Global Liver Proteomics of Rats Exposed for 5 Days to Phenobarbital Identifies Changes Associated with Cancer and with CYP Metabolism

Abstract

A global proteomics approach was applied to model the hepatic response elicited by the toxicologically well-characterized xenobiotic phenobarbital (PB), a prototypical inducer of hepatic xenobiotic metabolizing enzymes and a well-known nongenotoxic liver carcinogen in rats. Differential detergent fractionation two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry and systems biology modeling were used to identify alterations in toxicologically relevant hepatic molecular functions and biological processes in the livers of rats following a 5-day exposure to PB at 80 mg/kg/day or a vehicle control. Of the 3342 proteins identified, expression of 121 (3.6% of the total proteins) was significantly increased and 127 (3.8%) significantly decreased in the PB group compared to controls. The greatest increase was seen for cytochrome P450 (CYP) 2B2 (167-fold). All proteins with statistically significant differences from control were then analyzed using both Gene Ontology (GO) and Ingenuity Pathways Analysis (IPA, 5.0 IPA-Tox) for cellular location, function, network connectivity, and possible disease processes, especially as they relate to CYP-mediated metabolism and nongenotoxic carcinogenesis mechanisms. The GO results suggested that PB's mechanism of nongenotoxic carcinogenesis involves both increased xenobiotic metabolism, especially induction of the 2B subfamily of CYP enzymes, and increased cell cycle activity. Apoptosis, however, also increased, perhaps, as an attempt to counter the rising cancer threat. Of the IPA-mapped proteins, 41 have functions which are procarcinogenic and 14 anticarcinogenic according to the hypothesized nongenotoxic mechanism of imbalance between apoptosis and cellular proliferation. Twenty-two additional IPA nodes can be classified as procarcinogenic by the competing theory of increased metabolism resulting in the formation of reactive oxygen species. Since the systems biology modeling corresponded well to PB effects previously elucidated via more traditional methods, the global proteomic approach is proposed as a new screening methodology that can be incorporated into future toxicological studies.

Activation of CAR and PXR by Dietary, Environmental and Occupational Chemicals Alters Drug Metabolism, Intermediary Metabolism, and Cell Proliferation

“…In contrast to CAR that is primarily found in the cytosol [44], PXR is primarily found in the nucleus and inactive PXR may form repressor complexes with SMRT or SHP and histone deacetylases, and inhibit transcription [45, 46]. Ligand activation releases the co-repressors and initiates the recruitment of co-activators such as steroid receptor co-activator 1 (SRC-1) and histone acetylases that promote transcription [45–47] (Fig.…”

“…Barbituates have historically been correlated with liver tumor formation in murine experiments [173–175], and an increase in CAR expression in G1 phase is associated with cell proliferation [176]. Further, a global proteomics study following PB-treatment of wild-type rats found that a possible mechanism for carcinogenesis was through increased xenobiotic metabolism enzymes, especially CYP2B and other oxidoreductases, increased cell cycle activity, and a general induction of several pro-carcinogenic proteins [44]. PB was also prescribed to patients with hepatic injury due to its ability to decrease the time for hepatic regeneration, and CAR activation by PB or TCPOBOP has been correlated with hepatomegaly of the liver in response to toxicants.…”

“…Furthermore, cyclin B1, which induces progression from S to G2-phase, is also induced by CAR activation [168, 185]. A summary of the pro-carcinogenic genes induced by CAR is provided in Figure 3c, and Dai et al [44] provides a list of proteins effected by CAR activation. Therefore, the evidence suggests CAR activation induces a three-pronged attack that leads to increased cell cycle progression, DNA replication, and cell survival, ultimately leading to hepatomegaly of the liver and potentially hepatocarcinoma (HCC) (Fig.…”

Current methods of cytochrome P450 analysis

“…В ряде работ идентификация цитохромов Р450 была получена в рамках общего протеомного анализа [24][25][26]. Так, в работе [24] при протеомном анализе эндоплазматического ретикулума пораженных опухолью, индуцированной афлатоксином В1, тканей печени крыс обнаружено уменьшение экспрессии цитохромов Р450 по сравнению с контрольными образцами.…”

“…Так, в работе [24] при протеомном анализе эндоплазматического ретикулума пораженных опухолью, индуцированной афлатоксином В1, тканей печени крыс обнаружено уменьшение экспрессии цитохромов Р450 по сравнению с контрольными образцами. В работах [25,26] методами 2D-LC исследован эффект увеличения экспрессии цитохромов Р450 в печени крысы после введения животным фенобарбитала. В работе [26] показано, что при общем увеличении содержания цитохромов наибольший эффект наблюдается для изоформы CYP 2В2.…”