Toxicology in Vitro 23 (2009) 83–89

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Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

The mycotoxin patulin, modulates tight junctions in caco-2 cells

John McLaughlin a, Daniel Lambert a, Philip J. Padfield a, Julian P.H. Burt b, Catherine A. O’Neill a,*

a University of Manchester, Faculty of Medicine and Health Sciences, Section of Gastrointestinal Sciences, Stopford Building, Oxford Road, Manchester M13 9PT, UKb School of Informatics, Bangor University, Dean Street, Bangor, Gwynedd, LL57, 1UT, UK

a r t i c l e i n f o

Article history:Received 15 August 2008Accepted 16 October 2008Available online 29 October 2008

Keywords:Tight junctionPatulinMycotoxinOccludinZO-1Paracellular permeabilityClaudin

0887-2333/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.tiv.2008.10.009

* Corresponding author. Tel.: +44 161 275 1819; faE-mail address: catherine.a.oneill@Manchester.ac.u

a b s t r a c t

The mycotoxin patulin is a common contaminant of fruit. Here, we demonstrate that patulin reduces thebarrier properties of the intestinal cell line, caco-2 by specific effects on tight junction components.Within 5 h of exposure to 100 lM toxin, the transepithelial electrical resistance of caco-2 monolayerswas reduced by approximately 95% and the monolayer became more permeable to FITC-labelled dextransof 4–40 kDa. Immunoblotting revealed occludin proteolysis and a significant reduction in ZO-1 levels.Patulin had no influence on claudin levels but marked changes in their distribution were observed. Thesedata indicate that patulin decreases the barrier properties of caco-2 monolayers by modulation of thetight junction.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction Upon ingestion, patulin must first interact with the gastro-

Patulin is a secondary metabolite of fungi belonging to the gen-era Penicillium, Byssochylamys and Aspergillus (Gonzalez-Osnaya etal., 2007; Piemontese et al., 2005). The mycotoxin is mainly foundas a contaminant of fruit, particularly, apples and apple products,but has also been found in vegetables, stored cheese and cerealproducts (Lafont et al., 1990; Lopez-Diaz et al., 1996; Lopez-Diazand Flannigan, 1997). Patulin can be present in fruit products inthe lm range (Burda, 1992; Ritieni, 2003; Gonzalez-Osnaya et al.,1971). Therefore, given the high risk of exposure to this mycotoxin,its potential effects have been investigated by many authors. Invivo animal studies have demonstrated patulin to be mutagenic,immunosuppressive, neurotoxic and teratogenic (Ciegler et al.,1971; Pfeiffer et al., 1998; Roll et al., 1990). Different mechanismsto explain the toxicity of patulin have been suggested but its cellu-lar effects still remain contraversial. In some studies, patulin wasfound to inhibit key biosynthetic enzymes such as RNA polymerase(Arafat et al., 1985) and inhibition of the Na+ K+ ATPase has alsobeen observed (Riley and Showker, 1991). More recently, Mahfoudet al. (2002) suggested that patulin toxicity could be due to its highreactivity with SH groups in the active site of tyrosine phosphatase.Clearly, any modification of such an important enzyme could beexpected to have major implications for cells and tissues. However,although this is one likely mechanism of action, the toxin may alsohave additional modes of toxicity.

ll rights reserved.

x: +44 161 206 1495.k (C.A. O’Neill).

intestinal epithelium. Studies on animals have demonstratedthat patulin is rapidly absorbed and causes mucosal ulcerationand inflammation (Speijers et al., 1988; McKinley and Carlton,1980a,b). These observations, suggest that patulin can affect gas-trointestinal functions and it is one of the few mycotoxins whichappears to have direct effects on the gut. Using a rat model, McKin-ley et al. (1982) demonstrated that daily doses for two weeks at75% of the LD50 resulted in histopathological alterations to thegut including ulceration and inflammation. In separate studies,Speijers et al. (1988) demonstrated that rats given patulin at1 mM in drinking water developed ulceration of the gastrointesti-nal tract. Thus, it appears that a key feature of the interaction ofpatulin with the gastrointestinal tract is an inflammatory reaction.The mechanisms by which patulin has proinflammatory effects onthe gut is just beginning to be elucidated. A recent study byMahfoud et al. (2002) reported that 100 lM patulin is able todecrease the transepithelial electrical resistance (TEER), a markerof epithelial barrier function, in the gut epithelial cell line, caco-2(Mahfoud et al, 2002). Epithelial barrier function is provided forthe most part by tight junctions. These are multi-protein com-plexes at the most apical pole of the junctional complex in epithe-lial (and endothelial) cells. Tight junctions seal the paracellularpathway and limit transport by this route to small, hydrophilicmolecules and ions (reviewed in VanItallie and Anderson, 2006).Opening of epithelial tight junctions could potentially allow thetransport of luminal antigens and/or bacteria which could lead toan inflammatory reaction. However, the molecular mechanism(s)underlying the patulin-induced reduction in TEER has not beenfully investigated until now.

