Interactions of Mycotoxin Alternariol with Cytochrome P450 Enzymes and OATP Transporters

Alternariol (AOH) is an emerging mycotoxin produced by Alternaria strains. The acute toxicity of the mycotoxin is low; however, chronic exposure to AOH may result in the development of endocrine disruptor and/or carcinogenic effects. The toxicokinetic properties of AOH have barely been characterized. Therefore, in this study, we aimed to investigate its interactions with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters employing in vitro enzyme assays and OATP overexpressing cells, respectively. Our results demonstrated that AOH is a strong inhibitor of CYP1A2 (IC50 = 0.15 μM) and CYP2C9 (IC50 = 7.4 μM). Based on the AOH depletion assays in the presence of CYP enzymes, CYP1A2 is mainly involved, while CYP2C19 is moderately involved in the CYP-catalyzed biotransformation of the mycotoxin. AOH proved to be a strong inhibitor of each OATP transporter examined (IC50 = 1.9 to 5.4 μM). In addition, both direct and indirect assays suggest the involvement of OATP1B1 in the cellular uptake of the mycotoxin. These findings promote the deeper understanding of certain toxicokinetic interactions of AOH.

Cytochrome P450 (CYP) enzymes contribute to approximately 75% of the phase I biotransformation of xenobiotics [11]. The five human CYP isoforms, which catalyze the majority of these reactions, are CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 [11,12]. Since CYP-catalyzed oxidations typically affect the toxicokinetic and toxicodynamic properties of xenobiotics, these enzymes frequently play a major role in the detoxification or toxic activation of certain compounds. The interactions of some mycotoxins (including aflatoxin B1, ochratoxin A, and zearalenone) with CYP enzymes have been reported [13][14][15], while only limited data are available regarding AOH. Based on a previous study, AOH is biotransformed by CYP1A1 and 1A2 enzymes and the mycotoxin can increase the expression of CYP1A1 through the activation of aryl hydrocarbon receptor [16].
Solute carrier organic anion transporting polypeptides (OATPs), present in the cell membrane of epithelial and endothelial cells of the human body, are transporters that mediate the cellular uptake of large (>300 Da), negatively charged, or amphipathic organic molecules [17]. Steroid and thyroid hormones, bile acids, prostaglandins, and bilirubin are typical endogenous substrates of OATPs [18]. Furthermore, multispecific members of the OATP family (OATP1A2, OATP1B1, OATP1B3, and OATP2B1) also recognize various xenobiotics, such as drugs (e.g., statins and certain chemotherapeutics), food components (e.g., flavonoids), and toxins (e.g., α-amanitin and ochratoxin A) [19][20][21]. OATPs 1B1, 1B3, and 2B1 are expressed in human hepatocytes and contribute to the hepatic clearance of their substrates [22]. Furthermore, OATP2B1 and OATP1A2 appear in enterocytes and in the endothelial cells of the blood-brain barrier [23]. These multispecific OATPs are key players in the tissue uptake and clearance of drugs, nutrients, and xenobiotics. Previous studies demonstrated the interaction of the Aspergillus mycotoxin ochratoxin A with OATPs [21,24]; however, the effect of AOH on these carriers has not yet been characterized.
In this study, we aimed to investigate the interactions of AOH with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters employing in vitro assays with human recombinant enzymes and OATP overexpressing cells, respectively. Our results demonstrated that AOH is a potent inhibitor of CYP1A2, CYP2C9, and the OATPs tested. Furthermore, AOH seems to be a potential substrate for CYP1A2 and OATP1B1. Our findings contribute to the deeper understanding of certain toxicokinetic interactions of AOH.
Solvent controls were also applied in each experiment, DMSO concentrations did not exceed 0.13 v/v%. IC 50 values were calculated by sigmoidal fitting (Hill1), using the Origin (version 2018, OriginLab Corporation, Northampton, MA, USA) software.

Generation and Maintenance of OATP Overexpressing Cell Lines
A431 cell lines overexpressing human OATPs (1A2, 1B1, 1B3, or 2B1) and their mock transfected controls were generated earlier [29,30]. A431 cells expressed no or negligible OATPs compared to the overexpressing cell lines. Cells were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C, with 5% CO 2 . OATP expression was regularly monitored (based on the transport activity of transfected cells), and the cells were used up to 20 passages.

