ABSTRACT
The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10-200 microM ZEA showed efficacious metabolism of the toxin. alpha-zearalenol and beta-zearalenol were the measured preponderant metabolites (respectively 40.7+/-3.1% and 31.9+/-4.9% of total metabolites, after a 3h exposure to 10 microM ZEA), whereas ZEA-glucuronide and alpha-zearalenol glucuronide were less produced (respectively 8.2+/-0.9% and 19.1+/-1.3% of total metabolites, after a 3h exposure to 10 microM ZEA). Cell production of reduced metabolites was strongly inhibited by alpha-and beta-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited alpha-hydroxysteroid dehydrogenase type II and beta-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, alpha-zearalenol was preponderantly found at the basal side, whereas beta-zearalenol and both glucuronides were preferentially excreted at the apical side. As alpha-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.
Subject(s)
Estrogens, Non-Steroidal/metabolism , Mycotoxins/metabolism , Zearalenone/metabolism , 3-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , Biological Transport , Caco-2 Cells , Humans , Intestinal Neoplasms/metabolismABSTRACT
Deoxynivalenol (DON) is a major mycotoxic contaminant of cereal grains in Europe and North America. Human and animal contamination occurs mainly orally, and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. This study investigates the mechanisms of DON transepithelial transfer. Investigations using the human intestinal Caco-2 cell line showed a basal-to-apical polarized transport of the toxin. Both apical-basolateral (AP-BL) and basolateral-apical (BL-AP) transfers were time- and concentration-dependent, and not saturable between 5 and 30 microM DON. Arrhenius plot analysis revealed that transfer of 10 microM DON was temperature-dependent, with apparent activation energy E(a)=3.2 kcal mol(-1) in the AP-BL direction, and E(a)=10.4 kcal mol(-1) in the BL-AP direction. Intracellular DON accumulation was increased and DON efflux was decreased by ATP depletion, by P-glycoprotein inhibitor valspodar and by MRP2 inhibitor MK571, but not by BCRP inhibitor Ko143. Intracellular DON accumulation was then investigated using epithelial cell lines transfected with human P-glycoprotein or MRP2. This accumulation was decreased in LLCPK1-MDR1 and MDCKII-MRP2 cells, compared to wild-type cells, and the decrease could be reversed by valspodar or MK571. Taken together, these results suggest that DON is a substrate for both P-glycoprotein and MRP2.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Membrane Transport Proteins/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Trichothecenes/metabolism , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Biological Transport, Active , Caco-2 Cells , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Cyclosporins/pharmacology , Data Interpretation, Statistical , Dogs , Epithelium/metabolism , Flow Cytometry , Humans , LLC-PK1 Cells , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Multidrug Resistance-Associated Proteins/genetics , Propionates/pharmacology , Quinolines/pharmacology , Swine , Temperature , TransfectionABSTRACT
Mycotoxin nivalenol (NIV) is a natural contaminant of various cereal crops, animal feed and processed grains throughout the world. Human and animal contamination occurs mainly orally, and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. In this study, we investigated the mechanisms involved in NIV transepithelial transfer. The human intestinal Caco-2 cell line showed a basal-to-apical polarized transport of NIV. Using metabolic inhibitors and temperature-dependent experiments, we demonstrated that basolateral-apical (BL-AP) transfer of NIV involved an energy-dependent transport whereas apical-basolateral (AP-BL) transfer was governed by passive diffusion. NIV efflux was significantly decreased in the presence of the P-glycoprotein (P-gp) inhibitor valspodar, the multi-drug resistance-associated proteins (MRPs) inhibitor MK571, but was not modified by the breast cancer resistance protein (BCRP) inhibitor Ko143. Intracellular NIV accumulation was investigated using epithelial cell lines transfected with either human P-glycoprotein or MRP2. This accumulation was significantly decreased in LLCPK1/MDR1 and MDCKII/MRP2 cells, compared to wild-type cells, and this effect was reversed by valspodar and MK571, respectively. These in vitro results suggested that NIV was a substrate for both P-glycoprotein and MRP2. This interaction may play a key role in weak intestinal absorption of NIV and the mainly predominant excretion of NIV in faeces in animal studies.
