Probing the Interactions of 31 Mycotoxins with Xanthine Oxidase: Alternariol, Alternariol-3-Sulfate, and α-Zearalenol Are Allosteric Inhibitors of the Enzyme

Mycotoxins are frequent toxic contaminants in foods and beverages, causing a significant health threat. Interactions of mycotoxins with biotransformation enzymes (e.g., cytochrome P450 enzymes, sulfotransferases, and uridine 5′-diphospho-glucuronosyltransferases) may be important due to their possible detoxification or toxic activation during enzymatic processes. Furthermore, mycotoxin-induced enzyme inhibition may affect the biotransformation of other molecules. A recent study described the strong inhibitory effects of alternariol and alternariol-9-methylether on the xanthine oxidase (XO) enzyme. Therefore, we aimed to test the impacts of 31 mycotoxins (including the masked/modified derivatives of alternariol and alternariol-9-methylether) on XO-catalyzed uric acid formation. Besides the in vitro enzyme incubation assays, mycotoxin depletion experiments and modeling studies were performed. Among the mycotoxins tested, alternariol, alternariol-3-sulfate, and α-zearalenol showed moderate inhibitory actions on the enzyme, representing more than tenfold weaker impacts compared with the positive control inhibitor allopurinol. In mycotoxin depletion assays, XO did not affect the concentrations of alternariol, alternariol-3-sulfate, and α-zearalenol in the incubates; thus, these compounds are inhibitors but not substrates of the enzyme. Experimental data and modeling studies suggest the reversible, allosteric inhibition of XO by these three mycotoxins. Our results help the better understanding of the toxicokinetic interactions of mycotoxins.


Introduction
Mycotoxins are secondary metabolites of molds. Mycotoxin contamination in foods and beverages causes significant health threats worldwide [1]. Their typically high thermal stability and frequent occurrence in the food chain make the removal of mycotoxins very challenging [2,3]. Biotransformation enzymes (such as cytochrome P450 enzymes, sulfotransferases, and uridine 5 -diphospho-glucuronosyltransferases) play important roles in the toxic activation or detoxification of mycotoxins; in addition, the mycotoxin-induced inhibition of these enzymes may affect the biotransformation of other molecules [4].