Testing the Involvement of OATP1B1 in the Cellular Uptake of AOH
The A431 cell line overexpressing OATP1B1 or mock transfected control were trypsinized, and counted with automated cell counter (TC10, BIORAD, Hercules, CA, USA). The cells were washed with 1 mL buffer (pH 5.5; described above) and centrifuged for 5 min at 1500 g. After the supernatant was removed, 2 × 10 6 cells in 50 µL volume of the same buffer were handed out into separate Eppendorf tubes, then 50 µL of AOH solutions (diluted in buffer, pH 5.5) was added (final concentrations: 2, 5, or 10 µM). The cells were incubated for 15 min at 37 • C in shaking water bath. The reaction was stopped with 1 mL ice-cold PBS, after which the tubes were centrifuged for 5 min at 1500 g and 4 • C. The supernatant was removed, and the cells were washed with 1 mL ice-cold PBS, then the cell pellet was stored at −80 • C until analyses.
Before HPLC measurements, cell pellets were dissolved in 100 µL of 1 M NaOH solution with vigorous vortexing. After 15 min sonication, the solution was neutralized with 100 µL of 1 M HCl, then 200 µL acetonitrile was added. Samples were vortexed and centrifuged for 10 min at 14,000 g and 4 • C. Thereafter, the supernatant was directly analyzed with HPLC-FLD (see details in Section 2.7).

Testing AOH-OATP Interaction in Competitive Counterflow and Efflux Assays
The assays were performed as described recently [33]. Cells were seeded on 96-well plates one day prior to the measurement. After washing three times with PBS at room temperature, cells were incubated for 15 min with 8-acetoxy-1,3,6-trisulfopyrene (Ace, 5 µM). After this "preloading" stage, the supernatant was removed, and the cells were incubated with the same amount of the probe (Counterflow assay) or without the probe in buffer pH 5.5 (Efflux assay) in the absence and the presence of AOH for further 20 min. Fixed concentrations of AOH were selected based on the IC 50 values determined in the transport inhibition assay. Estrone-3-sulfate (E1S, 50 µM) and formaldehyde (PFA, 0.5%) served as reference substrate and inhibitor, respectively. Cells were washed three times with ice-cold PBS, then NaOH solution (0.1 N, 200 µL/well) was added. After 20 min, fluorescence was measured with an Enspire plate reader in Ex/Em wavelengths 460/510 nm. Bottom reading was applied with two horizontal (X) and two vertical (Y) points, measurement height was 3 mm with 200 flashes. Fluorescence was compared to the preloaded control incubated with Ace alone, considered as 100%.

Data Analyses
Data display mean and standard error of the mean (±SEM) values, derived from at least three independent experiments. Statistical significance (p < 0.05 and p < 0.01) was evaluated based on one-way ANOVA test followed by Tukey's post hoc test (version 21, IBM SPSS Statistics, Armonk, NY, USA).

Interaction of AOH with CYP Enzymes
The interactions of AOH with CYP enzymes were investigated employing 0 to 50 µM mycotoxin concentrations. AOH induced the concentration-dependent inhibition of the CYP enzymes tested, except CYP2D6-catalyzed dextromethorphan demethylation, which was not affected even by 50 µM AOH (Figure 1). It is not surprising because typically amines, which can be protonated under physiological circumstances, can interact with CYP2D6 [34]. Among the CYP enzymes examined, AOH proved to be the most potent inhibitor of CYP1A2 (IC 50 = 0.15 µM), causing statistically significant (p < 0.01) and close to complete inhibitions at 0.05 µM and 10 µM concentrations, respectively. Furthermore, the mycotoxin strongly inhibited the CYP2C9 enzyme (IC 50 = 7.4 µM), leading to more than an 80% decrease in the product formation at 50 µM concentration. At 5 µM concentration, AOH significantly (p < 0.01) inhibited CYP2C19; nevertheless, the highest AOH concentration applied (50 µM) caused only a 55% decrease in 4-hydroxymephenytoin production ( Figure 1). Finally, AOH inhibited CYP3A4 enzyme; however, the mycotoxin did not cause a further relevant decrease in the metabolite formation above 10 µM concentration (approximately 40% inhibition) (Figure 1).  To test the potential biotransformation of the mycotoxin by CYP enzymes, AOH was incubated with CYP1A2, 2C9, 2C19, 2D6, and 3A4. Even after a 120 min incubation, our results demonstrated no relevant changes in AOH levels in the presence of CYP2C9, CYP2D6, and CYP3A4. These incubates showed very similar data to the control (CYPnull) (Figure 2). CYP2C19 caused only a slight decrease in AOH levels, showing a statistically significant difference (p < 0.01) only after a 120 min incubation. However, CYP1A2 enzyme induced a concentration-dependent decrease in AOH concentrations, leading to a 16% and 25% depletion of AOH after 60 min and 120 min incubations, respectively (Figure 2).
Based on our current knowledge, this is the first study to examine the inhibitory action of AOH on CYP enzymes. Considering the above-listed data, AOH is a weak inhibitor of CYP2C19 and CYP3A4, while it exerts very strong and moderately strong inhibition on CYP1A2 and CYP2C9 enzymes, respectively ( Figure 1). Furthermore, based on these ob- Urolithins are colon metabolites of ellagitannins [35]; they have a very similar chemical structure to Alternaria mycotoxins. In agreement with our results, a previous study demonstrated the inhibitory effects of urolithins (e.g., urolithin A, B, and C) on CYP1A2 and CYP1B1 enzymes [36].
To test the potential biotransformation of the mycotoxin by CYP enzymes, AOH was incubated with CYP1A2, 2C9, 2C19, 2D6, and 3A4. Even after a 120 min incubation, our results demonstrated no relevant changes in AOH levels in the presence of CYP2C9, CYP2D6, and CYP3A4. These incubates showed very similar data to the control (CYPnull) (Figure 2). CYP2C19 caused only a slight decrease in AOH levels, showing a statistically significant difference (p < 0.01) only after a 120 min incubation. However, CYP1A2 enzyme induced a concentration-dependent decrease in AOH concentrations, leading to a 16% and 25% depletion of AOH after 60 min and 120 min incubations, respectively (Figure 2). Statistical evaluation was performed compared to the incubation with the same amount of CYPnull (15 mg/mL), which is a permeabilized and stabilized dried yeast powder (produced using an empty vector) and processed identically to CypExpress Cytochrome P450 products.

