Elsevier

Toxicology Letters

Volume 170, Issue 3, 15 May 2007, Pages 248-258
Toxicology Letters

Transepithelial transport of fusariotoxin nivalenol: Mediation of secretion by ABC transporters

https://doi.org/10.1016/j.toxlet.2007.03.012Get rights and content

Abstract

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.

Introduction

Nivalenol (NIV) (12,13-epoxy-3,4,7,15-tetrahydroxytrichothec-9-en-8-one) is a trichothecene mycotoxin produced by Fusarium species. This natural toxin occurs throughout the world in various cereal crops (wheat, maize, barley, oats and rye) and processed grains (malt, beer and bread) (Placinta et al., 1999, Eriksen and Alexander, 1998, Schollenberger et al., 2006). Owing to its stability during storage and food processing, and its resistance to high temperatures (Eriksen and Alexander, 1998), human exposure levels can exceed the temporary tolerable daily intake (t-TDI), particularly among vegans/macrobiotics (Gareis et al., 2003, Leblanc et al., 2005). Trichothecenes are potent inhibitors of protein synthesis (Ehrlich and Daigle, 1987) and are toxic to tissues with a high cell division rate, such as lymphoid organs and intestinal mucosa (Ueno, 1983; as review, Rotter et al., 1996). Chronic toxicity in animals includes feed refusal, vomiting, gastroenteritis and immunological dysfunction. Little is known about the toxic effects NIV in human, but Chinese epidemiological studies suggest a link between cancer of esophagus and gastric cardia and high levels of NIV in food (Hsia et al., 2004).

Studies on the toxicokinetics of NIV stress the interspecies variation in oral bioavailability (Onji et al., 1989, Hedman et al., 1997, Garaleviciene et al., 2002, Poapolathep et al., 2003). In pigs, NIV is slowly absorbed, most probably through the upper parts of the small intestine, and is mainly excreted unaltered in the faeces (Hedman et al., 1997). In rats, NIV is metabolically detoxified into de-epoxy-NIV, and excreted in urine and faeces (Onji et al., 1989). Human microflora seem to be unable to produce de-epoxidated metabolites (Sundstol-Eriksen and Pettersson, 2003), and the mechanisms underlying NIV absorption remain unknown.

As human and animal contamination occurs mainly by the oral route, the toxin has to pass through the intestinal epithelial barrier before causing potential health effects. It is then of great importance to study the mechanism of NIV absorption in the organism. Caco-2 cells have been widely used as a model of human intestinal epithelium for studies of intestinal xenobiotics absorption and metabolism (Artursson et al., 2001, Makhey et al., 1998). After the confluence, these cells exhibit morphological and functional similarities to intestinal enterocytes (Pinto et al., 1983). This model is also used for prediction of transport by ATPase binding cassette (ABC) transporters (Zhang et al., 2003, Makhey et al., 1998, Gutmann et al., 1999, Xia et al., 2005). However, the expression of multiple transporters in this cell model makes it difficult to design studies to elucidate the individual role of each transporter. In this context, cells transfected with human ABC transporters are useful tools for specifying the involvement of each transporter.

The present study was aimed at characterization of NIV transepithelial transfer using several epithelial cell lines: Caco-2 cells, LLCPK1 and MDCKII wild-type cells, LLCPK1 cells transfected with human P-glycoprotein (P-gp) and MDCKII cells transfected with multi-drug resistance-associated protein 2 (MRP2). Caco-2 cells were used to elucidate NIV intestinal transfer mechanisms in the absorptive (apical–basolateral) and secretory (basolateral–apical) directions. In addition, intracellular NIV accumulation in LLCPK1 and MDCKII cells overexpressing ABC transporters was assessed to evaluate the individual role of P-gp and MRP2 in NIV efflux.

Section snippets

Chemicals

Trisil-TBT was obtained from Perbio Science (Brebières, France). HPLC grade water prepared using a Milli-Q plus system (Millipore, Molsheim, France) was used for the gas chromatography analysis. Cell culture media and reagents came from Invitrogen (Cergy Pontoise, France). Fetal bovine serum was obtained from Perbio Science (Brebières, France). Culture flasks and plates came from Falcon (VWR International, Strasbourg, France). Immunostaining kits were purchased from Biomeda Corp., Microm

Cytotoxicity

A 6 h exposure of Caco-2 cell monolayers to NIV at a concentration range of 1–30 μM had no significant effect on their viability (Fig. 1A), and LDH leakage was not reduced in monolayers exposed to NIV concentrations up to 50 μM (Fig. 1B). After a 24 h exposure, a significant effect on cell viability and LDH leakage was observed from 30 μM NIV. According to these results it was decided to study the transepithelial transport of 5 μM NIV. Cell exposure to 5 μM NIV for 6 h showed neither a significant

Discussion

Dietary ingestion represents the most common route of human exposure to trichothecenes. The amount of NIV in cereals products varies considerably among world regions (from 20–60 μg/kg in France, to 584–1780 μg/kg in China) (Hsia et al., 2004), depending on weather and culture conditions (Placinta et al., 1999, Edwards, 2004). Estimated nivalenol intake of the French population is 88 ng/kg per day in adults, 163 ng/kg per day in children, and reaches 210 ng/kg per day in vegetarians (Leblanc et al.,

Acknowledgements

The authors thank Pr. Thierry Marchal for technical advice in immunocytochemistry, and Mr. Tim Greenland for his help in English editing.

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