84 J. McLaughlin et al. / Toxicology in Vitro 23 (2009) 83–89

Previously, we reported the modulation of the paracellularpathway by another food–borne mycotoxin, ochratoxin A. This tox-in was able to remove specific proteins from the tight junctioncomplex by a process involving oxidative events (McLaughlin etal., 2004; Lambert et al., 2007). In this study, we report that Patulinalters the properties of the tight junction in the model intestinalcell line caco-2, by a mechanism including proteolysis of specifictight junction components.

2. Methods

2.1. Materials

Cell culture media and supplements were purchased from invit-rogen (Paisley, UK). The anti-claudin, -occludin and -ZO-1 antibod-ies were purchased from Zymed (San Francisco, USA) and thehorseradish peroxidase conjugated secondary antibodies werefrom Bio-Rad (Hemel Hempstead, UK). FITC-labelled secondaryantibodies were purchased from Stratech Laboratories, Cambridge,UK. TranswellTM filters were purchased from Corning Life SciencesInc. (Appleton Woods, Birmingham, UK). All other reagents wereobtained from Sigma–Aldrich (Poole, UK) unless stated otherwise.

2.2. Cell culture

Caco-2 cells, obtained from the European cell Culture collection(ECACC No. 86010202, Porton Down, UK) were maintained in Dul-becco’s modified eagles medium (DMEM) containing high glucose(4.5 g/l), 10% Fetal calf serum, 2 mM Glutamine, 1% non-essentialamino-acids and 50 IU/ml penicillin/50 lg/ml streptomycin (Invit-rogen, Paisley, UK). These cells (passages 41–50) were cultured in75 cm2 T-flasks (Fisher, UK) at 37 �C in a 5% CO2, constant humidityenvironment with medium replaced three times a week. Monolay-ers were split (1:10) when they reached�80% confluency, typicallyonce per week.

2.3. Immunofluorescenct confocal microscopy

Caco-2 cells cultured on chamber slides (Nalge Nunc, IL) werefixed with 4% Paraformaldehyde for 10 min at room temperaturefor occludin, claudin 1 or ZO-1, or for 20 min in methanol at�20 �C for claudins 3 and 4. Cells were then washed three timesin tris buffered saline (TBS), treated with 0.5% Triton-x-100 inTBS for 5 min, washed in TBS three times and then blocked in 2%normal serum for 1 h at room temperature. Samples were incu-bated with the primary antibody (monoclonal anti-occludin, cloneOC-3F10, monoclonal anti-ZO-1, clone ZO-1-1A12, clone diluted at1:50, monoclonal anti-claudin 1, clone 2H10d10, monoclonal anti-claudin 4, clone 3E2C1 and polyclonal anti-claudin 3 diluted at1:100) diluted in TBS containing 2% serum) overnight at 4 �C.FITC-conjugated rabbit anti-mouse or goat anti-rabbit IgG wereused as the secondary antibodies. Cells were then washed threetimes in TBS. Images were captured using confocal laser scanningmicroscopy (Leica SP2 AOBS confocal scanning system). A galleryof 30 optical sections (1 lM) through the Z-plane was obtainedand composite images were processed using Confocal AssistantV4.02. X–Z sections were calculated using ‘Image J’ software.

2.4. Treatment of cells with patulin and matrixmetalloproteinaseinhibitors

Cell medium was replaced with phenol red free, fetal calf serumfree medium 24 h before the start of the experiment. Patulin wasdissolved in sterile distilled water. The toxin was added at a rangeof concentrations to either the apical or basolateral side of cells

growing in TranswellTM chambers. Zinc Sulphate or 1,10 phenan-throline were added at 0.25 mM to either side of cells for 15 minbefore the addition of patulin.

