Elsevier

Toxicology Letters

Volumes 140–141, 11 April 2003, Pages 465-476
Toxicology Letters

Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2)

https://doi.org/10.1016/S0378-4274(03)00043-2Get rights and content

Abstract

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is absorbed from small intestine and, in plasma, binds to serum albumin. Prolonged half-live results from reabsorption by proximal tubules and enterohepatic circulation. The mechanism whereby OTA crosses intestine was investigated by means of a cell culture system consisting of Caco-2 cells, as in vitro model of human intestinal epithelium. Cytotoxicity assays on proliferating Caco-2 cells showed that 0.4 μM OTA inhibits MTT reduction by 50%. Transepithelial transport and intracellular accumulation of OTA were studied in Caco-2 cells, differentiated in bicameral inserts. At pH 7.4, OTA is transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. Conditions closer to in vivo situation in duodenum (AP pH 6.0, BL pH 7.4) increase intracellular accumulation and transepithelial transport. AP to BL transport becomes higher than BL to AP transport, suggesting OTA absorption. Addition of serum albumin in BL compartment further increases OTA absorption across Caco-2 cells and suggests that in vivo OTA transport from serosal to luminal side of enterocytes is prevented, due to its binding to plasma proteins. Competition experiments showed that carrier systems for large neutral amino acids, H+/dipeptides cotransporter, organic anion (p-aminohippurate) carrier and organic anion transporter (oatp) are not implicated in OTA transport across Caco-2 cells, in contrast to what was reported in kidney and liver. AP and BL transport and intracellular accumulation of OTA are increased in the presence of non specific inhibitors of MRPs (indomethacin, genistein and probenecid) and of 1-chloro-2,4-dinitrobenzene (biotransformed into 2,4-dinitrophenyl-gluthatione, a specific inhibitor of MRPs), but are affected by verapamil, an inhibitor of P-gp. This suggests that the multidrug resistance-associated protein (MRP2) could be implicated in transepithelial transport. Therefore, absorption of OTA across the intestinal mucosa would be limited thanks to its excretion through MRP2 at the apical pole of enterocytes.

Introduction

Ochratoxin A (OTA), a mycotoxin mainly produced by two ubiquitous species of fungi, Aspergillus alutaceus and Penicillium verrucosum, contains a dihydroisocoumarin moiety linked to a l-phenylalanine residue. OTA is a frequent contaminant in various plant products and animal feeds and is also found in human and animal fluids and tissues (Pohland et al., 1992). OTA has a number of toxic effects in mammals, the most notable being nephrotoxicity. It has been implicated in the human Balkan Endemic Nephropathy and in the porcine nephropathy and is also immunosuppressive, teratogenic, genotoxic and carcinogenic (Steyn, 1993). Molecular toxicity would result from competition with phenylalanine for protein synthesis, promotion of lipid peroxidation, inhibition of mitochondrial ATP production, as well as production of DNA adducts (Dirheimer and Creppy, 1991).

Kumagai (1998) showed that OTA is absorbed from the jejunum of rats, even when its level is higher in plasma than in jejunal lumen and that OTA uptake increases when its pH is decreased. In different species including humans, 97–100% of the toxin is bound to plasma proteins (Hagelberg et al., 1989), which facilitates the passive diffusion of OTA from the digestive tract and, furthermore, retards its elimination, by limiting its transfer from the bloodstream to the hepatic and renal cells (Marquardt and Frohlich, 1992).

Since the kidney plays a key role in OTA toxicity (Steyn, 1993), the interaction of OTA with renal cells has been extensively investigated. Uptake of OTA across the apical membrane of different renal cells was shown to be partially mediated by the l-phenylalanine carrier and the H+-driven dipeptide carrier (Gekle et al., 1993, Schwerdt et al., 1997, Schwerdt et al., 1998, Zingerle et al., 1997) and by the organic anion carrier (Bahnemann et al., 1997, Welborn et al., 1998, Groves et al., 1999).

The 170 kDa P-glycoprotein (Pgp) and the 190 kDa multidrug resistance-associated protein (MRP) are two membrane proteins implicated in the active efflux of drugs and xenobiotics (Loe et al., 1996). They belong to the ABC superfamily of transport proteins and function as energy-dependent efflux pumps, decreasing free cellular concentrations of drugs and xenobiotics. Pgp transports mainly cationic compounds whereas MRP transports amphipatic anionic conjugates (GSH, glucuronide and sulfate conjugates) as well as unaltered lipophilic substances (Paul et al., 1996). Pgp as well as MRP2 are expressed at the apical pole in Caco-2 cells, while MRP1 would not be expressed and MRP3 would be present at the basolateral pole (Hirohashi et al., 2000, Walgren et al., 2000). These efflux pumps also show overlapping substrate specificity.

Absorption within the gastrointestinal tract is the first step governing the entry of OTA in bloodstream and, eventually, its tissue distribution. The objective of this study is to investigate the mechanism(s) implicated in the transport of OTA across the human intestinal mucosa. For this purpose, we used an in vitro model of the intestinal barrier, based on the cultivation of the Caco-2 cell line on microporous membranes (Halleux and Schneider, 1991). Our results show that OTA is absorbed across the Caco-2 cells monolayers and that this absorption would be limited thanks to its excretion through MRP, probably the MRP2 isoform, at the apical pole of Caco-2 cells.

Section snippets

Chemicals

Culture reagents were purchased from Invitrogen (Merelbeke, Belgium). Biochemicals were purchased from VWR Int. (Leuven, Belgium) or Sigma-Aldrich (Bornem, Belgium). [3H(G)]ochratoxin A (14.8 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA) and d-[1-14C]mannitol (56 mCi/mmol) from Amersham Life Sciences (Little Chalfont, UK).

Cell culture

Caco-2 cells (ATCC, Rockville, MD), between passages 36 and 55, were routinely grown as in Halleux and Schneider, 1991, Sergent-Engelen et al., 1993 in a

MRP expression in Caco-2 cells

Reverse transcription-PCR (RT-PCR) and agarose gel electrophoresis indicate that the expected 498 bp (MRP1 gene product) and 659 bp (MRP2) fragments are expressed in Caco-2 cells (Fig. 1A). Human liver was used as positive (MRP2) and negative (MRP1) controls. cDNA was treated with primers amplifying a 574 bp fragment of human β-actin, to control the integrity of the isolated RNA as well as the amount of cDNA used for each amplification. These results are in agreement with the low expression of

Discussion

The transepithelial transport of OTA across the human intestinal barrier has been investigated by using an in vitro model based on the cultivation of Caco-2 cells in a serum-free nutritive medium, on a microporous membrane, separating the apical pole from the basolateral one.

RT-PCR analysis indicates that, in our serum-free culture conditions, Caco-2 cells express the MRP1 and MRP2 genes, at the mRNA level, MRP2 expression being higher than that of MRP1, as already reported by Hirohashi et al.

Acknowledgements

We thank G. Schmitz-Drévillon for her technical support, Dr S. Ponchaut and E. Mignolet for their support in HPLC analysis of ochratoxin A, Prof. B. Knoops for his help in molecular biology techniques and Prof. E.E. Creppy (Université de Bordeaux II, France) for providing us with a sample of OTA hydroxylated derivatives.

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