Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2).
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 microM 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.
Berger
Valérie
V
Laboratoire de Biochimie cellulaire, Institut des Sciences de la Vie, Université catholique de Louvain, Place Louis Pasteur 1, B-1348, Louvain-la-Neuve, Belgium.
Gabriel
Anne-Françoise
AF
Sergent
Thérèse
T
Trouet
André
A
Larondelle
Yvan
Y
Schneider
Yves-Jacques
YJ
D001692:Biological Transport; D001711:Biotransformation; D018938:Caco-2 Cells; D002478:Cells, Cultured; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000187:drug effects* / Q000235:genetics; D009793:Ochratoxins / Q000493:pharmacokinetics / Q000633:toxicity*; D012333:RNA, Messenger / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018675:Toxicity Tests
D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009793:Ochratoxins; D012333:RNA, Messenger; C025589:ochratoxin A