Title: Characterization of the secreted proteome of rat hepatocytes cultured in collagen sandwiches.
Authors: Farkas, Dora; Bhat, Vadiraja B; Mandapati, Saraswathi; Wishnok, John S; Tannenbaum, Steven R
Published In Chem Res Toxicol, (2005 Jul)
Abstract: Analysis of proteins in biological samples opens up the possibility of discovering new markers of toxicity. The liver is one of the primary targets of drug-induced toxicity, and it also secretes many plasma proteins, which can be measured clinically. Most of the plasma proteins produced by the liver are secreted by hepatocytes, but there is little information regarding the protein profile secreted by these cells. The purpose of this study was to analyze the secreted proteome of primary rat hepatocytes in a collagen gel sandwich configuration by a gel-LC-MS/MS procedure. We identified over 600 peptides corresponding to more than 200 proteins. The protein profile included over 50 plasma proteins, suggesting that the cultured hepatocytes secrete many of the proteins that they produce in vivo. Our data also suggests that the hepatocytes are actively remodeling their environment, since we identified several structural extracellular matrix proteins as well as some proteins known to be secreted specifically during liver regeneration. We also identified two proteins, alpha1-antitrypsin and alpha2-macroglobulin, whose secretions appear to be down-regulated in cells exposed to aflatoxin B1. It was noted that a 15 nM dose of aflatoxin B1 led to substantially diminished levels of these proteins and that day 6 of incubation was the ideal time point for medium collection. These data suggest that proteins in the conditioned medium of hepatocyte sandwich culture might lead to the discovery of biomarkers for drug-induced chemical toxicity.
PubMed ID: 16022505
MeSH Terms: Aflatoxins/pharmacology; Albumins; Animals; Cell Culture Techniques/methods*; Cells, Cultured; Collagen/pharmacology*; Culture Media, Conditioned; Gene Expression Regulation/drug effects; Hepatocytes/metabolism*; Hepatocytes/secretion*; Male; Proteome/metabolism*; Proteome/secretion*; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization