-GlcA, and AOH-3-*O*-S was measurable, when AOH was incubated with differentiated Caco-2 cells. Furthermore, AOH-3-*O*-S, AOH-7-*O*-S, AOH-9-*O*-S were detected when AOH was incubated with rat liver cytosol in the presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Burkhardt et al. 2009; Lemke et al. 2016).

*Excretion* After oral administration, absorbed AOH was eliminated in the urine. Whereas metabolites were detected in urine and bile, unabsorbed AOH was mostly excreted unchanged in the feces (> 89%) (Puntscher et al. 2019a). Radiolabeled AOH was recovered with 84.5% of the dose

in feces, 9.3% in urine and 0.05% in exhaled air after application of 200 mg/kg b.w. to NMRI mice (Schuchardt et al. 2014).

Since a study with intravenous AOH administration has not been performed, clearance (CL) and half-life ($t_{1/2}$) could only be estimated from data after oral treatment. In NMRI mice dosed p.o. with unlabeled 200 mg/kg b.w., AOH blood levels were measured between 0.5 and 48 h (Schuchardt et al. 2014). The elimination $t_{1/2}$ was determined as 1.1 h in male and 9.2 h in female mice, and the respective mean residence times (MRT) as 2.3 h and 12.5 h. Using the given time and concentration data, the CL/F values were estimated as 1.07 mg/h/(µg/L) in males and 0.35 mg/h/(µg/L) in females. After consideration of the mouse bodyweights (mean values about 40 g in males and 35 g in females) and consideration of an oral bioavailability *F* < 10%, we predicted the CL after intravenous application as about 2.7 mL/(h*kg) for male and 1.0 mL/(h*kg) for female NMRI mice.

## Alternariol monomethyl ether (AME)

*Absorption* Oral administration of an average of 61.6 mg/kg b.w. radiolabeled AME (dissolved in olive oil) in male Sprague Dawley rats resulted in the absorption of less than 10% of the dose (Pollock et al. 1982). The majority of the radioactivity was excreted in feces, apparently mostly as unchanged AME. The authors described the low solubility of AME as an uncertainty and recommended exposure studies with AME included into the diet. When Sprague Dawley rats received an *Alternaria* culture extract (50 mg/kg b.w. p.o.) containing eleven *Alternaria* toxins (AME at 32.3 µg/kg b.w.), 2.6% of the dose were recovered as AME and 0.6% as AME-3-*O*-sulfate in urine after 24 h (Puntscher et al. 2019a). AME was not detected in plasma after 3 and 24 h. whereas a small amount of AME-3-*O*-sulfate was detected at 24 h (0.06%). Taken together, these data suggested a low bioavailability of AME after oral administration. In in vitro experiments using the Caco-2 monolayer, AME was unable to permeate across the monolayer after 6 h of exposure at 20 µM, and only the conjugates AME-3-*O*-GlcA, AME-7-*O*-GlcA, and AME-3-*O*-sulfate were detected in the basolateral compartment (Burkhardt et al. 2009). A $P_{app}$ value at 1 h for 20 µM AME was calculated as 10.3±4.9 10⁻⁶ cm/s for the sum of conjugated AME (Burkhardt et al. 2009).

*Distribution* After oral administration of radiolabeled AME in Sprague Dawley rats, the total radioactivity was measured after 1 and 3 days in a broad panel of tissues including blood, liver, kidney, lung, heart, spleen, pancreas, thymus, adrenals, brain, testes, prostate, stomach, stomach contents, cecum, cecal contents, abdominal fat, and subcutaneous fat (Pollock et al. 1982). After one day, the by far highest amounts were detected in stomach, caecum and caecum contents, whereas smaller amounts were found in fat, liver and lung. On day 3, absorbed radioactivity was only
metabolism experiments were carried out with specific human recombinant CYP isoforms, it could be shown that CYP1A1 had the greatest activity, followed by CYP1A2, CYP2C19 and CYP3A4 (Pfeiffer et al. 2008). From this profile, it was concluded that significant extrahepatic hydroxylation, e.g., in the lungs and esophagus, could be expected.

The incubation of AOH with hepatic and intestinal microsomes from rats, pigs, and humans in the presence of uridine diphosphate glucuronic acid (UDPGA) resulted in the production of two glucuronide conjugates, tentatively identified as AOH-3-*O*-GlcA and AOH-9-*O*-GlcA (Pfeiffer et al. 2009a). The formation rates differed among species, sexes, and tissues. While AOH-3-*O*-GlcA was the prevalent glucuronide in liver microsomes of female Sprague Dawley rats and sows, and in intestinal microsomes of sows and men, AOH-9-*O*-GlcA predominated in the liver and intestinal microsomes of male Sprague Dawley rats. In liver microsomes of male Wistar rats and men, both glucuronides occurred with about 50%. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) showed that AOH was a substrate for UGT1A9, 1A1, 1A8, 1A10, 1A7, 2B15, 2B7, 1A3, and 1A6 in decreasing order, but not for UGT1A4 (Pfeiffer et al. 2009a). Metabolism studies of 50 µM AOH in precision-cut rat liver slices for 24 h gave rise to the four OH-metabolites of AOH with 2-OH-AOH as the preferred isomer, in contrast to the result obtained in the rat liver microsome assay (Burkhardt et al. 2011). Further incubation of the hydroxylated metabolites in rat liver cytosol fortified with S-adenosyl-L-methionine (SAM) produced *O*-methylated metabolites, possibly formed by catalysis of catechol-O-methyltransferase. From hydrolysis experiments, it was concluded that the majority of the phase I metabolites were subsequently conjugated with glucuronate or sulfate (Burkhardt et al. 2011). In a preliminary study with bile duct-cannulated male Sprague Dawley rats dosed with about 6.5 mg/kg b.w. AOH by gavage, the presence of the four OH-AOH metabolites and several *O*-methyl ethers could be demonstrated (Burkhardt et al. 2011). Moreover, glucuronides and sulfates were indirectly determined by hydrolysis. However, the formation of the phase II metabolites AOH-3-*O*-GlcA, AOH-9-*O*-GlcA, and AOH-3-*O*-S was measurable, when AOH was incubated with differentiated Caco-2 cells. Furthermore, AOH-3-*O*-S, AOH-7-*O*-S, AOH-9-*O*-S were detected when AOH was incubated with rat liver cytosol in the presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Burkhardt et al. 2009; Lemke et al. 2016).

*Excretion* After oral administration, absorbed AOH was eliminated in the urine. Whereas metabolites were detected in urine and bile, unabsorbed AOH was mostly excreted unchanged in the feces (> 89%) (Puntscher et al. 2019a). Radiolabeled AOH was recovered with 84.5% of the dose

in feces, 9.3% in urine and 0.05% in exhaled air after application of 200 mg/kg b.w. to NMRI mice (Schuchardt et al. 2014).

Since a study with intravenous AOH administration has not been performed, clearance (CL) and half-life ($t_{1/2}$) could only be estimated from data after oral treatment. In NMRI mice dosed p.o. with unlabeled 200 mg/kg b.w., AOH blood levels were measured between 0.5 and 48 h (Schuchardt et al. 2014). The elimination $t_{1/2}$ was determined as 1.1 h in male and 9.2 h in female mice, and the respective mean residence times (MRT) as 2.3 h and 12.5 h. Using the given time and concentration data, the CL/F values were estimated as 1.07 mg/h/(µg/L) in males and 0.35 mg/h/(µg/L) in females. After consideration of the mouse bodyweights (mean values about 40 g in males and 35 g in females) and consideration of an oral bioavailability *F* < 10%, we predicted the CL after intravenous application as about 2.7 mL/(h*kg) for male and 1.0 mL/(h*kg) for female NMRI mice.

## Alternariol monomethyl ether (AME)

*Absorption* Oral administration of an average of 61.6 mg/kg b.w. radiolabeled AME (dissolved in olive oil) in male Sprague Dawley rats resulted in the absorption of less than 10% of the dose (Pollock et al. 1982). The majority of the radioactivity was excreted in feces, apparently mostly as unchanged AME. The authors described the low solubility of AME as an uncertainty and recommended exposure studies with AME included into the diet. When Sprague Dawley rats received an *Alternaria* culture extract (50 mg/kg b.w. p.o.) containing eleven *Alternaria* toxins (AME at 32.3 µg/kg b.w.), 2.6% of the dose were recovered as AME and 0.6% as AME-3-*O*-sulfate in urine after 24 h (Puntscher et al. 2019a). AME was not detected in plasma after 3 and 24 h. whereas a small amount of AME-3-*O*-sulfate was detected at 24 h (0.06%). Taken together, these data suggested a low bioavailability of AME after oral administration. In in vitro experiments using the Caco-2 monolayer, AME was unable to permeate across the monolayer after 6 h of exposure at 20 µM, and only the conjugates AME-3-*O*-GlcA, AME-7-*O*-GlcA, and AME-3-*O*-sulfate were detected in the basolateral compartment (Burkhardt et al. 2009). A $P_{app}$ value at 1 h for 20 µM AME was calculated as 10.3±4.9 10⁻⁶ cm/s for the sum of conjugated AME (Burkhardt et al. 2009).

*Distribution* After oral administration of radiolabeled AME in Sprague Dawley rats, the total radioactivity was measured after 1 and 3 days in a broad panel of tissues including blood, liver, kidney, lung, heart, spleen, pancreas, thymus, adrenals, brain, testes, prostate, stomach, stomach contents, cecum, cecal contents, abdominal fat, and subcutaneous fat (Pollock et al. 1982). After one day, the by far highest amounts were detected in stomach, caecum and caecum contents, whereas smaller amounts were found in fat, liver and lung. On day 3, absorbed radioactivity was only
measured in the fat, in line with the low absorption rate and
high lipophilicity of the toxin.

*Metabolism* The metabolite profiles observed in the same rat study showed that absorbed AME was extensively metabolized, giving rise to more hydrophilic molecules (Pollock et al. 1982). Moreover, *O*-demethylation of AME to AOH was suggested. After oral administration of the culture extract containing AME among other *Alternaria* toxins to rats, modified forms of AME were discovered in feces, urine and plasma. After 3 h, about 0.06% of the dose was present as AME-3-*O*-sulfate in plasma, and 0.01% in urine, which increased to 0.63% after 24 h (Puntscher et al. 2019b). The parent compound AME was not detected in plasma, but 0.1% of the dose was found in urine at 3 h and 2.6% at 24 h. In feces, the majority of the administered AME was recovered unchanged after 24 h, while traces of 4-OH-AME were detected at levels below the LOD and 2% of the dose as AME-3-*O*-S.