Cereals and corresponding products
Fusarium sp. Low toxicity [20] Deoxynivalenol DON
Xenoestrogen, endocrine disruptor [20][21][22] Xanthine oxidoreductase is a 300 kDa homodimer protein containing molybdenum cofactor, flavin adenine dinucleotide (FAD) site, and Fe 2 S 2 sites [23]. The enzyme is important in purine catabolism because it catalyzes the transformation of hypoxanthine to xanthine and then xanthine to uric acid [24]. Xanthine oxidoreductase designation means two interconvertible forms of the same enzyme, including xanthine dehydrogenase and xanthine oxidase (XO). Xanthine dehydrogenase can be reversibly or irreversibly converted to XO [25]. Typically, xanthine dehydrogenase is intracellularly presented, while XO is dominant in the extracellular water space [23]. High uric acid levels cause hyperuricemia which may result in the development of gout, cardiovascular diseases, and metabolic syndrome [26]. In addition, XO generates superoxide anion radicals and hydrogen peroxide, which may also be involved in the unpleasant impacts of high uric acid formation [27]. Allopurinol, a potent inhibitor of XO, is commonly used to treat hyperuricemia and gout [28]. Furthermore, XO is also an important enzyme in the biotransformation of certain drugs, such as 6-mercaptopurine (used in the treatment of cancer and autoimmune diseases) [29].
Previous studies demonstrated that aflatoxins and ochratoxins could cause the development of gout in certain animals [30]. AFB1-contaminated diet (for 14 days and 21 days) increased XO activity and uric acid levels in the serum of fish [31]. In rats, chronic exposure to ZEN significantly elevated XO activity in the liver and kidneys [32]. The upregulation of xanthine oxidoreductase was observed in the liver and the jejunum of chickens after three weeks of DON-contaminated feeding [33]. In contrast, fumonisins (FB1 + FB2) alone and in combination with DON (15 days of exposure) decreased the expression of xanthine dehydrogenase in the jejunum of broiler chickens [34]. These results suggest that certain mycotoxins may be able to increase the expression and/or the activity of xanthine oxidoreductase.
Typically, less information is available regarding the inhibitory effects of mycotoxins on XO. In a recent study, Fan et al. described that AOH (IC 50 = 0.2 µM) and AME (IC 50 = 0.5 µM) are strong inhibitors of XO, where these mycotoxins showed approximately tenfold higher inhibitory potency compared with allopurinol [35]. Considering these results and the relatively low acute toxicity of AOH and AME, Fan et al. suggested that AOH may be a potential lead compound in the development of new potent XO inhibitors [35]. Urolithins (colon metabolites of ellagitannins) are considered barely toxic compounds [36]. They have very similar chemical structure to Alternaria mycotoxins. In the study of Fan et al. [35], urolithins examined were much weaker inhibitors of XO than AOH or AME. In another paper, considerably weaker inhibitory actions of AOH (IC 50 = 15.5 µM) and AME (IC 50 = 60.5 µM) were described regarding the XO enzyme [37]. In addition, the consumption of wheat grain contaminated with Alternaria spp. did not affect XO activity in broiler chickens [38].
Importantly, XO is a major component in bovine milk [39], which is commonly contaminated with certain mycotoxins (e.g., AFM1, OTA, ZEN, and α-ZEL) [40]. Thus, mycotoxin-XO interactions can also be important from this point of view.
In this work, the interactions of 31 mycotoxins with the XO enzyme were examined by applying in vitro enzyme assays and modeling studies. We planned to test the effects of AOH, AME, and their masked/modified derivatives (AS, AG, AMS, and AMG) on XO-catalyzed uric acid formation to confirm the previously reported data and to find potentially stronger inhibitors among AOH derivatives. In addition, we also investigated the impacts of 25 other mycotoxins, including AFB1, AFB2, AFG1, AFG2, AFM1, STC, CPA, CIT, DHC, OTA, OTB, OTC, PAT, BEA, DON, FB1, T2, ZEN, α-ZEL, β-ZEL, ZAN, α-ZAL, β-ZAL, Z14S, and Z14Glz ( Table 1). The potential inhibitory actions of mycotoxins on XO, the reversibility of their inhibitory effects, and the XO-induced mycotoxin depletion were assessed. Experimental results and molecular modeling studies suggest the moderate, allosteric inhibition of XO by AOH, AS, and α-ZEL ( Figure 1).

Inhibitory Effects of Mycotoxins on Xanthine Oxidase Enzyme
The impacts of 20 µM mycotoxin concentrations were tested to evaluate the potential inhibitory effects of mycotoxins on XO-catalyzed xanthine oxidation. Results are demonstrated in Figure 2, where Alternaria, Aspergillus/Penicillium, and Fusarium toxins are presented in three separate panels.
AOH, AS, AG, and AMS induced a statistically significant decrease in metabolite formation, while AME and AMG caused only slight or no effects, respectively ( Figure 2A). Among the Alternaria mycotoxins tested, AOH and AS showed the strongest inhibitory actions (resulting in a 54% and 40% decrease in uric acid formation, respectively). AG and AMS induced approximately 10% inhibition; therefore, these mycotoxins can be considered weak inhibitors of XO. Interestingly, our results suggest negligible inhibition of the enzyme by AME, while in a previous report, AME and AOH showed similarly strong inhibitory effects [35]. Furthermore, AS caused a slightly weaker impact vs. AOH (Figure 2A). This result is in accordance with our previous observation that quercetin-3 -sulfate was also a similarly strong inhibitor of XO than the parent flavonoid quercetin [41]. In Figure 2B, the impacts of Aspergillus/Penicillium mycotoxins are presented: AFB1, AFB2, AFG1, AFM1, STC, CPA, CIT, DHC, OTA, OTB, OTC, and PAT did not affect the XO-catalyzed metabolite formation. AFG2 showed a statistically significant but weak inhibitory action (14%) on XO ( Figure 2B).
Among the Fusarium mycotoxins, BEA, DON, FB1, T2, β-ZEL, ZAN, α-ZAL, β-ZAL, Z14S, and Z14Glz did not influence the XO-catalyzed uric acid formation ( Figure 2C). However, ZEN induced a small decrease (10%), while α-ZEL caused a marked (54%) decrease in metabolite production. To the best of our knowledge, the inhibitory actions of ZEN and α-ZEL on the XO enzyme have not been previously reported. Nevertheless, in a recent in vivo study, chronic exposure to ZEN significantly elevated XO activity in the liver and kidneys of rats [32]. These observations suggest that ZEN and/or its metabolites may affect the expression of XO, which mechanism may overwrite the direct inhibitory actions noticed for ZEN and α-ZEL in the current study.
Based on sigmoidal fitting, the IC 50 values of AOH, AS, and α-ZEL were 10.6 µM, 15.0 µM, and 17.1 µM, respectively. Nevertheless, it is important to note that α-ZEL can produce close to complete inhibition at high concentrations; while for AOH and AS, we observed the lower plateau of the sigmoid curve around 35% metabolite formation. In the same assay, allopurinol showed much stronger inhibition on XO-catalyzed xanthine oxidation; its IC 50 value was 0.5 µM. These data demonstrated that AOH, AS, and α-ZEL are approximately 20-to 35-fold weaker inhibitors of XO compared with the positive control allopurinol. Therefore, our results do not support the previous study of Fan et al. [35], which suggested that AOH and AME are highly potent inhibitors of XO. This is surprising because, in our earlier studies with flavonoids and XO [41,42], we determined similar IC 50 values to other research groups. However, in regard to their inhibitory actions on XO enzyme, another study suggested higher IC 50 values of AOH (IC 50 = 15.5 µM) and AME (IC 50 = 60.5 µM) [37]. These data are in accordance with our findings, showing a similar IC 50 value of AOH determined in the current study and explaining why we did not notice relevant inhibitory effect of AME (20 µM). Nevertheless, the consumption of wheat grain contaminated with Alternaria spp. did not affect XO activity in broiler chickens [38], suggesting the minor in vivo relevance of the moderate inhibitory actions of Alternaria mycotoxins on XO.
In the following experiment, we examined the reversibility of mycotoxin-induced XO inhibition. Therefore, the XO enzyme was preincubated with AOH, AS, or α-ZEL (each 50 µM) for 10 min, then we started the reaction with the addition of the substrate (final concentrations: 5, 10, or 25 µM). Similar to the previous experiments, the reaction was stopped after 5 min incubation. In a concentration-dependent fashion, the higher levels of the substrate significantly increased the XO-catalyzed uric acid formation ( Figure 4). These results demonstrate that AOH, AS, and α-ZEL are reversible inhibitors of XO. AOH, ZEN, and their derivatives appear in the circulation (and likely in tissues), typically at nanomolar concentrations [8,21]. However, the therapeutic plasma concentrations of allopurinol (competitive inhibitor and false substrate of XO) and its metabolite oxipurinol (pseudo-irreversible inhibitor of XO) are approximately 40 µM together [43]. In addition, allopurinol and oxipurinol are highly potent inhibitors of XO. Considering the significantly higher IC 50 values of AOH, AS, and α-ZEL (IC 50 = 10.6-17.1 µM) compared with allopurinol (IC 50 = 0.5 µM) as well as the much lower concentrations of these mycotoxins in the body, it is very unlikely that AOH, AS, or α-ZEL can induce a clinically relevant decrease in uric acid levels. Based on these data, it is also reasonable to hypothesize that AOH, AS, and α-ZEL are not able to disrupt the XO-mediated biotransformation of 6mercaptopurine or other drugs (e.g., azathioprine). Considering the previous observations in animal studies [30][31][32][33][34], some mycotoxins may be able to induce the increased expression of XO, which likely have more in vivo relevance than the inhibitory effects noticed in the current study.