Interaction of AOH with OATP Transporters
First, the potential inhibitory impact of AOH on OATP transporters was examined, employing pyranine or sulforhodamine 101, previously documented as fluorescent test substrates [29,31]. As demonstrated in Figure 3, AOH proved to be a potent inhibitor of each OATP tested, showing the strongest inhibitory actions on OATP2B1 and OATP1B1 (IC50 values were 1.9 μM and 2.0 μM, respectively), while slightly lower effects were observed regarding OATP1B3 and OATP1A2 (IC50 values were 4.1 μM and 5.4 μM, respectively). Based on our current knowledge, this is the first study to examine the inhibitory action of AOH on CYP enzymes. Considering the above-listed data, AOH is a weak inhibitor of CYP2C19 and CYP3A4, while it exerts very strong and moderately strong inhibition on CYP1A2 and CYP2C9 enzymes, respectively ( Figure 1). Furthermore, based on these observations, we did not see the relevant involvement of CYP2C9, CYP2D6, and CYP3A4 in the metabolism of AOH. On the other hand, CYP2C19 and mainly CYP1A2 seem to be important CYP enzymes regarding the biotransformation of this mycotoxin (Figure 2). In an earlier study, the CYP-catalyzed biotransformation of AOH was examined using pooled human hepatic microsomes, suggesting the CYP1A-mediated metabolism of AOH with the minor involvement of CYP2C19 and CYP3A4 [38]. Our study confirms the importance of the CYP1A2 and CYP2C19 enzymes.

