International Journal of Molecular Sciences Article Interaction of Mycotoxin Alternariol with Serum Albumin Eszter Fliszár-Nyúl 1,2, Beáta Lemli 2,3,Sándor Kunsági-Máté 2,4, Luca Dellafiora 5 , Chiara Dall’Asta 5, Gabriele Cruciani 6,Gábor Peth˝o 1,7 and Miklós Poór 1,2,* 1 Department of Pharmacology, Faculty of Pharmacy, University of Pécs, Szigeti út 12, H-7642 Pécs; Hungary; [email protected] (E.F.-N.); [email protected] (G.P.) 2 János Szentágothai Research Centre, University of Pécs, Ifjúság útja 20, H-7642 Pécs; Hungary; [email protected] (B.L.); [email protected] (S.K.-M.) 3 Institute of Organic and Medicinal Chemistry, Medical School, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary 4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Pécs, Rókus utca 2, H-7642 Pécs, Hungary 5 Department of Food and Drug, University of Parma, Via G.P. 7 Usberti 17/A, 43124 Parma, Italy; luca.dellafi[email protected] (L.D.); [email protected] (C.D.) 6 Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy; [email protected] 7 Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary * Correspondence: [email protected]; Tel.: +3672-536-000 (ext. 35052) Received: 19 April 2019; Accepted: 8 May 2019; Published: 12 May 2019 Abstract: Alternariol (AOH) is a mycotoxin produced by Alternaria species. In vitro studies suggest the genotoxic, mutagenic, and endocrine disruptor effects of AOH, and an increased incidence of esophageal cancer has been reported related to higher AOH exposure. Human serum albumin (HSA) is the most abundant plasma protein in the circulation, it is able to affect toxicokinetic properties of numerous xenobiotics. HSA forms stable complexes with several mycotoxins, however, the interaction of AOH with albumin has not been examined. In this study, the complex formation of AOH with HSA was tested, employing fluorescence spectroscopy, ultrafiltration, and molecular modeling. Each spectroscopic measurement shows the formation of stable AOH-HSA complexes (K = 4 105 L/mol). Investigations with site markers (in spectroscopic and ultrafiltration models) as × well as modeling studies suggest that AOH occupies Sudlow’s site I as a high-affinity binding site in HSA. The binding affinity of AOH towards bovine, porcine, and rat albumins was also tested, suggesting that AOH binds to rat albumin with considerably higher affinity than other albumins tested. Our results demonstrate the strong interaction of AOH with serum albumins, suggesting the potential in vivo importance of these interactions. Keywords: alternariol; serum albumin; albumin-ligand interaction; binding site; fluorescence spectroscopy 1. Introduction Mycotoxins are secondary metabolites of filamentous fungi, exerting adverse effects in humans and animals [1,2]. Alternaria species contaminate unprocessed food (e.g., cereal crops, grape, tomato, oilseeds, and citrus fruits) [3–5] and processed products (e.g., apple juice, wine, and tomato paste) [6–8]. Alternaria molds are responsible for the post-harvest decay of agricultural products, even during refrigerated transport and storage, due to their ability to grow even at low temperature [9]. Alternariol (AOH; Figure1) is a dibenzo- α-pyrone mycotoxin produced by Alternaria species, it shows Int. J. Mol. Sci. 2019, 20, 2352; doi:10.3390/ijms20092352 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 2352 2 of 18 Int. J. Mol. Sci. 2019, 20, x 2 of 19 structural similarity with known xenoestrogens such as urolithins and genistein [10]. The acute toxicityacute toxicity of AOH of isAOH considered is considered to be low to [ 11be], andlow its[11], chronic and itsin vivochronictoxicity in vivo is also toxicity controversial. is also Increasedcontroversial. incidence Increased of incidence esophageal of esophageal cancer has canc beener reportedhas been reported in some in areas some with areas higher with higher AOH exposureAOH exposure [12,13 ].[12,13]. Genotoxic, Genotoxic, mutagenic, mutagenic, and endocrineand endocrine disruptor disruptor effects effects of AOH of AOH have alsohave beenalso describedbeen described in several in severalin vitro in vitrostudies. studies. AOH AOH inhibits inhibits the the catalytic catalytic activity activity of topoisomeraseof topoisomerase I and I and II enzymesII enzymes [5 ,[5,14,15].14,15]. Furthermore, Furthermore, Tiessen Tiessen et et al. al. [ 16[16]] demonstrated demonstrated thatthat AOHAOH (as(as aa pro-oxidant)pro-oxidant) can change the cellular redox status, however, no oxidativeoxidative stress-related DNA damage was observed in cell culture. AOH has weak estrogenic and androgen androgenicic/antiandrogenic/antiandrogenic effects, effects, however, the dietary exposure results in its relatively low plasma concentrations (5–10 nM) which makes questionable the in vivo relevance ofof thethe endocrineendocrine disruptordisruptor eeffectsffects ofof AOHAOH [[10,11,17,18].10,11,17,18]. Human serum albu albuminmin (HSA) is the most abundant plasma protein in the human circulation. HSA maintainsmaintains thethe oncotic oncotic pressure pressure of of blood blood regulating regulating the the volume volume of theof intravascularthe intravascular space, space, as well as aswell it hasas it bu hasffer, buffer, pseudo–enzymatic, pseudo–enzymatic, and antioxidant and antioxidant functions functions [19]. Furthermore, [19]. Furthermore, HSA formsHSA stableforms complexesstable complexes with several with several endogenous endogenous and exogenous and exogenous compounds, compounds, including including ions, fattyions, acids,fatty acids, drug molecules,drug molecules, and toxic and toxic xenobiotics xenobiotics [19–22 [19–22].]. The formationThe formation of high-a of high-affinityffinity HSA-ligand HSA-ligand complexes complexes can acanffect affect the membrane the membrane transport, transport, distribution, distribution, and elimination and elimination of ligand of moleculesligand molecules [23,24]. HSA[23,24]. contains HSA threecontains domains three domains (I–III), each (I–III), includes each includes two subdomains two subdomains (called (called A and A B). and Sudlow’s B). Sudlow’s site I site (SSI) I (SSI) and Sudlow’sand Sudlow’s site site II (SSII) II (SSII) are are well-known well-known drug drug binding binding sites sites in in HSA HSA [ 19[19,25],,25], however, however, recentrecent studiesstudies revealed the the importance importance of of Heme Heme binding binding site site (or (or FA1) FA1) [26]. [26 ].SSI SSI is an is anapolar apolar cavity cavity in subdomain in subdomain IIA IIAwhich which includes includes the thesole sole tryptophan tryptophan residue residue of ofHSA HSA (Trp-214), (Trp-214), SSII SSII is is located located in in subdomain subdomain IIIA, while the HemeHeme sitesite isis anan apolarapolar pocketpocket inin subdomainsubdomain IBIB [[19].19]. Previous studies highlighted that several mycotoxins (e.g., ochratoxins, citrinin, andand zearalenone) form highly stable complexes with HSA [[27–30].27–30]. However,However, based based on on our our current current knowledge, knowledge, the interaction the interaction of AOH of with AOH serum with albumin serum hasalbumin not yethas been not yet investigated. been investigated. Since the Since albumin-binding the albumin-binding of xenobiotics of xenobiotics may be may a relevant be a relevant detail regardingdetail regarding their toxicokinetics, their toxicokinetics, it is important it is import to determineant to determine the affinity the of formedaffinity complexes of formed and complexes identify theirand identify high-affi theirnity high-affinity binding site(s) binding on HSA. site(s) on HSA. In thisthis study,study, thethe interaction interaction of of AOH AOH with with albumin albumin was was investigated. investigated. The The binding binding constant constant (K )(K of) AOH-HSAof AOH-HSA complex complex was was quantified quantified based based on three on spectroscopicthree spectroscopic methods, methods, and binding and binding site of AOH site of in HSAAOH was in HSA determined was determined by site markers by site using markers spectroscopic using spectroscopic and ultrafiltration and ultrafiltration techniques. techniques. To get a deeper To insightget a deeper into the insight binding into position the ofbinding AOH, position modeling of studies AOH, were modeling performed. studies Furthermore, were performed. species diFurthermore,fferences of AOH-albuminspecies differences interaction of AOH-albumin were also testedinteraction with bovinewere also (BSA), tested porcine with (PSA),bovine and(BSA), rat (RSA)porcine serum (PSA), albumins. and rat Our (RSA) results serum demonstrate albumins. that AOHOur results forms high-ademonstrateffinity complexes that AOH with forms HSA (K = 4 105 L/mol), during which AOH occupies SSI as binding site. Stability of AOH-albumin high-affinity× complexes with HSA (K = 4 × 105 L/mol), during which AOH occupies SSI as binding complexessite. Stability are of similar AOH-albumin (regarding complexes HSA, BSA, are and simi PSA),lar (regarding while AOH HSA, binds BSA, to RSAand PSA), with significantlywhile AOH higherbinds to a ffiRSAnity with compared significantly to the otherhigher albumins affinity compared tested. to the other albumins tested. Figure 1. Chemical structure of alternariol. 2. Results 2. Results 2.1. Fluorescence Spectra of AOH in the Absence and Presence of HSA 2.1. Fluorescence