Mycotoxin Depletion Assays
AOH, AS, and α-ZEL showed significant inhibitory effects on XO; therefore, we examined whether these mycotoxins are simply inhibitors or if they are also substrates of the enzyme. AOH, AS, and α-ZEL were incubated for 0 min, 30 min, and 60 min in the presence of the same amount of XO (0.0012 U/mL) used in the enzyme inhibition studies. Nevertheless, we did not see any changes in the concentrations of these mycotoxins ( Table 2), suggesting that XO is not involved in the biotransformation of AOH, AS, and α-ZEL.
XO is a major constituent of bovine milk [39]. The simultaneous presence of XO and certain mycotoxins (e.g., AFM1, OTA, ZEN, and α-ZEL) [40] in milk also makes important mycotoxin-XO interactions, including the potential XO-mediated biotransformation. Our data suggest that XO is not involved in the metabolism of AOH, AS, and α-ZEL. Nevertheless, α-ZEL commonly contaminates bovine milk. Therefore, it is reasonable to hypothesize that α-ZEL appears in milk partly in XO-bound form, which may affect its release and absorption from the gastrointestinal tract. Table 2. AOH, AS, and α-ZEL levels (% ± SEM) in samples after 0 min, 30 min, and 60 min incubation with XO (0.0012 U/mL; initial mycotoxin concentration: 5 µM; n = 3). We started the reaction with the addition of XO.

Modeling Studies
During the 300 blind docking runs in total, none of the ligands bound to the binding pocket of xanthine [44]. Nevertheless, in the best ranks for each ligand tested, another binding pocket was found ( Figure 5A), which was originally described by Kuwabara et al. [45]. AOH (8th-rank binding mode; Figure 5B), AS (1st-rank binding mode; Figure 5C), and α-ZEL (2nd-rank binding mode; Figure 5D) interact with W336, which amino acid is located in an alternative binding pocket far from the xanthine binding site. Furthermore, modeling studies suggest the interaction of AOH and AS with K433. Among the three mycotoxins examined, AS has the most favorable ∆G binding value, followed by α-ZEL and AOH. In this alternative binding pocket, AS forms hydrophilic interactions with W336, R426, K433, FAD, S1225, K1228, and S1234, while α-ZEL showed hydrophobic interactions with L147, A338, I1229, and A1231.
Notably, in our present investigations, FAD and Fe 2 S 2 were included in XO, and their proper partial charge distributions were calculated. In the recent study of Fan et al. [35], another binding pocket of AOH was also suggested during the blind docking calculations; nevertheless, this earlier evaluation was performed with the exclusion of FAD and Fe 2 S 2 .
Considering the results of incubation assays, mycotoxin depletion experiments, and modeling studies, AOH, AS, and α-ZEL seem to be allosteric inhibitors of the XO enzyme. It is also supported by the previous report of Fan et al. [35], where the non-competitive inhibitory mechanisms of AOH and AME were described.

Conclusions
In summary, the effects of 31 mycotoxins were examined on XO-catalyzed uric acid formation. Based on the in vitro enzyme incubation assays, mycotoxin depletion experiments, and molecular modeling studies, AOH, AS, and α-ZEL proved to be moderate allosteric inhibitors of XO. Our results also demonstrated that AOH, AS, and α-ZEL are inhibitors but not substrates of the enzyme. The above-listed observations make the suitability of AOH as a leading compound in the development of new XO inhibitors questionable, even if the structural modification results in the decreased toxicity of a derivative. Considering the typically nanomolar concentrations of mycotoxins in the circulation, it is reasonable to hypothesize that AOH, AS, and α-ZEL are not able to produce a clinically relevant decrease in uric acid levels and cannot interfere with the pharmacokinetics of drugs biotransformed by XO (e.g., 6-mercaptopurine). Because α-ZEL is a frequent contaminant in bovine milk, it is reasonable to hypothesize that this mycotoxin partly appears in milk in XO-bound form. Our results promote the deeper understanding of mycotoxin-XO interactions.