The oxidative metabolism of AME was investigated
in vitro in rat liver S9 fraction with NADPH regenerat-
ing system, showing extensive phase I metabolism includ-
ing *O*-demethylation to AOH (Pollock et al. 1982). Using
hepatic microsomes from rats, humans and pigs, the incu-
bation of 50 µM AME for 40 min resulted in the formation
of six hydroxylated metabolites, 2-OH-AME, 4-OH-AME,
8-OH-AME, 10-OH-AME, dihydroxy-AME and methyl-
hydroxy-AME, as well as demethylation to AOH (Pfeiffer
et al. 2007). The authors found species differences in the
oxidative metabolism capacity of AME, with the total for-
mation ratio of hydroxylated metabolites varying from 295
in rats to 559 and 679 pmol/min/nmol CYP P450 in pigs
and humans, respectively. Moreover, 8-OH-AME was the
major hydroxylation product in rat (50%) and pig micro-
somes (40%), whereas 2-OH-AME was the major oxidative
metabolite (40%) in humans. The relative amount of AOH
formed was about 20% in all three species. In a study inves-
tigating the specific activities of human recombinant CYP
P450 for AME hydroxylation, CYP1A1 showed the highest
transformation capacity, followed by CYP1A2, 2C19, and
3A4 (Pfeiffer et al. 2008). Hydroxylation at C-2 and C-4 was
preferred over hydroxylation at C-8 for most CYPs. CYP3A4
was the only isoform mainly generating 8-OH-AME.

In glucuronidation assays performed in hepatic and intes-
tinal microsomes of rats, pigs and humans, the major con-
jugation product formed was AME-3-*O*-GlcA, and AME-7-
*O*-GlcA was formed to a lesser extent (Pfeiffer et al. 2009a).
From the UGTs investigated, UGT1A1, 1A3, 1A7, 1A8,
1A9, and 1A10 produced both glucuronides, while UGT2B7
and UGT2B15 only formed AME-3-*O*-GlcA. UGT1A4 and
1A6 were not able to conjugate AME (Pfeiffer et al. 2009a).

AME sulfation was studied in rat liver cytosol with the
addition of PAPS, leading to the formation of AME-3-*O*-S
and AME-7-*O*-S (Burkhardt et al. 2009). In the same study,

AME conjugation was examined in Caco-2 cells, and AME-3-*O*-GlcA, AME-7-*O*-GlcA, and AME-3-*O*-S were identified after incubation for 2 h, with AME-3-*O*-GlcA as the by far most predominant metabolite. In precision-cut rat liver slices incubated for 24 h with AME (50–200 µM), the four monohydroxylated metabolites 2-OH-AME, 4-OH-AME, 8-OH-AME, and 10-OH-AME were produced (Burkhardt et al. 2011), thus confirming the results from the in vivo experiments. Moreover, methylation products of several metabolites were detectable. Hydroxylation at C-8 was the preferred reaction. Most of the phase I metabolites were further conjugated with glucuronic acid or sulfate.

*Excretion* Due to the poor absorption in the gastrointestinal tract, AME is mainly excreted unchanged in feces. In Sprague Dawley rats exposed orally to radiolabeled AME, 85% of the total dose was recovered in feces, mostly excreted on the first day (Pollock et al. 1982). Urine accounted for about 7%, while almost 2% were expired as CO2 from the lungs. After oral administration of the mixed *Alternaria* toxin extract to rats, absorbed AME was eliminated after 24 h mainly as the parent compound in the urine (> 2.8), and unabsorbed AME was excreted in the feces (> 89%) (Puntscher et al. 2019a). When the feces of 12-month-old Nigerian infants were analyzed for mycotoxins, almost all samples contained AME, indicating chronic low-level exposure (Krausová et al. 2022).

Altenuene (ALT)

To our knowledge, data on the absorption or distribution
of ALT are not available, while data on metabolism are
described below. Data on ADME parameters of the struc-
tural analog isoALT are not available.

*Metabolism* ALT underwent oxidative metabolism, with ca. 25% and 8% metabolic conversion in Dawley rat and human liver microsomes, respectively, when 1 mg microsomal protein was incubated with 50 µM ALT at 37 °C for 40 min (Pfeiffer et al. 2009b). The pattern of ALT oxidative metabolism indicated that 8-OH-ALT was the major metabolite (more than 70%) formed in different species, followed by 10-OH-ALT and 4-OH-ALT with different ratios (Pfeiffer et al. 2009b).

In precision-cut rat liver slices incubated for 24 h at 37 °C with 200 µM ALT, ca. 40% ALT remained unchanged, 50% was conjugated as glucuronide and 10% was oxidized (Pfeiffer et al. 2009b). Moreover, this study showed that the major oxidative metabolite, 8-OH-ALT, was mainly glucuronidated (75%), whereas 4-OH and 10-OH-ALT were minor metabolites (Pfeiffer et al. 2009b). ALT was preferentially hydroxylated by CYP2C19, followed by 2C9 and 2D6, and the main metabolite was the 8-OH-ALT (Pfeiffer et al. 2008).

Oral administration to rats of a complex *Alternaria* cul-
ture extract (50 mg/kg b.w.) containing 11 known toxins
measured in the fat, in line with the low absorption rate and
high lipophilicity of the toxin.

*Metabolism* The metabolite profiles observed in the same rat study showed that absorbed AME was extensively metabolized, giving rise to more hydrophilic molecules (Pollock et al. 1982). Moreover, *O*-demethylation of AME to AOH was suggested. After oral administration of the culture extract containing AME among other *Alternaria* toxins to rats, modified forms of AME were discovered in feces, urine and plasma. After 3 h, about 0.06% of the dose was present as AME-3-*O*-sulfate in plasma, and 0.01% in urine, which increased to 0.63% after 24 h (Puntscher et al. 2019b). The parent compound AME was not detected in plasma, but 0.1% of the dose was found in urine at 3 h and 2.6% at 24 h. In feces, the majority of the administered AME was recovered unchanged after 24 h, while traces of 4-OH-AME were detected at levels below the LOD and 2% of the dose as AME-3-*O*-S.

The oxidative metabolism of AME was investigated
in vitro in rat liver S9 fraction with NADPH regenerat-
ing system, showing extensive phase I metabolism includ-
ing *O*-demethylation to AOH (Pollock et al. 1982). Using
hepatic microsomes from rats, humans and pigs, the incu-
bation of 50 µM AME for 40 min resulted in the formation
of six hydroxylated metabolites, 2-OH-AME, 4-OH-AME,
8-OH-AME, 10-OH-AME, dihydroxy-AME and methyl-
hydroxy-AME, as well as demethylation to AOH (Pfeiffer
et al. 2007). The authors found species differences in the
oxidative metabolism capacity of AME, with the total for-
mation ratio of hydroxylated metabolites varying from 295
in rats to 559 and 679 pmol/min/nmol CYP P450 in pigs
and humans, respectively. Moreover, 8-OH-AME was the
major hydroxylation product in rat (50%) and pig micro-
somes (40%), whereas 2-OH-AME was the major oxidative
metabolite (40%) in humans. The relative amount of AOH
formed was about 20% in all three species. In a study inves-
tigating the specific activities of human recombinant CYP
P450 for AME hydroxylation, CYP1A1 showed the highest
transformation capacity, followed by CYP1A2, 2C19, and
3A4 (Pfeiffer et al. 2008). Hydroxylation at C-2 and C-4 was
preferred over hydroxylation at C-8 for most CYPs. CYP3A4
was the only isoform mainly generating 8-OH-AME.

In glucuronidation assays performed in hepatic and intes-
tinal microsomes of rats, pigs and humans, the major con-
jugation product formed was AME-3-*O*-GlcA, and AME-7-
*O*-GlcA was formed to a lesser extent (Pfeiffer et al. 2009a).
From the UGTs investigated, UGT1A1, 1A3, 1A7, 1A8,
1A9, and 1A10 produced both glucuronides, while UGT2B7
and UGT2B15 only formed AME-3-*O*-GlcA. UGT1A4 and
1A6 were not able to conjugate AME (Pfeiffer et al. 2009a).

AME sulfation was studied in rat liver cytosol with the
addition of PAPS, leading to the formation of AME-3-*O*-S
and AME-7-*O*-S (Burkhardt et al. 2009). In the same study,

AME conjugation was examined in Caco-2 cells, and AME-3-*O*-GlcA, AME-7-*O*-GlcA, and AME-3-*O*-S were identified after incubation for 2 h, with AME-3-*O*-GlcA as the by far most predominant metabolite. In precision-cut rat liver slices incubated for 24 h with AME (50–200 µM), the four monohydroxylated metabolites 2-OH-AME, 4-OH-AME, 8-OH-AME, and 10-OH-AME were produced (Burkhardt et al. 2011), thus confirming the results from the in vivo experiments. Moreover, methylation products of several metabolites were detectable. Hydroxylation at C-8 was the preferred reaction. Most of the phase I metabolites were further conjugated with glucuronic acid or sulfate.

*Excretion* Due to the poor absorption in the gastrointestinal tract, AME is mainly excreted unchanged in feces. In Sprague Dawley rats exposed orally to radiolabeled AME, 85% of the total dose was recovered in feces, mostly excreted on the first day (Pollock et al. 1982). Urine accounted for about 7%, while almost 2% were expired as CO2 from the lungs. After oral administration of the mixed *Alternaria* toxin extract to rats, absorbed AME was eliminated after 24 h mainly as the parent compound in the urine (> 2.8), and unabsorbed AME was excreted in the feces (> 89%) (Puntscher et al. 2019a). When the feces of 12-month-old Nigerian infants were analyzed for mycotoxins, almost all samples contained AME, indicating chronic low-level exposure (Krausová et al. 2022).

Altenuene (ALT)

To our knowledge, data on the absorption or distribution
of ALT are not available, while data on metabolism are
described below. Data on ADME parameters of the struc-
tural analog isoALT are not available.

*Metabolism* ALT underwent oxidative metabolism, with ca. 25% and 8% metabolic conversion in Dawley rat and human liver microsomes, respectively, when 1 mg microsomal protein was incubated with 50 µM ALT at 37 °C for 40 min (Pfeiffer et al. 2009b). The pattern of ALT oxidative metabolism indicated that 8-OH-ALT was the major metabolite (more than 70%) formed in different species, followed by 10-OH-ALT and 4-OH-ALT with different ratios (Pfeiffer et al. 2009b).

