Proteomic investigation into N-nitrosomorpholine mediated changes in rat liver

Proteomic approaches are widely explored to evaluate their usefulness for detecting early toxicological endpoints and for gaining insight into the mechanisms behind the toxic response. In order to relate changes in protein expression to conventional endpoints of toxicity, a common animal model of chemical hepatocarcinogenesis was used. N-nitrosomorpholine (NNM) at 20 mg/kg body weight was applied to young adult male Wistar rats for 7 weeks to induce hepatocarcinogenesis. After 18 weeks of exposure-free period, animals were killed and left liver lobes were prepared for histopathological and proteomic investigations. Liver tissue from 5 animals of each treatment group (vehicle-control + NNM group) was analyzed. Proteins were separated using 2D electrophoresis. Gels were visualized using ruthenium II tris and ProExpress¶TM imaging platform. Gel images were analyzed using ProteinMine¶TM 2D image analysis software. Spot values were statistically evaluated by Impressionist¶TM 2D data analysis software. 7 upregulated and 27 downregulated spots were detected in livers of treated animals. 31 spots were found in gels of the NNM-treated group only, 17 spots were restricted to gels of the control group. Differentially expressed spots were excised from gels mechanically and proteins were identified by common MS methods. Results of histopathology demonstrated a significantly increased number of focal preneoplastic and benign neoplastic lesions in livers of treated specimens. GST-p, a common marker for neoplastic tissues, was found to be highly expressed in livers of the treatment group, as confirmed by histological staining. Other upregulated spots included L-plastin, chloride intracellular channel protein 1, elongation factor-2, P-47 and aflatoxin B1 aldehyde reductase, which is suspected to play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Among downregulated spots senescence marker protein-30 was found, which may cause dysregulation of Ca-dependent pathways. Among others, transaldolase, serotransferrin precursor, catalase, ubiquitin carboxyl-terminal hydrolase 14 and 3-oxo-5-beta-steroid 4-dehydrogenase were detected only in the NNM-treated group.