Interaction of AOH with OATP Transporters
First, the potential inhibitory impact of AOH on OATP transporters was examined, employing pyranine or sulforhodamine 101, previously documented as fluorescent test substrates [29,31]. As demonstrated in Figure 3, AOH proved to be a potent inhibitor of each OATP tested, showing the strongest inhibitory actions on OATP2B1 and OATP1B1 (IC 50 values were 1.9 µM and 2.0 µM, respectively), while slightly lower effects were observed regarding OATP1B3 and OATP1A2 (IC 50 values were 4.1 µM and 5.4 µM, respectively).
First, the potential inhibitory impact of AOH on OATP transporters was examined, employing pyranine or sulforhodamine 101, previously documented as fluorescent test substrates [29,31]. As demonstrated in Figure 3, AOH proved to be a potent inhibitor of each OATP tested, showing the strongest inhibitory actions on OATP2B1 and OATP1B1 (IC50 values were 1.9 μM and 2.0 μM, respectively), while slightly lower effects were observed regarding OATP1B3 and OATP1A2 (IC50 values were 4.1 μM and 5.4 μM, respectively). To determine whether AOH is a potential transported substrate of OATP1B1, AOH uptake was investigated in OATP expressing and mock transfected A431 cells. In these To determine whether AOH is a potential transported substrate of OATP1B1, AOH uptake was investigated in OATP expressing and mock transfected A431 cells. In these experiments, cells were incubated with increasing concentrations of AOH, then the cellular mycotoxin concentration was quantified by HPLC-FLD. OATP1B1 expressing cells showed higher cellular AOH levels compared to mock cells (Figure 4), suggesting the OATP1B1mediated transport of the mycotoxin. However, likely due to the rapid and significant membrane association of the mycotoxin, we also found relatively high levels of AOH in the mock cells. Therefore, to confirm the involvement of OATP1B1 in the cellular uptake of AOH, further experiments were performed. experiments, cells were incubated with increasing concentrations of AOH, then the cellular mycotoxin concentration was quantified by HPLC-FLD. OATP1B1 expressing cells showed higher cellular AOH levels compared to mock cells (Figure 4), suggesting the OATP1B1-mediated transport of the mycotoxin. However, likely due to the rapid and significant membrane association of the mycotoxin, we also found relatively high levels of AOH in the mock cells. Therefore, to confirm the involvement of OATP1B1 in the cellular uptake of AOH, further experiments were performed. Recently, a novel method, termed as competitive counterflow (CCF), was established that can distinguish transported substrates and non-transported inhibitors of OATP2B1 or OATP1A2 [39,40]. The method is based on the exchanger function of OATPs, by which even large organic substrates can be swapped between the opposite sites of the cell membrane. Lately, using the fluorogenic substrate 8-acetoxy-1,3,6-trisulfopyrene (Ace), we developed the CCF for OATP1B1 [33]. The method comprises two steps. First, the cells are loaded with Ace to a steady state (by the function of OATP1B1). Next, the investigated Recently, a novel method, termed as competitive counterflow (CCF), was established that can distinguish transported substrates and non-transported inhibitors of OATP2B1 or OATP1A2 [39,40]. The method is based on the exchanger function of OATPs, by which even large organic substrates can be swapped between the opposite sites of the cell membrane. Lately, using the fluorogenic substrate 8-acetoxy-1,3,6-trisulfopyrene (Ace), we developed the CCF for OATP1B1 [33]. The method comprises two steps. First, the cells are loaded with Ace to a steady state (by the function of OATP1B1). Next, the investigated compound (AOH in the current study) is added. The addition of a substrate (E1S used here as a reference substrate) at high enough amounts (ten-fold of its IC 50 value) triggers the efflux of the probe, leading to a decrease in the fluorescence signal in the CCF (Figure 5, top) and not inhibiting efflux ( Figure 5, bottom). On the other hand, non-transported inhibitors (we used formaldehyde, a chemical crosslinker here) blocks the efflux of the probe (Figure 5, bottom) and there is no change in the fluorescence in the CCF. Figure 5 indicates that AOH added in excess (already at two-fold of its IC 50 ) generated a decrease in the fluorescence signal in the CCF, while not inhibiting efflux. These observations indicate that AOH is a potential substrate of OATP1B1.
Metabolites 2023, 13, x FOR PEER REVIEW 9 of 12 E1S ( Figure 5). These observations strongly suggest that AOH is not only an inhibitor but also a substrate of the OATP1B1 transporter.

Conclusions
In summary, CYP enzymes and OATP transporters are involved in the pharmacokinetics and/or toxicokinetics of several drugs and xenobiotics. Since toxicokinetic interactions of mycotoxin AOH have barely been characterized, in the current study, we aimed to investigate its interactions with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters. Considering the low oral bioavailability of AOH as well as its typically nanomolar concentrations in the circulation [4,9,10], it seems Only limited information is available regarding the interactions of mycotoxins with OATP transporters. Nevertheless, based on previous studies, certain OATPs may have significant involvement in the tissue uptake and accumulation of mycotoxins ochratoxin A [21,24] and deoxynivalenol [41]. In our study, AOH showed strong, concentrationdependent inhibitory action on each OATP transporter tested (IC 50 = 1.9 to 5.4 µM). Therefore, the potential involvement of OATP1B1 in the cellular uptake of the mycotoxin was also examined. We measured higher concentrations of AOH in OATP1B1 expressing vs. the mock cells (Figure 4). In addition, counterflow and efflux assays were performed, where we could distinguish non-transported inhibitors and transported substrates of OATP1B1 [33]. In these experiments, AOH behaved similarly to the reference substrate E1S ( Figure 5). These observations strongly suggest that AOH is not only an inhibitor but also a substrate of the OATP1B1 transporter.

Conclusions
In summary, CYP enzymes and OATP transporters are involved in the pharmacokinetics and/or toxicokinetics of several drugs and xenobiotics. Since toxicokinetic interactions of mycotoxin AOH have barely been characterized, in the current study, we aimed to investigate its interactions with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters. Considering the low oral bioavailability of AOH as well as its typically nanomolar concentrations in the circulation [4,9,10], it seems to be unlikely that AOH can considerably interfere with the CYP-catalyzed biotransformation and/or the OATP-mediated transport of drugs. However, AOH concentration may be significantly higher in the intestinal tract than in the circulation, suggesting the possible inhibition of OATP2B1-mediated absorption of certain compounds. In addition, the involvement of OATPs in the tissue uptake of mycotoxin AOH may have toxicological importance. Considering the endocrine disruptor effects of AOH [4,7], further studies are reasonable to test the potential role of OATP transporters in the uptake of the mycotoxins into the target organs.