In precision-cut rat liver slices incubated for 24 h at 37 °C with 200 µM ALT, ca. 40% ALT remained unchanged, 50% was conjugated as glucuronide and 10% was oxidized (Pfeiffer et al. 2009b). Moreover, this study showed that the major oxidative metabolite, 8-OH-ALT, was mainly glucuronidated (75%), whereas 4-OH and 10-OH-ALT were minor metabolites (Pfeiffer et al. 2009b). ALT was preferentially hydroxylated by CYP2C19, followed by 2C9 and 2D6, and the main metabolite was the 8-OH-ALT (Pfeiffer et al. 2008).

Oral administration to rats of a complex *Alternaria* cul-
ture extract (50 mg/kg b.w.) containing 11 known toxins
(ALT at 39.2 µg/kg b.w.) showed that ALT was not detected in plasma and urine at 3 and 24 h (Puntscher et al. 2019b), and that only a small amount of ALT was detected in the feces, 7% of the dose at 24 h, indicating a huge pre-systemic or systemic metabolism of ALT in mice. These results are in accordance with two screening studies in the Beijing general population, which showed that ALT was either not detected or detected in only 0.3% of the analyzed urine samples (Fan et al. 2021; Qiao et al. 2022).

*Excretion* After oral administration in rats, ALT seems to be extensively metabolized, and only 7% of the parent compound was found in the feces after 24 h (Puntscher et al. 2019b).

## Tenuazonic acid (TeA)

*Absorption* After oral administration to pigs or broiler chickens, TeA was completely absorbed with a mean $T_{max}$ of 0.32 h in pigs and 2.60 h in chickens as measured by LC-MS/MS (Fraeyman et al. 2017). A study in human volunteers showed that 90% of the ingested TeA was excreted in urine in 24 h, indicating a high bioavailability (Asam et al. 2013). Similar data were obtained in rats, where 20% and 87% of the ingested TeA were found in the urine at 3 and 24 h, respectively, and a low level of the toxin was found in plasma (3 h, 0.22%; 24 h, 0.02%) (Puntscher et al. 2019b).

*Distribution* Oral administration of a complex *Alternaria* culture extract (50 mg/kg body weight) to Sprague Dawley rats containing 11 known toxins (TeA at 29.848 µg/kg bw) showed that TeA was detected in plasma (1,504 ± 853 and 139 ± 228 ng/mL, corresponding to 0.22% and 0.02% after 3 and 24 h, respectively) and urine (2,242.8 ± 957.5 and 860.0 ± 267.3 µg/mL corresponding to 20% and 87% after 3 and 24 h, respectively), suggesting systemic exposure (Puntscher et al. 2019b).

*Metabolism* In vitro data on the metabolism of TeA are not yet available. However, considering an in vivo study in human volunteers, TeA is apparently not metabolized. The toxin was excreted unchanged in urine in rats and mice (Asam et al. 2013; Puntscher et al. 2019a).

*Excretion* TeA was eliminated more slowly in broiler chickens (mean $t_{1/2}$ 0.55 h in pigs vs. 2.45 h in chickens after oral administration), showing a significantly lower estimated total body clearance (mean CL/F = 446.1 mL/h/kg in pigs vs. 59.2 mL/h/kg in chickens after oral administration) (Fraeyman et al. 2017). Urine is the major excretion route of TeA in humans, rats and mice (Asam et al. 2013; Puntscher et al. 2019b).

## Altersetin (AST)

*Absorption* Oral administration of a complex *Alternaria* culture extract (50 mg/kg b.w.) containing 11 known toxins

(AST at 919 µg/kg bw) in Sprague Dawley rats showed that low levels of AST were detected at 3 and 24 h in plasma (24.3 ± 8.9 and 16.8 ± 12.7 ng/mL, respectively, corresponding to 0.12% and 0.08%), while the AST concentration in urine was below the LOQ (Puntscher et al. 2019b).

No data are available on the distribution or metabolism of AST (in vitro or in vivo), while the few data on excretion are described below.

*Excretion* Due to matrix effects, only semi-quantitative data are available for the excretion of AST in feces. After oral administration to Sprague Dawley rats of a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins (AST at 919 µg/kg bw), AST was excreted in feces with 0.4% and 45% of the dose after 3 and 24 h, respectively (Puntscher et al. 2019b).

## Tentoxin (TEN)

*Absorption* Oral administration of a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins (TEN at 1.2 µg/kg bw) to rats resulted in the detection of TEN at low levels in urine, with 0.6 ng/mL (0.1%) and 0.3 ng/mL (0.9%) at 3 and 24 h, respectively, but not in plasma (Puntscher et al. 2019b).

*Distribution* No data are available.

*Metabolism* TEN is extensively and rapidly metabolized by rat liver microsomes into two major metabolites, a N-demethylated and a hydroxylated metabolite (Delaforge et al. 1997; Perrin et al. 2011). No data are available on the in vivo metabolism.

*Excretion* Oral administration of a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins (TEN at 1.2 µg/kg bw) to rats showed that 45% TEN was found in the feces at 24 h (Puntscher et al. 2019b).

## Perylene quinones

*Altertoxin I and II (ATX-I and II)* An in vitro study by Fleck et al. (2014a, b) investigated the intestinal absorption and metabolism of *Alternaria*-derived perylene quinones in the Caco-2 Transwell® system. After apical administration of 10 µM ATX-I, approx. 59% and 6% of the initial toxin amount were recovered, respectively, in the apical and basolateral compartments after 30 min. The total recovery was not significantly different from the cell-free control setup and metabolites were not detectable using LC-DAD-MS, implying that ATX-I was not metabolized in this experiment. The relatively high apparent permeability coefficient ((10.1 ± 2.4) × 10⁻⁶ cm/s at 30 min) of ATX-I indicated considerable transepithelial transfer and a potentially high absorption rate into the systemic circulation.

Under comparable conditions, ATX-II underwent partly reductive de-epoxidation yielding ATX-I. This outcome is
(ALT at 39.2 µg/kg b.w.) showed that ALT was not detected in plasma and urine at 3 and 24 h (Puntscher et al. 2019b), and that only a small amount of ALT was detected in the feces, 7% of the dose at 24 h, indicating a huge pre-systemic or systemic metabolism of ALT in mice. These results are in accordance with two screening studies in the Beijing general population, which showed that ALT was either not detected or detected in only 0.3% of the analyzed urine samples (Fan et al. 2021; Qiao et al. 2022).

*Excretion* After oral administration in rats, ALT seems to be extensively metabolized, and only 7% of the parent compound was found in the feces after 24 h (Puntscher et al. 2019b).

## Tenuazonic acid (TeA)

*Absorption* After oral administration to pigs or broiler chickens, TeA was completely absorbed with a mean $T_{max}$ of 0.32 h in pigs and 2.60 h in chickens as measured by LC-MS/MS (Fraeyman et al. 2017). A study in human volunteers showed that 90% of the ingested TeA was excreted in urine in 24 h, indicating a high bioavailability (Asam et al. 2013). Similar data were obtained in rats, where 20% and 87% of the ingested TeA were found in the urine at 3 and 24 h, respectively, and a low level of the toxin was found in plasma (3 h, 0.22%; 24 h, 0.02%) (Puntscher et al. 2019b).

*Distribution* Oral administration of a complex *Alternaria* culture extract (50 mg/kg body weight) to Sprague Dawley rats containing 11 known toxins (TeA at 29.848 µg/kg bw) showed that TeA was detected in plasma (1,504 ± 853 and 139 ± 228 ng/mL, corresponding to 0.22% and 0.02% after 3 and 24 h, respectively) and urine (2,242.8 ± 957.5 and 860.0 ± 267.3 µg/mL corresponding to 20% and 87% after 3 and 24 h, respectively), suggesting systemic exposure (Puntscher et al. 2019b).

*Metabolism* In vitro data on the metabolism of TeA are not yet available. However, considering an in vivo study in human volunteers, TeA is apparently not metabolized. The toxin was excreted unchanged in urine in rats and mice (Asam et al. 2013; Puntscher et al. 2019a).

*Excretion* TeA was eliminated more slowly in broiler chickens (mean $t_{1/2}$ 0.55 h in pigs vs. 2.45 h in chickens after oral administration), showing a significantly lower estimated total body clearance (mean CL/F = 446.1 mL/h/kg in pigs vs. 59.2 mL/h/kg in chickens after oral administration) (Fraeyman et al. 2017). Urine is the major excretion route of TeA in humans, rats and mice (Asam et al. 2013; Puntscher et al. 2019b).

## Altersetin (AST)

*Absorption* Oral administration of a complex *Alternaria* culture extract (50 mg/kg b.w.) containing 11 known toxins

(AST at 919 µg/kg bw) in Sprague Dawley rats showed that low levels of AST were detected at 3 and 24 h in plasma (24.3 ± 8.9 and 16.8 ± 12.7 ng/mL, respectively, corresponding to 0.12% and 0.08%), while the AST concentration in urine was below the LOQ (Puntscher et al. 2019b).

No data are available on the distribution or metabolism of AST (in vitro or in vivo), while the few data on excretion are described below.

*Excretion* Due to matrix effects, only semi-quantitative data are available for the excretion of AST in feces. After oral administration to Sprague Dawley rats of a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins (AST at 919 µg/kg bw), AST was excreted in feces with 0.4% and 45% of the dose after 3 and 24 h, respectively (Puntscher et al. 2019b).

## Tentoxin (TEN)

*Absorption* Oral administration of a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins (TEN at 1.2 µg/kg bw) to rats resulted in the detection of TEN at low levels in urine, with 0.6 ng/mL (0.1%) and 0.3 ng/mL (0.9%) at 3 and 24 h, respectively, but not in plasma (Puntscher et al. 2019b).

*Distribution* No data are available.

*Metabolism* TEN is extensively and rapidly metabolized by rat liver microsomes into two major metabolites, a N-demethylated and a hydroxylated metabolite (Delaforge et al. 1997; Perrin et al. 2011). No data are available on the in vivo metabolism.

*Excretion* Oral administration of a complex *Alternaria* culture extract (50 mg/kg body weight) containing 11 known toxins (TEN at 1.2 µg/kg bw) to rats showed that 45% TEN was found in the feces at 24 h (Puntscher et al. 2019b).

