nivalenol and thiazolyl-blue

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON<15-ADON≈DON

Topics: Algorithms; Caco-2 Cells; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Humans; Intestinal Mucosa; Mycotoxins; Tetrazolium Salts; Thiazoles; Trichothecenes

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.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Transporters; Biological Transport, Active; Caco-2 Cells; Cell Line; Chromatography, Gas; Electrochemistry; Epithelium; Humans; Immunohistochemistry; L-Lactate Dehydrogenase; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Mycotoxins; Propionates; Quinolines; Temperature; Tetrazolium Salts; Thiazoles; Trichothecenes

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 microM for NIV to 70 microM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

Topics: Apoptosis; Bromodeoxyuridine; Cell Cycle; Cell Differentiation; Cell Division; Cell Membrane; Cell Physiological Phenomena; Cell Survival; Cycloheximide; DNA Replication; Dose-Response Relationship, Drug; Flow Cytometry; Fumonisins; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Mitochondria; Mycotoxins; Tetrazolium Salts; Thiazoles; Trichothecenes; Trypan Blue