2.5. MTT assay

3-{4,5-dimethylthiazol-2-yl}diphenyltetrazolium bromide(MTT) was prepared as a stock solution of 5 mg/ml in phosphatebuffered saline.

Caco-2 cells were grown to 100% confluence in 96 well platesand then treated with Patulin for up to 12 h as described above.Treatment medium was then replaced by medium containing10% (v/v) MTT stock solution. Plates were incubated for 4 h at37 �C after which time the medium was replaced by dimethylsulphoxide. Plates were shaken to dissolve the purple formazanproduct and the absorbance of each well at 570 nm was read usinga Bio-Tek EL340 plate reader (Bio-Tek Instruments Inc., VT, USA).Cell viability was expressed as

Cell viability ¼ Treated wells A570=Untreated wells A570:

2.6. Statistical analysis

Data are expressed as mean ± standard deviation. Statisticalanalysis was performed using non-parametric Mann–Whitneywith p < 0.05 considered statistically significant. All experimentswere conducted n > 5.

2.7. Other methods

Preparation of total cell extract, SDS–PAGE, measurement ofparacellular permeability and measurement of TEER were carriedout exactly as described in McLaughlin et al. (2004).

3. Results

Initially, the effects of patulin on the TEER were studied. Patulinwas added to either the apical or basolateral side of the TranswellTM

chamber at a concentration of 100 lM and the TEER of the mono-layer was measured over 5 h (Fig. 1A). When mycotoxin was addedapically, a decrease in TEER was observed that was statistically sig-nificant after 1 h (69 ± 6% of control, p < 0.03, n = 8, Fig. 1A, s). Thereduction in TEER was such that at 5 h an approximately 95%reduction in TEER was observed. The TEER of untreated monolay-ers remained constant throughout this time period at approxi-mately 3000 X cm2 (Fig. 1A, d). When Patulin was added to thebasolateral side of the cells, a similar decline in TEER was observed,although the kinetics were slower (Fig. 1A, .). Patulin also exhib-ited dose-dependent effects on the TEER at 5 h. 100 lM mycotoxinadded apically produced the largest effect (Fig. 1B) but even at10 lM, the toxin reduced the TEER by around 50% of that of controlcells (Fig. 1B).

Mahfoud et al. (2002) demonstrated that Patulin was not toxicto caco-2 cells at 100 lM. To confirm this we examined the effectsof 100 lM Patulin on cell viability using the MTT assay. After 12 hincubation with the toxin, caco-2 monolayers remained confluentand, although very leaky, were found to be 100% viable (n = 5,Fig. 1C). In addition, when we observed patulin treated vs. un-treated cells by phase contrast microscopy, we could observe nodifference in the morphology of the cells (data not shown). Thisis in agreement with the study of Mahfoud et al. (2002) and ledus to speculate that the effect of patulin on TEER was specificrather than due to general toxicity and disruption of the monolayerintegrity.

To confirm an effect on the paracellular pathway, we examinedthe transport of FITC-labelled dextrans of different molecular

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Fig. 1. Effect of patulin on the transepithelial electrical resistance and viability of caco-2 cells. (A) Caco-2 cells were plated on 12 mm Transwell filters. 100 lM Patulin wasadded to either the apical (s) or basolateral (.) chamber and the electrical resistance was measured over time. Significant decreases in resistance were observed within 1 h(69 ± 6% of control values, p < 0.03, n = 8). (B) The effects of patulin on TEER are dose-dependent in caco-2 cells. (C) Caco-2 cells growing on 6 well culture plates were treatedfor up to 12 h with 100 lM patulin and the viability of the monolayer measured using the MTT assay. In control, untreated cells ( ) the viability was found to be virtually100% (n = 5). After treatment with 100 lM mycotoxin ( ), no change in the viability was observed and the cells were also fully viable (n = 5).