## Perylene quinones

*Altertoxin I and II (ATX-I and II)* An in vitro study by Fleck et al. (2014a, b) investigated the intestinal absorption and metabolism of *Alternaria*-derived perylene quinones in the Caco-2 Transwell® system. After apical administration of 10 µM ATX-I, approx. 59% and 6% of the initial toxin amount were recovered, respectively, in the apical and basolateral compartments after 30 min. The total recovery was not significantly different from the cell-free control setup and metabolites were not detectable using LC-DAD-MS, implying that ATX-I was not metabolized in this experiment. The relatively high apparent permeability coefficient ((10.1 ± 2.4) × 10⁻⁶ cm/s at 30 min) of ATX-I indicated considerable transepithelial transfer and a potentially high absorption rate into the systemic circulation.

Under comparable conditions, ATX-II underwent partly reductive de-epoxidation yielding ATX-I. This outcome is
in line with the knowledge of ATX-II being more reactive
due to its epoxide moiety. The transformation to ATX-I con-
tributed to the notable decrease of the ATX-II concentration
on the apical side of the Caco-2 system over time, which
did not result from transepithelial transport alone, since the
apparent permeability coefficient ((0.6 ± 0.2) × 10⁻⁶ cm/s
after 30 min) was calculated to be significantly lower than
that of ATX-I. Moreover, a noteworthy loss of the overall
ATX-II level was determined in the cell-containing experi-
ment compared to the cell-free control setup (Fleck et al.
2014a, b). In addition to the conversion to ATX-I, potential
reactions with other macromolecules including DNA must
be considered. Moreover, due to the epoxide moiety, ATX-II
is likely to react also with SH-groups as already demon-
strated for glutathione and related thiols (Fleck et al. 2014a,
b; Jarolim et al. 2017).

In a pilot study with four Sprague Dawley rats orally
administrated with an *Alternaria* culture extract (50 mg/kg
b.w.) containing 11 known toxins including 149 µg ATX-I
and 212 mg ATX-II, Puntscher et al. (2019b) found that
unchanged ATX-I was mainly excreted fecally (15%), and
to a lesser extent in the urine (up to 0.5%). In contrast, the
ATX-II levels were below the detection limit in all analyzed
matrices (urine, feces, plasma). This finding matched with
the in vitro results from Fleck et al. (2014a, b) and indicated
toxin loss by adsorption, chemical reaction, or metabolism.
Moreover, in the presence of potential reaction partners, the
loss of detectable “free” ATX-II is considerably rapid, as
has been shown for plant-based anthocyanidin delphinidin
(Aichinger et al. 2018).

A more extensive in vivo study in fourteen Sprague
Dawley rats per group administered orally with the same
*Alternaria* culture extract containing eleven known toxins
resulted in comparable results (Puntscher et al. 2019a).
ATX-I was measurable in all matrices (<0.05% of the dose
found in urine and plasma, <4% in feces), which pointed to a
certain bioavailability of this toxin. Analogously to the pilot
study, ATX-II was not detected in any of the analyzed sam-
ples. When a different group of rats was exposed to isolated
ATX-II, ATX-I was determined in all samples, suggesting
that de-epoxidation of ATX-II also occurred in vivo. How-
ever, it must be pointed out, that the formation of ATX-I
covers only a small proportion of the overall loss of detect-
able ATX-II.

The challenges of recovering perylene quinones from
in vivo samples illustrate the lack of toxicokinetic data.
The conversion of ATX-II to ATX-I represents a relevant
metabolic pathway, taking place not only in vitro (Caco-2,
HCT 116, HepG2, and V79 cells (Fleck et al. 2014a, b) but
also potentially in vivo (Puntscher et al. 2019a). However,
the enzymes catalyzing the de-epoxylation have not been

identified yet. As shown for the trichothecene T-2 toxin,
de-epoxylation by intestinal microbiota is possible. A simi-
lar mechanism might be relevant for PQs. As mentioned
before, ATX-II can also form conjugates with glutathione,
which was shown under cell-free conditions, leading to a
less genotoxic adduct than the parent toxin. Nevertheless,
ATX-II-glutathione or subsequent metabolites such as
ATX-II-cysteine or ATX-II-mercapturic acid have not been
described in the literature as metabolites of ATX-II in any
in vitro model and/or the in vivo rat study by Puntscher et al.
(2019a).

*Alterperylenol (ALP) and stemphyltoxin III (STTX-III)*

Fleck et al. (2014a, b) also examined the intestinal absorp-
tion of ALP (or alteichin-ALTCH) and STTX-III in the
Caco-2 Transwell® system. ALP might be formed by the
fungus or might arise from de-epoxidation of STTX-III.
Thirty minutes post-administration, about 48% and 4% of
the initial ALP amount (10 µM) were recovered from the
apical and basolateral side, respectively. The relatively high
apparent permeability coefficient ((7.0 ± 1.5) × 10⁻⁶ cm/s at
30 min) indicated good intestinal absorption although only
low toxin levels were measurable in the basolateral compart-
ment. The recovery of ALP from the Caco-2 system was
lower than from a cell-free control setup. Potential ALP
metabolites were not detected in the LC-DAD-MS analysis.

When the same in vitro system was used to explore the
transepithelial transfer of STTX-III (10 µM) the toxin was
undetectable on the basolateral side after 30 min. Never-
theless, low levels of its de-epoxidation product ALP were
found in both compartments. The authors concluded that
STTX-III was poorly absorbed from the gut and would pri-
marily affect the digestive tract after ingestion, whereas ALP
could reach the systemic circulation. The reduced recov-
ery of STTX-III and ALP from cell-containing assays as
compared to the cell-free control setup indicated additional
interactions with the cells (Fleck et al. 2014a, b).

The pilot study of Puntscher et al. (2019b) in four
Sprague Dawley rats orally administered with an *Alternaria*
culture extract (50 mg/kg b.w.) containing eleven known
toxins including 189 µg ALT and 315 µg STTX-III found
lower fecal (3%) and urinary (up to 0.2%) excretion rates
for ALP than ATX-I. Analogously to ATX-II, STTX-III was
not detectable in any of the study samples. The low recov-
eries supported the in vitro findings of Fleck et al. (2014a,
b) pointing to toxin loss by adsorption, chemical reaction,
degradation, or metabolism. In the extended study with 14
Sprague Dawley rats receiving the same *Alternaria* culture
extract containing eleven known toxins (Puntscher et al.
2019a), again only low ALP values were measured in all
matrices (<0.03% recovery in urine and plasma, <5% in
feces), whereas STTX-III was not detectable in the analyzed
samples.
in line with the knowledge of ATX-II being more reactive
due to its epoxide moiety. The transformation to ATX-I con-
tributed to the notable decrease of the ATX-II concentration
on the apical side of the Caco-2 system over time, which
did not result from transepithelial transport alone, since the
apparent permeability coefficient ((0.6 ± 0.2) × 10⁻⁶ cm/s
after 30 min) was calculated to be significantly lower than
that of ATX-I. Moreover, a noteworthy loss of the overall
ATX-II level was determined in the cell-containing experi-
ment compared to the cell-free control setup (Fleck et al.
2014a, b). In addition to the conversion to ATX-I, potential
reactions with other macromolecules including DNA must
be considered. Moreover, due to the epoxide moiety, ATX-II
is likely to react also with SH-groups as already demon-
strated for glutathione and related thiols (Fleck et al. 2014a,
b; Jarolim et al. 2017).

In a pilot study with four Sprague Dawley rats orally
administrated with an *Alternaria* culture extract (50 mg/kg
b.w.) containing 11 known toxins including 149 µg ATX-I
and 212 mg ATX-II, Puntscher et al. (2019b) found that
unchanged ATX-I was mainly excreted fecally (15%), and
to a lesser extent in the urine (up to 0.5%). In contrast, the
ATX-II levels were below the detection limit in all analyzed
matrices (urine, feces, plasma). This finding matched with
the in vitro results from Fleck et al. (2014a, b) and indicated
toxin loss by adsorption, chemical reaction, or metabolism.
Moreover, in the presence of potential reaction partners, the
loss of detectable “free” ATX-II is considerably rapid, as
has been shown for plant-based anthocyanidin delphinidin
(Aichinger et al. 2018).

A more extensive in vivo study in fourteen Sprague
Dawley rats per group administered orally with the same
*Alternaria* culture extract containing eleven known toxins
resulted in comparable results (Puntscher et al. 2019a).
ATX-I was measurable in all matrices (<0.05% of the dose
found in urine and plasma, <4% in feces), which pointed to a
certain bioavailability of this toxin. Analogously to the pilot
study, ATX-II was not detected in any of the analyzed sam-
ples. When a different group of rats was exposed to isolated
ATX-II, ATX-I was determined in all samples, suggesting
that de-epoxidation of ATX-II also occurred in vivo. How-
ever, it must be pointed out, that the formation of ATX-I
covers only a small proportion of the overall loss of detect-
able ATX-II.

The challenges of recovering perylene quinones from
in vivo samples illustrate the lack of toxicokinetic data.
The conversion of ATX-II to ATX-I represents a relevant
metabolic pathway, taking place not only in vitro (Caco-2,
HCT 116, HepG2, and V79 cells (Fleck et al. 2014a, b) but
also potentially in vivo (Puntscher et al. 2019a). However,
the enzymes catalyzing the de-epoxylation have not been

identified yet. As shown for the trichothecene T-2 toxin,
de-epoxylation by intestinal microbiota is possible. A simi-
lar mechanism might be relevant for PQs. As mentioned
before, ATX-II can also form conjugates with glutathione,
which was shown under cell-free conditions, leading to a
less genotoxic adduct than the parent toxin. Nevertheless,
ATX-II-glutathione or subsequent metabolites such as
ATX-II-cysteine or ATX-II-mercapturic acid have not been
described in the literature as metabolites of ATX-II in any
in vitro model and/or the in vivo rat study by Puntscher et al.
(2019a).

*Alterperylenol (ALP) and stemphyltoxin III (STTX-III)*

Fleck et al. (2014a, b) also examined the intestinal absorp-
tion of ALP (or alteichin-ALTCH) and STTX-III in the
Caco-2 Transwell® system. ALP might be formed by the
fungus or might arise from de-epoxidation of STTX-III.
Thirty minutes post-administration, about 48% and 4% of
the initial ALP amount (10 µM) were recovered from the
apical and basolateral side, respectively. The relatively high
apparent permeability coefficient ((7.0 ± 1.5) × 10⁻⁶ cm/s at
30 min) indicated good intestinal absorption although only
low toxin levels were measurable in the basolateral compart-
ment. The recovery of ALP from the Caco-2 system was
lower than from a cell-free control setup. Potential ALP
metabolites were not detected in the LC-DAD-MS analysis.

