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

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.

Proinflammatory mesenchymal effects of the non-genotoxic hepatocarcinogen phenobarbital: a novel mechanism of antiapoptosis and tumor promotion

“…We have shown in previous and the present study that the NGC PB is able to induce multiple alterations in the hepatic mesenchyme, which is reflected not only by altered transcriptome profiles but also by deregulated patterns of intracellular as well as secreted proteins (36). PB treatment elevated transcript levels of chemokines of the CC-, CXC-type, of the TNF, TGFß and interleukin superfamily.…”

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…The literature search resulted in 59 papers for “genotoxicity & 3D in vitro model”, 73 papers for “genotoxicity & advanced in vitro model”, 134 papers for “genotoxicity & high throughput”, eight papers for “genotoxicity & high throughput & nanomaterials” [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ] and six papers for “genotoxicity & high throughput & nanoparticles” (4 are the same as for “genotoxicity & high throughput & nanomaterials”) [ 16 , 17 , 18 , 19 , 21 , 22 ], seven papers for “genotoxicity & organ on chip“ [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], seven papers for “genotoxicity & 3D models & nanoparticles” [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ], 11 papers for “genotoxicity & 3D models & nanomaterials” [ 30 , 31 , 33 , 36 , 37 , 38 , 39 , 40 , 41 ] for the period of 20 years (2000–2020), whereby the year 2020 was considered only from January to August. ( Table 1 ).…”

“…The literature search resulted in 59 papers for "genotoxicity & 3D in vitro model", 73 [16][17][18][19]21,22], seven papers for "genotoxicity & organ on chip" [23][24][25][26][27][28][29], seven papers for "genotoxicity & 3D models & nanoparticles" [30][31][32][33][34][35][36], 11 papers for "genotoxicity & 3D models & nanomaterials" [30,31,33,[36][37][38][39][40][41] for the period of 20 years (2000-2020), whereby the year 2020 was considered only from January to August. (Table 1).…”

Serum NMR metabolomics uncovers multiple metabolic changes in phenobarbital-treated dogs

“…Circulatory glutamine is largely synthesized in the liver by glutamine synthetase, which catalyzes the glutamine synthesis from glutamate and ammonia (Listrom et al, 1997 ; van Straaten et al, 2006 ). Phenobarbital treatment has been associated with the down-regulation of hepatic glutamine synthetase (Klepeisz et al, 2013 ), which most probably explains why in our study, samples with lower glutamine concentrations were more likely to belong to the phenobarbital-treated group. Glutamine plays an important role in multiple cellular functions in various organ systems, the CNS being one of them (Curi et al, 2005 ).…”