J. McLaughlin et al. / Toxicology in Vitro 23 (2009) 83–89 85

weights through caco-2 monolayers exposed to patulin for 5 h. Ta-ble 1 shows an approximately 10� increase in the paracellular fluxof 4 kDa dextrans in treated cells vs control cells, (n = 6). Previouslywe demonstrated that caco-2 cells do not normally permit passageof significant amounts of higher molecular weight dextrans(Mclaughlin et al., 2004). However, upon treatment with patulin,the monolayers also became permeable to small amounts of 10,20 and 40 kDa dextran species (Table 1).

Modulation of barrier properties is often mirrored by changes inspecific TJ protein components. We therefore, examined whether

Table 1Patulin increases the permeability of caco-2 monolayers to FITC-dextrans. Caco-2monolayers were treated with 100 lM patulin for 5 h. After this time 4,10,20 or40 kDa FITC-dextran was added to the apical side of the chamber and their flux to thebasolateral chamber measured after 4 h. Patulin caused an approximately 10-foldincrease in the paracellular transport of 4 kDa dextrans (p < 0.006, n = 6) in treatedcells vs. control cells. The paracellular pathway was also available to 10, 20 and40 kDa dextrans in treated but not control cells (n = 6).

FITC-dextranspecies (kDa)

Permeability of controlcells (ng/ml)

Permeability of treatedcells (ng/ml)

4 82.5 (±12.3) 1005.9 (±78.6)10 None detected 43.7 (±7.8)20 None detected 31.5 (±5.6)40 None detected 9.4 (±0.8)

the patulin-induced reduction of TEER could be due to changes inthe expression of particular TJ proteins.

In untreated caco-2 cells the majority of occludin resolves as asingle band at approximately 65 kDa (Fig. 2A). However, afterexposure of the cells to Patulin on either the apical or basolateralside, occludin resolves as two bands, the high molecular weightband at 65 kDa (HMW) plus a second band of slightly lower molec-ular weight (�51 kDa – LMW, Fig. 2A). In addition, there is a tem-poral progression in the appearance of LMW occludin, such that by5 h a significant proportion of the occludin exists as the LMW form(Fig. 2A and B). This shift between the two forms was variable be-tween experiments and between 30–50% of the occludin signalwas present in the LMW form at the end of 5 h. In addition, densi-tometry revealed a reduction in the total occludin signal with time(Fig. 2C). However, the loss of total occludin immunoreactivity wasalso variable between experiments. Typically, between 50–70% ofthe total occludin signal remained after 5 h exposure to patulin.This variability in absolute levels between experiments was notdue to differences in different batches of patulin or cells and ap-peared to be a genuine feature of the experimental system.

ZO-1 is a second member of the TJ complex that resolves asmultiple bands when analysed by immunoblotting due to phos-phorylation (Van Itallie et al., 1995; Antonetti et al., 1999). Whencells treated with Patulin were examined with respect to ZO-1, arapid decline was observed in the total levels of this protein. By3 h virtually no protein could be detected (Fig. 3A and B). Interest-

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Fig. 2. Patulin has effects on specific tight junction proteins. Confluent caco-2monolayers growing on TranswellTM filters were treated with patulin for up to 5 h.The cells were harvested into buffer containing SDS and the protein extractanalysed by SDS–PAGE followed by immunoblotting using antibodies to occludin,ZO-1 and claudins. (A) At t = 0, occludin appears as a single band of molecularweight 65 kDa (HMW). However, after treatment with patulin, a second lowermolecular weight (LMW) band is also evident. (B) Densitometry revealed that by5 h the level of LMW occludin is approximately 30% of the total signal (n = 5). (C)However, the total occludin signal also decreases and by 5 h only approximately60% of the signal remains when compared to control untreated cells (p = 0.023,n = 5).

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Fig. 3. Patulin causes loss of ZO-1 from cells. Confluent caco-2 monolayers weretreated with patulin for up to 5 h and then harvested into lysis buffer. The proteinextract was analysed by immunoblotting using antibodies against ZO-1 andclaudins 1, 3 or 4. (A) At t = 0, ZO-1 resolves as a major band at �200 kDa.However, over time the signal diminishes such that by 4 h only approximately 10%of the initial signal remains when analysed by densitometry (B) p = 0.02, n = 5. By5 h, no signal could be detected at all. (C) By contrast, the cellular levels of claudin 1were unaffected by treatment with patulin and the signal obtained remainedunchanged throughout the course of the experiment (n = 5). Similar results werealso obtained for claudins 3 and 4 (data not shown).