When the same in vitro system was used to explore the
transepithelial transfer of STTX-III (10 µM) the toxin was
undetectable on the basolateral side after 30 min. Never-
theless, low levels of its de-epoxidation product ALP were
found in both compartments. The authors concluded that
STTX-III was poorly absorbed from the gut and would pri-
marily affect the digestive tract after ingestion, whereas ALP
could reach the systemic circulation. The reduced recov-
ery of STTX-III and ALP from cell-containing assays as
compared to the cell-free control setup indicated additional
interactions with the cells (Fleck et al. 2014a, b).

The pilot study of Puntscher et al. (2019b) in four
Sprague Dawley rats orally administered with an *Alternaria*
culture extract (50 mg/kg b.w.) containing eleven known
toxins including 189 µg ALT and 315 µg STTX-III found
lower fecal (3%) and urinary (up to 0.2%) excretion rates
for ALP than ATX-I. Analogously to ATX-II, STTX-III was
not detectable in any of the study samples. The low recov-
eries supported the in vitro findings of Fleck et al. (2014a,
b) pointing to toxin loss by adsorption, chemical reaction,
degradation, or metabolism. In the extended study with 14
Sprague Dawley rats receiving the same *Alternaria* culture
extract containing eleven known toxins (Puntscher et al.
2019a), again only low ALP values were measured in all
matrices (<0.03% recovery in urine and plasma, <5% in
feces), whereas STTX-III was not detectable in the analyzed
samples.
<table><caption>**Table 8** Phase I and phase II metabolites of AOH and AME</caption><thead><tr><th>Metabolic reaction</th><th>Metabolite of AOH</th><th>Metabolite of AME</th><th>Enzyme isoform</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Demethylation</td><td rowspan="4">-</td><td rowspan="4">alternariol (AOH)</td><td>Rat liver post-mitochondrial supernatant with NADPH</td><td>Pollock et al. (1982)</td></tr><tr><td>Adult male Sprague Dawley rats (detected in urine and feces)</td><td></td></tr><tr><td>Homogenized liver samples of gilts with NADPH</td><td>Olsen and Visconti (1988)</td></tr><tr><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="10">Hydroxylation</td><td rowspan="2">2-hydroxy-alternariol (2-OH-AOH)</td><td rowspan="2">2-hydroxy-alternariol monomethyl ether (2-OH-AME)</td><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>Precision-cut liver slices</td><td>Burkhardt et al. (2011)</td></tr><tr><td rowspan="2">4-hydroxy-alternariol (4-OH-AOH)</td><td rowspan="2">4-hydroxy-alternariol monomethyl ether (4-OH-AME)</td><td>Male and female NMRI mice (detected in blood)</td><td>Schuchardt et al. (2014)</td></tr><tr><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="2">8-hydroxy-alternariol (8-OH-AOH)</td><td rowspan="2">8-hydroxy-alternariol monomethyl ether (8-OH-AME)</td><td>Precision-cut rat liver slices</td><td>Burkhardt et al. (2011)</td></tr><tr><td>Male and female NMRI mice (detected in blood)</td><td>Schuchardt et al. (2014)</td></tr><tr><td rowspan="2">10-hydroxy-alternariol (10-OH-AOH)</td><td rowspan="2">10-hydroxy-alternariol monomethyl ether (10-OH-AME)</td><td>Male Sprague Dawley rats (detected in feces)</td><td>Puntscher et al. (2019a)</td></tr><tr><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="4">Methylation and hydroxylation</td><td rowspan="2">dihydroxy-alternariol monomethyl ether ((OH)<sub>2</sub>-AME)</td><td rowspan="2">-</td><td>Precision-cut liver slices</td><td>Burkhardt et al. (2011)</td></tr><tr><td>Rat, pig and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="2">methyl-hydroxy-alternariol (Me-OH-AOH)</td><td rowspan="2">methyl-hydroxy-alternariol monomethyl ether (Me-OH-AME)</td><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>Incubation of purified hydroxy metabolites with rat liver cytosol and SAM</td><td>Burkhardt et al. (2011)</td></tr></tbody></table>

<figure><img src="image_14.png" /></figure>
<table><caption>**Table 8** Phase I and phase II metabolites of AOH and AME</caption><thead><tr><th>Metabolic reaction</th><th>Metabolite of AOH</th><th>Metabolite of AME</th><th>Enzyme isoform</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Demethylation</td><td rowspan="4">-</td><td rowspan="4">alternariol (AOH)</td><td>Rat liver post-mitochondrial supernatant with NADPH</td><td>Pollock et al. (1982)</td></tr><tr><td>Adult male Sprague Dawley rats (detected in urine and feces)</td><td>Olsen and Visconti (1988)</td></tr><tr><td>Homogenized liver samples of gilts with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="10">Hydroxylation</td><td rowspan="2">2-hydroxy-alternariol (2-OH-AOH)</td><td rowspan="2">2-hydroxy-alternariol monomethyl ether (2-OH-AME)</td><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>Precision-cut liver slices</td><td>Burkhardt et al. (2011)</td></tr><tr><td rowspan="2">4-hydroxy-alternariol (4-OH-AOH)</td><td rowspan="2">4-hydroxy-alternariol monomethyl ether (4-OH-AME)</td><td>Male and female NMRI mice (detected in blood)</td><td>Schuchardt et al. (2014)</td></tr><tr><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="2">8-hydroxy-alternariol (8-OH-AOH)</td><td rowspan="2">8-hydroxy-alternariol monomethyl ether (8-OH-AME)</td><td>Precision-cut rat liver slices</td><td>Burkhardt et al. (2011)</td></tr><tr><td>Male and female NMRI mice (detected in blood)</td><td>Schuchardt et al. (2014)</td></tr><tr><td rowspan="2">10-hydroxy-alternariol (10-OH-AOH)</td><td rowspan="2">10-hydroxy-alternariol monomethyl ether (10-OH-AME)</td><td>Male Sprague Dawley rats (detected in feces)</td><td>Puntscher et al. (2019a)</td></tr><tr><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="4">Methylation and hydroxylation</td><td rowspan="2">dihydroxy-alternariol monomethyl ether ((OH)<sub>2</sub>-AME)</td><td rowspan="2">-</td><td>Precision-cut liver slices</td><td>Burkhardt et al. (2011)</td></tr><tr><td>Rat, pig and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td rowspan="2">methyl-hydroxy-alternariol (Me-OH-AOH)</td><td rowspan="2">methyl-hydroxy-alternariol monomethyl ether (Me-OH-AME)</td><td>Rat, pig, and human liver microsomes with NADPH</td><td>Pfeiffer et al. (2007)</td></tr><tr><td>Incubation of purified hydroxy metabolites with rat liver cytosol and SAM</td><td>Burkhardt et al. (2011)</td></tr></tbody></table>