Xanthine Oxidase Assay
The in vitro XO assay was carried out as was previously reported [41,42]. Xanthine The same experimental design was applied with the following modifications to test the reversibility of the mycotoxin-induced inhibition of XO. In these experiments, incubates contained mycotoxin (50 µM), the enzyme (0.0012 U/mL), and increasing concentrations of xanthine (5, 10, or 25 µM). XO was preincubated (10 min, 700 rpm, 37 • C) with the mycotoxins; thereafter, the incubation (5 min, 700 rpm, 37 • C) was started with the addition of xanthine. Other experimental details remained unchanged.
After the concentrations of uric acid (c uric acid ) and xanthine (c xanthine ) were quantified in the samples, we calculated the rate of metabolite formation (R). R (%) = 100 × c uric acid / (c uric acid + c xanthine ), Then the R values of control samples were used as the bases of comparison (100%) when the inhibitory actions of mycotoxins were examined: Uric acid formation (%) = 100 × R inhibitor / R control , where R inhibitor and R control are the metabolite formation rates in the presence and absence of the inhibitor, respectively. IC 50 values were determined with sigmoidal (Hill1) fitting employing the OriginPro 8 program using these data (OriginLab Corporation, Northampton, MA, USA).

Mycotoxin Depletion Assays
In order to test the potential XO-catalyzed biotransformation of AOH, AS, and α-ZEL, these mycotoxins (each 5 µM) were incubated with XO enzyme (0.0012 U/mL) for 0 min, 30 min, and 60 min. Incubations were performed in the absence of xanthine. Other experimental details were the same as described in Section 4.2. Mycotoxin levels in the supernatants were quantified with HPLC-FLD (see details in Section 4.4).

Modeling Studies
The structures of AOH, AS, and α-ZEL were built in Maestro (Schrödinger, Maestro Schrödinger Release 2020-4). The energy minimization of the ligands was carried out with OpenBabel [49], using a steepest descent and a conjugate gradient algorithm. Gasteiger-Marsilli partial charges [50] were assigned to the ligand atoms in AutoDock Tools [51]. Flexibility was allowed on the ligands at all active torsions.
Atomic coordinates of XO were obtained from the Protein Data Bank (PDB) with PDB code 3eub [44], similar to our earlier study [41]. The amino acids of the target molecule were equipped with polar hydrogen atoms and Gasteiger-Marsilli partial charges in AutoDock Tools. The geometry and partial charges of the non-amino acid molecules, as the flavine-adenine dinucleotide (FAD), molibdopteroate, and the Fe 2 S 2 inorganic cluster were calculated by MOPAC [52] with a PM7 parametrization [53], and a gradient norm of 0.001. The reduced form of FAD was used, according to Kuwabara et al. [45].
Ligands were docked to XO using AutoDock 4.2.6 [51]. The number of grid points was set to 126 × 126 × 126 at a 0.850 A grid spacing. A blind docking [54,55] investigation was carried out, where the docking box covered the whole surface of the target molecule. The Lamarckian genetic algorithm was used for the global search. A hundred docking runs were executed for each ligand, and the resulting ligand conformations were ranked by their free energy [56]. The lower rank means the higher calculated free energy (∆G binding ). The docked ligand conformations demonstrated were employed for subsequent evaluations [57].

Statistical Analyses
The mean and standard error of the mean (± SEM) values are demonstrated in the figures and tables. Statistical evaluations (p < 0.05 and p < 0.01) were executed by applying one-way ANOVA and Tukey's post hoc test with SPSS Statistics software (IBM, Armonk, NY, USA).