Study of the gastrointestinal biotransformation of zearalenone in a Caco-2 cell culture system with liquid chromatographic methods.

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the detection of zearalenone (ZON), alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) in in vitro biological samples. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of ZON, alpha-ZOL, beta-ZOL, alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL) in in vitro biological samples. Zearalanone (ZAN) was used as internal standard in both methods. The limit of detection/limit of quantitation (LOD/LOQ) values for ZON, alpha-ZOL and beta-ZOL were 2/7, 2/7 and 4/13 microg l(-1), respectively, for the HPLC-FLD method. For the LC-MS/MS method LOD/LOQ values for ZON, alpha-ZOL, beta-ZOL, alpha-ZAL and beta-ZAL were 6/20, 5/17, 4/14, 9/30 and 6/19 microg l(-1), respectively. Within-day and between-day precision were less then 11 and 14%, respectively for the HPLC-FLD method, and both less then 20% for the LC-MS/MS method. The recovery of ZON and its metabolites ranged between 73 and 89% for the HPLC-FLD method and between 69 and 112% for the LC-MS/MS method. The methods were used for the detection of the compounds in in vitro biological samples, obtained with human intestinal Caco-2 cells culture experiments. The 8-days post-confluent Caco-2 cells were treated with ZON or a mixture of ZON and imazalil (IMA). After an incubation time of 24 h the samples were analysed with the HPLC-FLD method. Neither ZON nor its derivatives were detected in the samples. The disappearance of ZON could possibly point out the formation of phase II metabolites like glucuronide conjugates. Therefore, samples were pretreated with beta-glucuronidase before LC-MS/MS analysis. The LC-MS/MS results showed that ZON, alpha-ZOL and beta-ZOL could only be detected in the beta-glucuronidase pretreated samples. This confirmed the formation of glucuronide conjugates and the hydroxylation of ZON during the incubation with Caco-2 cells.

Liquid chromatographic methods for biotransformation studies of ochratoxin A.

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OTalpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco-2 cell culture experiments and in in vivo urine samples from sheep. A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed and validated for the detection of OTA and its metabolites OTalpha, 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OTalpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 microg/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 microg/L, respectively for the LC-MS/MS method. Within-day and between-day precision were both <12% for the HPLC-FLD method, and <10% for the LC-MS/MS method. The recovery of OTA and its metabolites ranged between 71 and 111% for the HPLC-FLD method and between 79 and 110% for the LC-MS/MS method. In the first experiment only OTA was added to the Caco-2 cells while in the second experiment 3-methylcholanthrene (3MC) was also present in the cell culture systems. Besides OTA, which was recovered in all the samples, an unknown compound was also observed in the second experiment. When 3MC was added, the results showed that the OTA concentration in the basolateral samples was decreased by 50%. The methods were also implemented for the analysis of urine samples of sheep, fed increasing amounts of OTA. With the HPLC-FLD method it could be concluded that the concentration of OTA and OTalpha increased according to ingested amounts of OTA, with OTalpha being the most abundant compound. The results obtained with the LC-MS/MS method confirmed these results.

Binding and endocytosis of a monoclonal antibody to a high molecular weight human milk fat globule membrane-associated antigen by cultured MCF-7 breast carcinoma cells.

The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, the label migrates as two bands with apparent molecular weights of about 350,000 and 400,000. These bands disappear, or their molecular weight is affected, after treatment of the cells with cycloheximide or of cell lysates with trypsin, Pronase, or neuraminidase but not treatment of the immunoprecipitate with endoglycosidase F. This suggests that these antigens are glycoproteins with O-linked oligosaccharides containing sialic acid in the epitope. By analogy, they should be similar, if not identical, to those recognized by the monoclonal antibodies designated HMFG1 (H. Burchell, H. Durbin, and J. Taylor-Papadimitriou, J. Immunol., 131:508-513, 1983) and DF3 (H. Sekine, T. Ohno, and D.W. Kufe, J. Immunol., 135:3610-3615, 1985). Binding at 4 degrees C of the 3H-labeled antibody by MCF-7 cells indicates the specific attachment of about 1.2 X 10(6) IgG molecules per cells with a Kd of about 14 nM. At 37 degrees C, cells take up the 3H-labeled antibody in amounts much higher than the binding capacity. In addition to cell-associated material, labeled digestion products are released into the culture medium. Cell fractionation by differential centrifugation and isopycnic equilibration on sucrose gradient indicates that the bulk of cell-associated antibody is distributed like the marker enzyme of lysosomes. Although the total uptake of the antibody by the cells is unaffected by either 50 microM chloroquine or 3 micrograms/ml cycloheximide, the release of digestion products is completely inhibited by chloroquine. Antigen-antibody dissociation is pH dependent, since, respectively, 50 and 84% of membrane-bound antibody are released during washing at pH 4.6 and 4.1.(ABSTRACT TRUNCATED AT 400 WORDS)