<figure><img src="image_16.png" /></figure>
<table><caption>**Table 8** (continued)</caption><thead><tr><th>Metabolic reaction</th><th>Metabolite of AOH</th><th>Metabolite of AME</th><th>Enzyme isoform</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Glucuronidation</td><td>alternariol-3-O-glucuronide (AOH-3-O-GlcA)</td><td>alternariol monomethyl ether-3-O-glucuronide (AME-3-O-GlcA)</td><td>Caco-2 cell line<br>Hepatic and intestinal microsomes of humans, pigs and rats with UDPGA</td><td>Burkhardt et al. (2009)<br>Pfeiffer et al. (2009b)</td></tr><tr><td>-</td><td>alternariol monomethyl ether-7-O-glucuronide (AME-7-O-GlcA)</td><td>Caco-2 cell line<br>Liver and intestinal microsomes of humans, pigs and rats with UDPGA</td><td>Burkhardt et al. (2009)<br>Pfeiffer et al. (2009b)</td></tr><tr><td>alternariol-9-O-glucuronide (AOH-9-O-GlcA)</td><td>-</td><td>Caco-2 cell line<br>Hepatic and intestinal microsomes of humans, pigs and rats with UDPGA</td><td>Burkhardt et al. (2009)<br>Pfeiffer et al. (2009a)</td></tr><tr><td>-</td><td>alternariol monomethyl ether glucuronides</td><td>Homogenized liver and intestinal samples of gilts with UDPGA</td><td>Olsen and Visconti (1988)</td></tr><tr><td rowspan="3">Sulfation</td><td>alternariol-3-O-sulfate (AOH-3-O-S)</td><td>alternariol monomethyl ether-3-O-sulfate (AME-3-O-S)</td><td>Caco-2 cell line<br>Male Sprague Dawley rats (AOH-3-O-S found in urine, AME-3-O-S detected in urine, plasma, and feces)</td><td>Burkhardt et al. (2009)<br>Puntscher et al. (2019a)</td></tr><tr><td>alternariol-7-O-sulfate (AOH-7-O-S)</td><td>alternariol monomethyl ether-7-O-sulfate (AME-7-O-S)</td><td>Rat liver cytosol with PAPS</td><td>Burkhardt et al. (2009)</td></tr><tr><td>alternariol-9-O-sulfate (AOH-9-O-S)</td><td>-</td><td>Rat liver cytosol with PAPS</td><td>Burkhardt et al. (2009)</td></tr><tr><td colspan="5"><strong>Studies performed with single human enzyme isoforms</strong></td></tr><tr><td rowspan="6">Hydroxylation</td><td>2-hydroxy-alternariol (2-OH-AOH)</td><td>2-hydroxy-alternariol monomethyl ether (2-OH-AME)</td><td>CYP isoforms 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4</td><td>Pfeiffer et al. (2008)</td></tr><tr><td>-</td><td>-</td><td>CYP3A5</td><td></td></tr><tr><td>4-hydroxy-alternariol (4-OH-AOH)</td><td>4-hydroxy-alternariol monomethyl ether (4-OH-AME)</td><td>CYP2E1</td><td></td></tr><tr><td>-</td><td>-</td><td>CYP isoforms 1A1, 1A2, 1B1, 2A6, 2C9, 2C19, 2D6, 3A4</td><td></td></tr><tr><td>8-hydroxy-alternariol (8-OH-AOH)</td><td>8-hydroxy-alternariol monomethyl ether (8-OH-AME)</td><td>CYP isoforms 2B6, 3A5</td><td></td></tr><tr><td>10-hydroxy-alternariol (10-OH-AOH)</td><td>10-hydroxy-alternariol monomethyl ether (10-OH-AME)</td><td>CYP isoforms 1A2, 2C19, 2D6, 2E1, 3A4</td><td></td></tr></tbody></table>
<table><caption>**Table 8** (continued)</caption><thead><tr><th>Metabolic reaction</th><th>Metabolite of AOH</th><th>Metabolite of AME</th><th>Enzyme isoform</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Glucuronidation</td><td>alternariol-3-O-glucuronide (AOH-3-O-GlcA)</td><td>alternariol monomethyl ether-3-O-glucuronide (AME-3-O-GlcA)</td><td>Caco-2 cell line<br>Hepatic and intestinal microsomes of humans, pigs and rats with UDPGA</td><td>Burkhardt et al. (2009)<br>Pfeiffer et al. (2009b)</td></tr><tr><td>-</td><td>alternariol monomethyl ether-7-O-glucuronide (AME-7-O-GlcA)</td><td>Caco-2 cell line<br>Liver and intestinal microsomes of humans, pigs and rats with UDPGA</td><td>Burkhardt et al. (2009)<br>Pfeiffer et al. (2009b)</td></tr><tr><td>alternariol-9-O-glucuronide (AOH-9-O-GlcA)</td><td>-</td><td>Caco-2 cell line<br>Hepatic and intestinal microsomes of humans, pigs and rats with UDPGA</td><td>Burkhardt et al. (2009)<br>Pfeiffer et al. (2009a)</td></tr><tr><td>-</td><td>alternariol monomethyl ether glucuronides</td><td>Homogenized liver and intestinal samples of gilts with UDPGA</td><td>Olsen and Visconti (1988)</td></tr><tr><td rowspan="3">Sulfation</td><td>alternariol-3-O-sulfate (AOH-3-O-S)</td><td>alternariol monomethyl ether-3-O-sulfate (AME-3-O-S)</td><td>Caco-2 cell line<br>Male Sprague Dawley rats (AOH-3-O-S found in urine, AME-3-O-S detected in urine, plasma, and feces)</td><td>Burkhardt et al. (2009)<br>Puntscher et al. (2019a)</td></tr><tr><td>alternariol-7-O-sulfate (AOH-7-O-S)</td><td>alternariol monomethyl ether-7-O-sulfate (AME-7-O-S)</td><td>Rat liver cytosol with PAPS</td><td>Burkhardt et al. (2009)</td></tr><tr><td>alternariol-9-O-sulfate (AOH-9-O-S)</td><td>-</td><td>Rat liver cytosol with PAPS</td><td>Burkhardt et al. (2009)</td></tr><tr><td colspan="5"><strong>Studies performed with single human enzyme isoforms</strong></td></tr><tr><td rowspan="6">Hydroxylation</td><td>2-hydroxy-alternariol (2-OH-AOH)</td><td>2-hydroxy-alternariol monomethyl ether (2-OH-AME)</td><td>CYP isoforms 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4</td><td>Pfeiffer et al. (2008)</td></tr><tr><td>-</td><td>-</td><td>CYP3A5</td><td></td></tr><tr><td>4-hydroxy-alternariol (4-OH-AOH)</td><td>4-hydroxy-alternariol monomethyl ether (4-OH-AME)</td><td>CYP2E1</td><td></td></tr><tr><td>-</td><td>-</td><td>CYP isoforms 1A1, 1A2, 1B1, 2A6, 2C9, 2C19, 2D6, 3A4</td><td></td></tr><tr><td>8-hydroxy-alternariol (8-OH-AOH)</td><td>8-hydroxy-alternariol monomethyl ether (8-OH-AME)</td><td>CYP isoforms 2B6, 3A5</td><td></td></tr><tr><td>10-hydroxy-alternariol (10-OH-AOH)</td><td>10-hydroxy-alternariol monomethyl ether (10-OH-AME)</td><td>CYP isoforms 1A2, 2C19, 2D6, 2E1, 3A4</td><td></td></tr></tbody></table>
<table><caption>**Table 8** (continued)</caption><thead><tr><th>Metabolic reaction</th><th>Metabolite of AOH</th><th>Metabolite of AME</th><th>Enzyme isoform</th><th>References</th></tr></thead><tbody><tr><td rowspan="3">Glucuronidation</td><td>alternariol-3-O-glucuronide (AOH-3-O-GlcA)</td><td>alternariol monomethyl ether-3-O-glucuronide (AME-3-O-GlcA)</td><td>UGT isoforms 1A1, 1A3, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15</td><td rowspan="3">Pfeiffer et al. (2009a)</td></tr><tr><td></td><td></td><td>UGT1A6</td></tr><tr><td>alternariol-9-O-glucuronide (AOH-9-O-GlcA)</td><td></td><td>UGT isoforms 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15</td></tr><tr><td></td><td></td><td>alternariol monomethyl ether-7-O-glucuronide (AME-7-O-GlcA)</td><td>UGT isoforms 1A1, 1A3, 1A7, 1A8, 1A9, 1A10</td><td></td></tr></tbody></table>

## Impact of gut microbiota on the toxicokinetics of *Alternaria* mycotoxins

The gut microbiota has been known to participate in the detoxification processes of xenobiotics introduced through the diet. Despite this, few studies have focused on the potential impact of gut microbiota on the toxicokinetics of *Alternaria* mycotoxins. When a naturally occurring mixture of *Alternaria* toxins (containing AOH, AME, ALT, TeA, TEN, ATX-I, ATX-II, ALP, STTX-III, ALS, and AST) was anaerobically incubated for 24 h with 14 human gut bacterial strains, a bacteria-independent complete loss of the epoxide-carrying *Alternaria* mycotoxins ATX-II and STTX-III was found (Crudo et al. 2021a). In contrast, ALT, TeA, TEN, and ATX-I were almost completely recovered after 24-h incubation with the different bacterial strains, whereas the recoveries of AOH, AME, ALP, ALS, and AST were significantly decreased. In addition to the incomplete recovery, the *Alternaria* mycotoxins AOH, AME, AST and ALP were found to partially accumulate within the various bacterial pellets, especially in those of Gram-negative microbiota. However, the adsorption by bacteria was not investigated in the study (Crudo et al. 2020). Nevertheless, the recoveries were also reduced after short-term incubations of AOH, AME, ATX-II, ALP and STTX-III mixture with human fecal slurries. Finally, recovery percentages ranging from 70 to 85% were reported after the incubation of AOH with *Escherichia coli* DH5α and *Lactobacillus plantarum* BFE5092 (Lemke et al. 2016).

Taken together, these results suggest that gut microbiota might contribute to the reduction of the free absorbable proportion of *Alternaria* mycotoxins introduced through the diet.

## Summary of toxicokinetic and biotransformation studies

The available toxicokinetic data on *Alternaria* toxins (Table 8) showed that the oral bioavailabilities are generally

low (AOH, AME, AST) except for TeA, which has high bio-availability in humans and rats. The low oral bioavailabilities could result from a high hepatic first pass since the intestinal resorption appeared to be considerably high as found in experiments with Caco-2 cells ($P_{app}$).

The distribution of *Alternaria* toxins is not well studied. Whereas AOH is widely distributed in mice, showing sex-dependent differences, AME is mostly distributed in liver, lung and fat, and TeA seems to be poorly distributed. The fraction unbound in plasma ($F_{u,p}$) is low for AOH and unknown for the other toxins.

In vitro and in vivo studies in different species indicated that AOH and AME are extensively metabolized in liver fractions, mostly to form hydroxylated metabolites. Comparably, TEN is extensively metabolized in vitro by microsomal fractions of rats. ALT is minimally metabolized in vitro by conjugation, and TeA is excreted unchanged in human urine (see Fig. 2).

The majority of the *Alternaria* toxins are excreted via the feces (AOH, AME, AST, and TEN), whereas urine is the major excretion route of TeA. AOH and AME, the most studied (in vitro and in vivo) of these toxins seem to have the same toxicokinetic behavior, i.e., a good intestinal resorption with a high hepatic metabolism while ALT has a low hepatic metabolism. For AST and TEN, this behavior is much more uncertain since the scientific literature is much poorer. Lastly, for TeA the situation is clearer since there are human data available.

With respect to perylene quinones, the presented in vitro and in vivo studies implied the probability of ATX-I and ALP being partially absorbed from the intestinal lumen in contrast to ATX-II and STTX-III. The latter mycotoxins underwent reductive de-epoxidation in Caco-2 cells, whereas there was no indication of the metabolism of ATX-I and ALP. Altogether, the low recoveries determined in vitro and in vivo highlight the urgent need for a better understanding of the fate of *Alternaria*-derived perylene quinones in the human body. One conceivable explanation might be an interaction with the gut microbiota (Adhikari et al. 2017;
<table><caption>**Table 8** (continued)</caption><thead><tr><th>Metabolic reaction</th><th>Metabolite of AOH</th><th>Metabolite of AME</th><th>Enzyme isoform</th><th>References</th></tr></thead><tbody><tr><td rowspan="3">Glucuronidation</td><td>alternariol-3-O-glucuronide (AOH-3-O-GlcA)</td><td>alternariol monomethyl ether-3-O-glucuronide (AME-3-O-GlcA)</td><td>UGT isoforms 1A1, 1A3, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15</td><td rowspan="3">Pfeiffer et al. (2009a)</td></tr><tr><td></td><td></td><td>UGT1A6</td></tr><tr><td>alternariol-9-O-glucuronide (AOH-9-O-GlcA)</td><td></td><td>UGT isoforms 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15</td></tr><tr><td></td><td></td><td>alternariol monomethyl ether-7-O-glucuronide (AME-7-O-GlcA)</td><td>UGT isoforms 1A1, 1A3, 1A7, 1A8, 1A9, 1A10</td><td></td></tr></tbody></table>

## Impact of gut microbiota on the toxicokinetics of *Alternaria* mycotoxins

The gut microbiota has been known to participate in the detoxification processes of xenobiotics introduced through the diet. Despite this, few studies have focused on the potential impact of gut microbiota on the toxicokinetics of *Alternaria* mycotoxins. When a naturally occurring mixture of *Alternaria* toxins (containing AOH, AME, ALT, TeA, TEN, ATX-I, ATX-II, ALP, STTX-III, ALS, and AST) was anaerobically incubated for 24 h with 14 human gut bacterial strains, a bacteria-independent complete loss of the epoxide-carrying *Alternaria* mycotoxins ATX-II and STTX-III was found (Crudo et al. 2021a). In contrast, ALT, TeA, TEN, and ATX-I were almost completely recovered after 24-h incubation with the different bacterial strains, whereas the recoveries of AOH, AME, ALP, ALS, and AST were significantly decreased. In addition to the incomplete recovery, the *Alternaria* mycotoxins AOH, AME, AST and ALP were found to partially accumulate within the various bacterial pellets, especially in those of Gram-negative microbiota. However, the adsorption by bacteria was not investigated in the study (Crudo et al. 2020). Nevertheless, the recoveries were also reduced after short-term incubations of AOH, AME, ATX-II, ALP and STTX-III mixture with human fecal slurries. Finally, recovery percentages ranging from 70 to 85% were reported after the incubation of AOH with *Escherichia coli* DH5α and *Lactobacillus plantarum* BFE5092 (Lemke et al. 2016).

Taken together, these results suggest that gut microbiota might contribute to the reduction of the free absorbable proportion of *Alternaria* mycotoxins introduced through the diet.

## Summary of toxicokinetic and biotransformation studies

The available toxicokinetic data on *Alternaria* toxins (Table 8) showed that the oral bioavailabilities are generally

low (AOH, AME, AST) except for TeA, which has high bio-availability in humans and rats. The low oral bioavailabilities could result from a high hepatic first pass since the intestinal resorption appeared to be considerably high as found in experiments with Caco-2 cells ($P_{app}$).

The distribution of *Alternaria* toxins is not well studied. Whereas AOH is widely distributed in mice, showing sex-dependent differences, AME is mostly distributed in liver, lung and fat, and TeA seems to be poorly distributed. The fraction unbound in plasma ($F_{u,p}$) is low for AOH and unknown for the other toxins.

In vitro and in vivo studies in different species indicated that AOH and AME are extensively metabolized in liver fractions, mostly to form hydroxylated metabolites. Comparably, TEN is extensively metabolized in vitro by microsomal fractions of rats. ALT is minimally metabolized in vitro by conjugation, and TeA is excreted unchanged in human urine (see Fig. 2).

The majority of the *Alternaria* toxins are excreted via the feces (AOH, AME, AST, and TEN), whereas urine is the major excretion route of TeA. AOH and AME, the most studied (in vitro and in vivo) of these toxins seem to have the same toxicokinetic behavior, i.e., a good intestinal resorption with a high hepatic metabolism while ALT has a low hepatic metabolism. For AST and TEN, this behavior is much more uncertain since the scientific literature is much poorer. Lastly, for TeA the situation is clearer since there are human data available.

With respect to perylene quinones, the presented in vitro and in vivo studies implied the probability of ATX-I and ALP being partially absorbed from the intestinal lumen in contrast to ATX-II and STTX-III. The latter mycotoxins underwent reductive de-epoxidation in Caco-2 cells, whereas there was no indication of the metabolism of ATX-I and ALP. Altogether, the low recoveries determined in vitro and in vivo highlight the urgent need for a better understanding of the fate of *Alternaria*-derived perylene quinones in the human body. One conceivable explanation might be an interaction with the gut microbiota (Adhikari et al. 2017;
<figure><img src="image_15.png" /><figcaption>**Fig. 2** Summary on the metabolism of selected *Alternaria* toxins. The center picture of *Alternaria alternata* was kindly provided by Roman Labuda</figcaption></figure>

Crudo et al. 2021a). Overall, little is known about the metabolic pathways of these toxins, let alone their toxicokinetic parameters. Even less data are available for other perylene quinones occurring in *Alternaria* strains such as ATX-III and stemphylperylenol. Although data on *Alternaria* toxins

are lacking, evidence from other mycotoxins indicates that adverse health effects caused by mycotoxins including *Alternaria* toxins are likely to be dependent on the combined alimentary and respiratory exposure.
<figure><img src="image_17.png" /><figcaption>**Fig. 2** Summary on the metabolism of selected *Alternaria* toxins. The center picture of *Alternaria alternata* was kindly provided by Roman Labuda</figcaption></figure>

Crudo et al. 2021a). Overall, little is known about the metabolic pathways of these toxins, let alone their toxicokinetic parameters. Even less data are available for other perylene quinones occurring in *Alternaria* strains such as ATX-III and stemphylperylenol. Although data on *Alternaria* toxins

are lacking, evidence from other mycotoxins indicates that adverse health effects caused by mycotoxins including *Alternaria* toxins are likely to be dependent on the combined alimentary and respiratory exposure.
## Conclusion and identification of data gaps

Several *Alternaria* toxins have a cytotoxic and mutagenic potential, some already at nanomolar concentrations. However, existing contradictions in the in vitro studies reported indicate that further testing, if applicable, according to OECD guidelines is necessary to close the existing data gaps. Furthermore, in vitro studies strongly argue for endocrine disruptive and immunotoxic effects of several *Alternaria* toxins. However, respective in vivo studies are still missing. For determining safety levels for the combined dietary, respiratory, and dermal exposure that are risks to human health, further data are needed using well-characterized test items.

Critical data gaps comprise the toxicological relevance of perylene quinones formed by *Alternaria* species, especially those with reactive epoxide moieties. So far, these compounds are rarely found in food but are well known to be formed by fungi. The fate of these compounds in plants, during food processing and digestion remains to be elucidated.

Of note, most published studies have been performed with toxins isolated from fungal culture. Thus, purity of the applied toxin preparations is of utmost importance for the interpretation of the results, since traces of potent co-occurring toxins might lead to false structure-activity conclusions. It is the intention of the PARC project to close some of these data gaps on the hazards of *Alternaria* toxins and a program of studies is ongoing.

**Acknowledgements** The European Partnership for the Assessment of Risks from Chemicals has received funding from the European Union's Horizon Europe research and innovation program under Grant Agreement No 101057014 and has received co-funding of the authors' institutions. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.

**Funding** Open access funding provided by University of Vienna.

## Declarations

**Conflict of interest** The authors declare that they have no conflict of interest.

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Aichinger G, Beisl J, Marko D (2017) Genistein and delphinidin antagonize the genotoxic effects of the mycotoxin alternariol in human colon carcinoma cells. *Mol Nutr Food Res* 61(2):10. https://doi.org/10.1002/mnfr.201600462

Aichinger G, Puntscher H, Beisl J, Kütt ML, Warth B, Marko D (2018) Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II. *Toxicol Lett* 284:136-142. https://doi.org/10.1016/j.TOXLET.2017.12.002

Aichinger G, Krüger F, Puntscher H, Preindl K, Warth B, Marko D (2019) Naturally occurring mixtures of *Alternaria* toxins: anti-estrogenic and genotoxic effects in vitro. *Arch Toxicol* 93(10):3021-3031. https://doi.org/10.1007/s00204-019-02545-z

Aichinger G, Živná N, Varga E, Crudo F, Warth B, Marko D (2020a) Microfiltration results in the loss of analytes and affects the in vitro genotoxicity of a complex mixture of *Alternaria* toxins. *Mycotoxin Res* 36:399-408. https://doi.org/10.1007/s12550-020-00405-9

Aichinger G, Dellafiora L, Pantazi F, Del Favero G, Galaverna G, Dallasta C, Marko D (2020b) *Alternaria* toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid *in silico/in vitro* study. *Arch Toxicol* 94(6):2225-2237. https://doi.org/10.1007/s00204-020-02746-x

Aichinger G, Pantazi F, Marko D (2020c) Combinatory estrogenic effects of bisphenol A in mixtures with alternariol and zearalenone in human endometrial cells. *Toxicol Lett* 319:242-249. https://doi.org/10.1016/j.TOXLET.2019.10.025

Aichinger G, Grgic D, Beisl J, Crudo F, Warth B, Varga E, Marko D (2022a) N-acetyl cysteine alters the genotoxic and estrogenic properties of *Alternaria* toxins in naturally occurring mixtures. *Emerg Contamin* 8:30-38. https://doi.org/10.1016/j.emcon.2021.12.004

Aichinger G, Pahlke G, Puntscher H, Groestlinger J, Grabher S, Braun D, Tillmann K, Plasenzotti R, Del Favero G, Warth B, Höger H, Marko D (2022b) Markers for DNA damage are induced in the rat colon by the *Alternaria* toxin altertoxin-II, but not a complex extract of cultured *Alternaria alternata*. *Front Toxicol* 4:10. https://doi.org/10.3389/FTOX.2022.977147

An YH, Zhao TZ, Miao J, Liu GT, Zheng YZ, Xu YM, Van Etten RL (1989) Isolation, identification, and mutagenicity of alternariol monomethyl ether. *J Agric Food Chem* 37(5):1341-1343. https://doi.org/10.1021/jf00089a029

Asam S, Habler K, Rychlik M (2013) Determination of tenuazonic acid in human urine by means of a stable isotope dilution assay. *Anal Bioanal Chem* 405(12):4149-4158. https://doi.org/10.1007/s00216-013-6793-5

Balázs A, Faisal Z, Csepregi R, Kőszegi T, Kriszt B, Szabó I, Poór M (2021) *In vitro* evaluation of the individual and combined cytotoxic and estrogenic effects of zearalenone, its reduced metabolites, alternariol, and genistein. *Int J Mol Sci* 22(12):10. https://doi.org/10.3390/ijms22126281

Beerweiler CC, Masanetz RK, Schaub B (2023) Asthma and allergic diseases: Cross talk of immune system and environmental
**Conclusion and identification of data gaps**

Several *Alternaria* toxins have a cytotoxic and mutagenic potential, some already at nanomolar concentrations. However, existing contradictions in the in vitro studies reported indicate that further testing, if applicable, according to OECD guidelines is necessary to close the existing data gaps. Furthermore, in vitro studies strongly argue for endocrine disruptive and immunotoxic effects of several *Alternaria* toxins. However, respective in vivo studies are still missing. For determining safety levels for the combined dietary, respiratory, and dermal exposure that are risks to human health, further data are needed using well-characterized test items.

Critical data gaps comprise the toxicological relevance of perylene quinones formed by *Alternaria* species, especially those with reactive epoxide moieties. So far, these compounds are rarely found in food but are well known to be formed by fungi. The fate of these compounds in plants, during food processing and digestion remains to be elucidated.

Of note, most published studies have been performed with toxins isolated from fungal culture. Thus, purity of the applied toxin preparations is of utmost importance for the interpretation of the results, since traces of potent co-occurring toxins might lead to false structure–activity conclusions. It is the intention of the PARC project to close some of these data gaps on the hazards of *Alternaria* toxins and a program of studies is ongoing.

**Acknowledgements** The European Partnership for the Assessment of Risks from Chemicals has received funding from the European Union's Horizon Europe research and innovation program under Grant Agreement No 101057014 and has received co-funding of the authors' institutions. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.

