Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

Philip Klepeisz,Sandra Sagmeister,Christopher Gerner,Bettina Grasl-Kraupp,Verena Haudek-Prinz,Melanie Pichlbauer,Simon Afford

doi:10.1371/journal.pone.0076137

Abstract

Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

Highlights

Screening assays, which enable the early detection of potential carcinogenic activities, are of crucial importance for safe drug development strategies
Cell isolation was performed by liver perfusion followed by separation into hepatocytes (HCs) and non-parenchymal cells (NPCs)
Amongst known marker proteins for endothelial cells we identified endothelial cell-specific molecule 1, endothelial nitric oxide synthase and septin-2, while MMP-3 and various collagens are characteristic for the stellate cells

Summary

Introduction

Screening assays, which enable the early detection of potential carcinogenic activities, are of crucial importance for safe drug development strategies. Chemical compounds may cause cancer by directly affecting DNA and are called genotoxic carcinogens. This type of compounds is detectable as such by the application of well-established in vitro assays, such as Ames bacterial reverse mutation assay, mammalian forward mutation assays and detection of chromosomal aberrations. DNA directly are called non-genotoxic carcinogens (NGCs) [1]. In contrast to genotoxic carcinogens, there are no sufficiently accurate and validated short-term assays that may allow detection of NGCs [2,3,4,5,6]. Employed assays necessitate long-term rodent carcinogenicity assays causing high efforts, costs and time requirement as major drawbacks. In order to overcome these problems, deeper insights into NGC-relevant mechanisms are urgently required, which may be obtained by the application of a screening technology such as proteome profiling

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