86 J. McLaughlin et al. / Toxicology in Vitro 23 (2009) 83–89

ingly, the level of claudins 1 did not change over the same timeperiod following treatment with patulin (Fig. 3C and D). Identicalresults were also obtained for claudins 3 and 4 whose levels alsoremained unchanged by treatment with patulin (data not shown).

Loss of ZO-1 and occludin might be reasonably expected tocause other changes to the TJ. Therefore, we calculated a Z-seriesof confocal images of caco-2 cells stained for occludin, ZO-1 orclaudin isoforms 1, 3 and 4. In untreated cells the tight junctionproteins were appropriately localised in a characteristic ‘chickenwire’ pattern consistent with their distribution in tight junctions(Fig. 4 ‘control’). In cells treated with patulin for 5 h (Fig. 5 ‘Trea-ted’) clear differences in the staining patterns were noted. Foroccludin (Fig. 4 ‘occludin’) the staining became more punctuate

and less intense consistent with a loss of protein form the cell.For claudins 1,3 and 4 the staining pattern became quite diffusecompared to the controls and large gaps were also observed tohave appeared in the ‘chicken wire’ pattern (Fig. 4 ‘Treated’). ForZO-1 the effects of the toxin were quite extreme and we could de-tect little fluorescence, probably due to its rapid removal from thecells upon treatment with the toxin (Fig. 4 ‘ZO-1’).

Appearance of a second lower molecular weight species ofoccludin has been documented by several authors. In some studies,this is due to proteolysis of occludin by matrix metalloproteinases(MMPs) (Lohmann et al., 2004; Wachtel et al., 1999). Since caco-2cells have been demonstrated to produce MMPs (Halvorsen et al.,2000; Wong et al., 2001), we investigated whether these enzymescould be inducing occludin proteolysis in response to patulin.

Zn2+ and 1,10 phenanthroline are inhibitors of MMPs (Lohmannet al., 2004; Wachtel et al., 1999). We investigated whether theseinhibitors could affect patulin-mediated changes to occludin incaco-2 cells. Confluent caco-2 cells growing on TranswellTM filters

Fig. 4. Patulin disrupts tight junction architecture. Caco-2 cells grown to confluenceon chamber slides were treated for 5 h with 100 lM Patulin. The cells were thenfixed and stained using analysed using antibodies to occludin claudin 1, claudin 3,claudin 4 and ZO-1. The left hand panels (control) represent untreated cells and theright hand panels (treated) represent patulin treated cells. All samples were viewedby confocal microscopy and a Z-series of images calculated for each. The toxin hasmajor effects on the localisation of claudins but does not affect their absolute levels.By contrast, the intensity of the signal for occludin and ZO-1 is reduced in treatedcells.

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Fig. 5. Inhibitors of matrixmetalloproteinases partially prevent generation of LMWoccludin. Cells growing on TranswellTM filters were either untreated, (lane 1) treatedwith patulin alone (lane 2) or pre-treated with phenanthroline (lane 3, A and E) or zincsulphate (lane 3, C and F) for 15 min before the addition of patulin. All the cells werethen incubated for 5 h then harvested and subjected to immunoblot using either ananti-occludin (A and C) or an anti-ZO-1 (E and F) antibody. The percentages of HMWvs LMW occludin were then calculated using densitometry (B and D). In untreatedcells, 100% of the occludin resolves as a single band corresponding to HMW occludin( ). In cells treated with patulin occludin resolves as two bands, the HMW ( ) andLMW ( ) form. Densitometry showed that HMW occludin accounted for approxi-mately 60% of the total signal. However in cells pre-treated with phenanthrolineHMW occludin predominates and is approximately 90% of the total signal (B, n = 5,p = 0.032). Zinc sulphate also protects HMW occludin to some degree and HMWoccludin represents approximately 70% of the total signal compared to cells treatedwith patulin alone (n = 5, p = 0.045). By contrast, matrix metalloproteinase inhibitorsdo not protect the cells from loss of ZO-1 and this is lost from the cells by treatmentwith patulin or treatment with patulin plus MMP inhibitors.