**Funding** Open access funding provided by University of Vienna.

**Declarations**

**Conflict of interest** The authors declare that they have no conflict of interest.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Bensassi F, Gallerne C, Sharaf El Dein O, Hajlaoui MR, Bacha H, Lemaire C (2012) Cell death induced by the *Alternaria* mycotoxin Alternariol. Toxicol in Vitro 26(6):915-923. https://doi.org/10.1016/j.tiv.2012.04.014

Bensassi F, Gallerne C, Sharaf El Dein O, Rabeh Hajlaoui M, Bacha H, Lemaire C (2015) Combined effects of alternariols mixture on human colon carcinoma cells. Toxicol Mech Methods 25(1):56-62. https://doi.org/10.3109/15376516.2014.985354

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Chiesi C, Fernandez-Blanco C, Cossignani L, Font G, Ruiz MJ (2015) Alternariol-induced cytotoxicity in Caco-2 cells. protective effect of the phenolic fraction from virgin olive oil. Toxicon 93:103-111. https://doi.org/10.1016/j.toxicon.2014.11.230

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Crudo F, Aichinger G, Mihajlovic J, Dellafiora L, Varga E, Puntscher H, Warth B, Dall'Asta C, Berry D, Marko D (2020) Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of *Alternaria* toxins *in vitro*. Arch Toxicol 94(10):3541. https://doi.org/10.1007/S00204-020-02831-1

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Crudo F, Aichinger G, Dellafiora L, Kiss E, Mihajlovic J, Del Favero G, Berry D, Dall'Asta C, Marko D (2022) Persistence of the antagonistic effects of a natural mixture of *Alternaria* mycotoxins

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Demaegdt H, Daminet B, Evrard A, Scippo ML, Muller M, Pussemier L, Callebaut A, Vandermeiren K (2016) Endocrine activity of mycotoxins and mycotoxin mixtures. Food Chem Toxicol 96:107-116. https://doi.org/10.1016/j.FCT.2016.07.033

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202249981

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Bensassi F, Gallerne C, Sharaf El Dein O, Hajlaoui MR, Bacha H, Lemaire C (2012) Cell death induced by the *Alternaria* mycotoxin Alternariol. Toxicol in Vitro 26(6):915-923. https://doi.org/10.1016/j.tiv.2012.04.014

Bensassi F, Gallerne C, Sharaf El Dein O, Rabeh Hajlaoui M, Bacha H, Lemaire C (2015) Combined effects of alternariols mixture on human colon carcinoma cells. Toxicol Mech Methods 25(1):56-62. https://doi.org/10.3109/15376516.2014.985354

Brugger EM, Wagner J, Schumacher DM, Koch K, Podlech J, Metzler M, Lehmann L (2006) Mutagenicity of the mycotoxin alternariol in cultured mammalian cells. Toxicol Lett 164(3):221-230. https://doi.org/10.1016/j.TOXLET.2006.01.001

Burkhardt B, Pfeiffer E, Metzler M (2009) Absorption and metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in Caco-2 cells *in vitro*. Mycotoxin Res 25(3):149-157. https://doi.org/10.1007/S12550-009-0022-2/METRICS

Burkhardt B, Wittenauer J, Pfeiffer E, Schauer UMD, Metzler M (2011) Oxidative metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in precision-cut rat liver slices *in vitro*. Mol Nutr Food Res 55(7):1079-1086. https://doi.org/10.1002/MNFR.201000487

Cephus JY, Gandhi VD, Shah R, Brooke Davis J, Fuseini H, Yung JA, Zhang J, Kita H, Polosukhin VV, Zhou W, Newcomb DC (2021) Estrogen receptor-α signaling increases allergen-induced IL-33 release and airway inflammation. Allergy Eur J Allergy Clin Immunol 76(1):255-268. https://doi.org/10.1111/ALL.14491

Chiesi C, Fernandez-Blanco C, Cossignani L, Font G, Ruiz MJ (2015) Alternariol-induced cytotoxicity in Caco-2 cells. protective effect of the phenolic fraction from virgin olive oil. Toxicon 93:103-111. https://doi.org/10.1016/j.toxicon.2014.11.230

Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, Smith CC, Štětina R (2008) The comet assay: topical issues. Mutagenesis 23(3):143-151. https://doi.org/10.1093/mutage/gem051

Crudo F, Varga E, Aichinger G, Galaverna G, Marko D, Dall'Asta C, Dellafiora L (2019) Co-occurrence and combinatorial effects of *Alternaria* mycotoxins and other xenobiotics of food origin: current scenario and future perspectives. Toxins. https://doi.org/10.3390/toxins11110640

Crudo F, Aichinger G, Mihajlovic J, Dellafiora L, Varga E, Puntscher H, Warth B, Dall'Asta C, Berry D, Marko D (2020) Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of *Alternaria* toxins *in vitro*. Arch Toxicol 94(10):3541. https://doi.org/10.1007/S00204-020-02831-1

Crudo F, Aichinger G, Mihajlovic J, Varga E, Dellafiora L, Warth B, Dall'Asta C, Berry D, Marko D (2021a) *In vitro* interactions of *Alternaria* mycotoxins, an emerging class of food contaminants, with the gut microbiota: a bidirectional relationship. Arch Toxicol 95(7):2533-2549. https://doi.org/10.1007/s00204-021-03043-x

Crudo F, Barilli A, Mena P, Rotoli BM, Rio DD, Dall'Asta C, Dellafiora L (2021b) An *in vitro* study on the transport and phase II metabolism of the mycotoxin alternariol in combination with the structurally related gut microbial metabolite urolithin C. Toxicol Lett 340:15-22. https://doi.org/10.1016/j.TOXLET.2021.01.007

Crudo F, Aichinger G, Dellafiora L, Kiss E, Mihajlovic J, Del Favero G, Berry D, Dall'Asta C, Marko D (2022) Persistence of the antagonistic effects of a natural mixture of *Alternaria* mycotoxins

on the estrogen-like activity of human feces after anaerobic incubation. Toxicol Lett 1(358):88-99. https://doi.org/10.1016/j.toxlet.2022.01.015

Dall'Asta C, Cirlini M, Falavigna C (2014) Mycotoxins from *Alternaria*: Toxicological implications. Adv Mol Toxicol 8:107-121. https://doi.org/10.1016/B978-0-444-63406-1.00003-9

Davis VM, Stack ME (1991) Mutagenicity of stemphyltoxin III, a metabolite of *Alternaria alternata*. Appl Environ Microbiol 57(1):180-182. https://doi.org/10.1128/AEM.57.1.180-182.1991

Davis VM, Stack ME (1994) Evaluation of alternariol and alternariol methyl ether for mutagenic activity in *Salmonella typhimurium*. Appl Environ Microbiol 60(10):3901-3902. https://doi.org/10.1128/AEM.60.10.3901-3902.1994

Del Favero G, Hohenbichler J, Mayer RM, Rychlik M, Marko D (2020) Mycotoxin altertoxin II induces lipid peroxidation connecting mitochondrial stress response to NF-κB inhibition in THP-1 macrophages. Chem Res Toxicol 33(2):492-504. https://doi.org/10.1021/acs.chemrestox.9b00378

Delaforge M, Andre F, Jaouen M, Dolgos H, Benech H, Gomis JM, Noel JP, Cavelier F, Verducci J, Aubagnac JL, Liebermann B (1997) Metabolism of tentoxin by hepatic cytochrome P-450 3A isozymes. Eur J Biochem 250(1):150-157. https://doi.org/10.1111/j.1432-1033.1997.00150.x

Dellafiora L, Warth B, Schmidt V, Del Favero G, Mikula H, Fröhlich J, Marko D (2018) An integrated *in silico/in vitro* approach to assess the xenostrogenic potential of *Alternaria* mycotoxins and metabolites. Food Chem 248:253-261. https://doi.org/10.1016/j.FOODCHEM.2017.12.013

Demaegdt H, Daminet B, Evrard A, Scippo ML, Muller M, Pussemier L, Callebaut A, Vandermeiren K (2016) Endocrine activity of mycotoxins and mycotoxin mixtures. Food Chem Toxicol 96:107-116. https://doi.org/10.1016/j.FCT.2016.07.033

den Hollander D, Holvoet C, Demeyere K, De Zutter N, Audenaert K, Meyer E, Croubels S (2022) Cytotoxic effects of alternariol, alternariol monomethyl-ether, and tenuazonic acid and their relevant combined mixtures on human enterocytes and hepatocytes. Front Microbiol. https://doi.org/10.3389/fmicb.2022.849243

Dong Z, Liu G, Dong Z, Qlan Y, An Y, Miao J, Zhen Y (1987) Induction of mutagenesis and transformation by the extract of *Alternaria alternata* isolated from grains in Linxian, China. Carcinogenesis 8(7):989-991. https://doi.org/10.1093/carcin/8.7.989

Dong L, Kim HJ, Cao TQ, Liu Z, Lee H, Ko W, Kim YC, Sohn JH, Kim TK, Yim JH, Lee DS, Oh H (2021) Anti-inflammatory effects of metabolites from Antarctic fungal strain *Pleosporales* sp. Sf-7343 in hacat human keratinocytes. Int J Mol Sci 22(18):10. https://doi.org/10.3390/ijms22189674

EFSA on Contaminants in the Food Chain (2011) Scientific Opinion on the risks for animal and public health related to the presence of *Alternaria* toxins in feed and food. EFSA J 9(10):2407. https://doi.org/10.2903/j.efsa.2011.2407. (97 pp)

EFSA Scientific Committee (2012) Scientific Opinion on Exploring options for providing advice about possible human health risks based on the concept of Threshold of Toxicological Concern (TTC). EFSA J. https://doi.org/10.2903/J.EFSA.2012.2750

EFSA, Arcella D, Eskola M, Gómez Ruiz JA (2016) Dietary exposure assessment to *Alternaria* toxins in the European population. EFSA J. https://doi.org/10.2903/J.EFSA.2016.4654

EFSA Scientific Committee, More SJ, Bampidis V, Benford D, Bragard C, Halldorsson TI, Hernández-Jerez AF, Hougaard Bennekou S, Koutsoumanis KP, Machera K, Naegeli H, Nielsen SS, Schlatter JR, Schrenk D, Silano V, Turck D, Younes M, Gundert-Remy U, Kass GEN, Wallace HM (2019) Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment. EFSA J. https://doi.org/10.2903/J.EFSA.2019.5708

European Commission (2022) Commission Recommendation (EU) 2022/553 of 5 April 2022 on monitoring the presence
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