Characterization of the Secreted Proteome of Rat Hepatocytes Cultured in Collagen Sandwiches

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, α1-antitrypsin and α2-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.