J. McLaughlin et al. / Toxicology in Vitro 23 (2009) 83–89 87

were incubated with either phenanthroline (0.25 mM) or zinc sul-phate (0.25 mM) for 15 min in serum free medium. The mediumwas then replaced with fresh medium containing patulin(100 lM) and either phenanthroline or zinc sulphate at the sameconcentrations. Cells were then harvested and analysed by westernblot using antibodies to occludin. At 0.25 mM, both inhibitors pre-vented proteolysis to some degree although neither fully protectedHMW occludin. 1,10 phenanthroline appeared to provide betterprotection against occludin proteolysis than zinc sulphate andapproximately 90% of the total occludin signal remained as the

88 J. McLaughlin et al. / Toxicology in Vitro 23 (2009) 83–89

HMW form following treatment with phenanthroline (Fig. 5A (lane3) and B). At higher concentrations, both inhibitors caused signifi-cant disturbance to the adhesion of the cells (data not shown). Bycontrast, neither Zinc2+ nor 1,10 phenanthroline could protect thecells from loss of ZO-1 immunoreactivity and this was lost fromthe cells rapidly after exposure to patulin (Fig. 5 E and F, lane 3).We were unable to investigate the effects of the inhibitors on TEERas they both significantly affected this property when applied tothe cells.

4. Discussion

In this study, we have examined the effects of a food–bornemycotoxin, Patulin, on the tight junction in the model intestinalcell line caco-2.

A previous study by others demonstrated that patulin-induceda decrease in the TEER of cultured caco-2 monolayers. However,the molecular mechanism behind this observation was not fullyinvestigated. In particular, it was not clear whether this reductionin TEER was due to changes in barrier function or in plasma mem-brane properties. The initial finding from our study is that Patulinis able to induce a rapid decrease in the barrier function of caco-2cell monolayers: as well as a time-dependent decrease in the TEER,patulin is able to increase the permeability of the monolayer toparacellular tracers. Caco-2 cells do not normally permit paracellu-lar passage of anything larger than 4 kDa (Mclaughlin et al., 2004).However, in the presence of patulin, even 40 kDa dextrans cancross the monolayer although the amounts are small. Increasedpermeability could be due to non-specific cytotoxicity and distur-bance of the monolayer. However, the observation that patulin didnot influence either cell morphology or cell viability at the concen-trations used argues strongly against this. Previously, we demon-strated fluxes of dextrans in response to complete opening oftight junctions using EGTA (Lambert et al., 2005). By comparison,the dextran fluxes observed in this study are significantly smallerthan those elicited by EGTA. Taken together these data indicatethat patulin causes a loosening of the tight junction rather than awholesale destruction.

The kinetics of the patulin-induced reduction in TEER are differ-ent depending on whether the toxin is added to the apical or baso-lateral side of the monolayer. Although the mechanism by whichpatulin gains entry into the cell is not known, it may rely on spe-cific transporters located within the cell membranes. The differ-ences in sensitivity to the toxin may possibly reflect polarity inthe composition of the apical and basolateral membranes ofcaco-2 cells with respect to transporter expression.

We have shown that the mechanism by which Patulin induces areduction in barrier function involves altering the expression lev-els and/or distribution of several tight junction proteins. Bothoccludin and ZO-1 are lost from the cell when analysed either bywestern blotting or by immunofluorescent microscopy. By con-trast, the distribution of claudins 1, 3 and 4 is severely affectedby patulin although the absolute levels of these proteins are unaf-fected as judged by western blotting. Since ZO-1 and occludin formthe link between the tight junction and the cytoskeleton, it is per-haps not surprising that the patulin-induced effects on these pro-teins lead to changes in the overall architecture of the tightjunction.

Numerous studies using cytokines, hormones and growth fac-tors have demonstrated the relationship between ZO-1 abundanceand tightness of the junction. In addition, a number of pathogensand their toxins have also been demonstrated to modify ZO-1 lev-els and epithelial permeability. For example, Entamoeba histolyticahas been demonstrated to displace ZO-1 from the junction in T84intestinal cells, leading to rapid degradation of the protein and de-

crease in the barrier properties of T84 cells (Leroy et al., 2000).Occludin has also been demonstrated to be involved in the integ-rity of tight junction barrier function in several studies (Balda etal., 1996, 2000). Therefore it seems reasonable to conclude thatthe loss of these proteins is at least in part responsible for thereduction in barrier function.

The effects of patulin on occludin appear to be proteolytic sincea second, lower molecular weight fragment of occludin is gener-ated in response to the toxin. Occludin proteolysis may involveMMPs because inhibitors of these protect occludin from patulin-mediated cleavage at least to some degree. At higher concentra-tions these inhibitors were toxic to the cells so unfortunately, wewere not able to demonstrate complete protection of occludin bythese inhibitors. Therefore, we cannot completely rule out other,perhaps proteolytic events leading to the generation of LMWoccludin. Unfortunately, it was not possible to determine whetherMMP inhibitors also protect the TEER from the effects of patulinbecause both inhibitors reduced the TEER of caco-2 monolayers(data not shown).

MMP mediated proteolysis of occludin has been observed inother studies (Lohmann et al., 2004; Wachtel et al., 1999). The tyro-sine phosphatase inhibitor, phenylarsine oxide, also produces aproteolyic fragment of occludin similar in size (51 kDa) to theone we observe in response to patulin (Lohmann et al., 2004). Inthis study, the mechanism of phenylarsine oxide induced occludinproteolysis was via MMPs. Interestingly, Mahfoud et al. (2002) sug-gested that patulin is a tyrosine phosphatase inhibitor since its ef-fects can be mimicked and potentiated by phenylarsineoxide.Mahfoud et al. (2002) attributed the inhibition of tyrosine phos-phatase to the particular efficacy with which patulin binds thiolgroups which are known to exist in the active site of tyrosine phos-phatases. At present it is not clear how phenylarsineoxide activatesMMPs to bring about occludin proteolysis. However, whatever thismechanism may be, it would appear from our study, that patulinmay operate by a similar mechanism.

Treatment of caco-2 cells with patulin results in reductions incellular ZO-1 levels and the overall levels of occludin. These effectsare not observed when cells are treated with tyrosine phosphataseinhibitors. Lohmann et al. (2004) observed no overall loss of occlu-din following treatment of cells with phenylarsineoxide and thelevels of ZO-1 also remained unchanged. The observation that cellstreated with patulin have reduced immunoreactivity for bothoccludin and ZO-1 suggests that patulin has other mechanisms oftight junction disruption alongside occludin proteolysis. Tightjunctions are well documented to be regulated by kinases and bothZO-1 and occludin undergo phosphorylation (Andreeva et al.,2001; Antonetti et al., 1999). Some serine and threonine phospho-rylases are known to be regulated by tyrosine phosphatases so itseems likely that the proposed tyrosine phosphatase inhibiting ef-fects of patulin may have further, currently unknown effects per-haps on signalling molecules which regulate tight junctions andother cellular functions. A further possible mechanism to explainthe effects of patulin is inhibition of protein synthesis. Patulinhas been demonstrated to downregulate protein synthesis in othersystems (Arafat and Musa, 1995; Miura et al., 1993). The observa-tion that cellular levels of occludin and ZO-1 decrease while clau-dins remain constant, could be a feature of differences in half livesof these proteins. Relatively few studies have investigated half livesof tight junction proteins and the results obtained from differentauthors are variable (Chen et al., 2000; Guo et al., 2005; Van Itallieet al., 2004). Chen et al. (2000) observed half lives of 11 h and 5 hfor occludin and ZO-1, respectively in MDCK cells. Guo et al. (2005)on the other hand, found a half life for occludin of 120 min in anintestinal epithelial cell line. Therefore it seems that the half lifemay vary depending on the cell type. However, in general it ap-pears that claudins may have longer half lives than either occludin

J. McLaughlin et al. / Toxicology in Vitro 23 (2009) 83–89 89

or ZO-1 and half lives as long as 14 h have been quoted for certainclaudin isoforms (Van Itallie et al., 2004).

In summary, the mycotoxin, patulin causes a rapid reduction incaco-2 barrier function by perturbation of the tight junction com-plex. This may well explain some of the observed in vivo effects ofthis toxin on the gastrointestinal tract.

Acknowledgement

The authors wish to thank all members of the GastrointestinalSciences group, University of Manchester, for contributing to help-ful discussions.

This work was funded by the The Digestive Disorders Founda-tion (Grant Ref. RA/3), North Manchester Fund.

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