Changes in global gene and protein expression during early mouse liver carcinogenesis induced by non-genotoxic model carcinogens oxazepam and Wyeth-14,643

Abstract

We hypothesized that the mouse liver tumor response to non-genotoxic carcinogens would involve some common early gene and protein expression changes that could ultimately be used to predict chemical hepatocarcinogenesis. In order to identify a panel of genes to test, we analyzed global differences in gene and protein expression in livers from B6C3F1 mice following dietary treatment with two rodent carcinogens, the benzodiazepine anti-anxiety drug oxazepam (2500 p.p.m.) and the hypolipidemic agent Wyeth (Wy)-14,643 (500 p.p.m.) compared with livers from untreated mice. Male mice were exposed for 2 weeks and 1, 3 or 6 months to oxazepam or Wy-14,643 in an age-matched study design. By histopathological evaluation, no liver preneoplastic foci or tumors were detected at 6 months in treated or control groups. By cDNA microarray analysis [NIEHS Mouse Chip (8700 genes); n = 3 individual livers/group, four hybridizations/sample], expression of 36 genes or 220 genes were changed relative to control livers following 6 months of oxazepam or Wy-14,643 treatment, respectively. To obtain a more comprehensive picture of gene/protein expression changes, we also conducted a proteomics study by 2D-gel electrophoresis followed by matrix assisted laser desorption/ionization-mass spectrometry on cytoplasmic, nuclear, and microsomal subcellular fractions of the same liver samples utilized for the cDNA microarray analysis. Real-time PCR, western blot analysis and immunohistochemistry were utilized for validation and to expand the results to other time points. Cyp2b20, growth arrest- and damage-inducible gene beta (Gadd45beta), tumor necrosis factor alpha-induced protein 2 and insulin-like growth factor binding protein 1 (Igfbp5) genes and proteins were upregulated by oxazepam, and Cyp2b20, Cyclin D1, proliferating cell nuclear antigen, Igfbp5, Gadd45beta and cell death-inducing DNA fragmentation factor alpha subunit-like effector A exhibited higher expression after Wy-14,643 treatment. Most of these genes/proteins were also deregulated at 2 weeks. There appeared to be more distinct than common changes in the expression of carcinogenesis-related genes/proteins between the two compounds, suggesting that the major carcinogenic pathways are different for these compounds and may be distinct for different chemical classes.

Biokinetics and Subchronic Toxic Effects of Oral Arsenite, Arsenate, Monomethylarsonic Acid, and Dimethylarsinic Acid in v-Ha-ras Transgenic (Tg.AC) Mice

“…Other notable alterations in gene expression were the overexpression of IGFBP-1 and suppression of insulin-like growth factor 1 (IGF-1). Chronic exposure to nongenotoxic chemicals such as oxazepam and Wyeth-14,643 increased the expression of IGFBP-1 in a time-dependent manner (Iida et al 2003), and overexpression of IGFBP-1 was also seen in transplacental arsenic-induced HCC and tumor-surrounding tissues ). Dysregulation of the IGF axis has been implicated in liver tumor formation and progression (Scharf et al 2001).…”

How Proteomics Can Assist in the Detection and Avoidance of Adverse Drug Reactions

“…After exposure to N-nitrosomorpholine, the expression of malignancy-associated proteins such as caspase-8, vimentin and Rho GDP dissociation inhibitor were seen after 3 weeks of drug treatment whilst transformed cells were seen only after 18 weeks [55]. Similarly, many protein expression changes seen after 6 months of exposure to oxazepam and Wyeth-14,643 were already seen after exposure for 2 weeks [54]. It was notable that, whilst both oxazepam and Wyeth-14,643 are carcinogenic in rodents, they each caused changes in different groups of proteins, suggesting that they may have different mechanisms of carcinogenesis.…”

“…Typically, drugs are tested over an extended time in rodents to investigate these effects and to assist in predicting the likelihood of adverse time-related effects in humans. Time course studies of drugs and carcinogens known to cause liver cancer in rodents (oxazepam, Wyeth-14,643 and Nnitrosomorpholine), have recently been combined with proteomic analyses [54,55]. In these studies, 2-D PAGE revealed dramatic changes in protein expression in the liver prior to the onset of carcinogenesis.…”

Proteomics for the early detection and treatment of hepatocellular carcinoma

“…To investigate proteins involved in peroxisome proliferation, Chu et al (2002) compared the protein profiles of acyl-CoA oxidase-deficient and peroxisome proliferator-treated mouse liver with SEL-DI-TOF-MS. With 2D-PAGE-MS, Zeindl-Eberhart et al (2001, 2004 compared the protein profiles of rat HCC tissues induced by genotoxic or non-genotoxic carcinogens and identified aldose reductase-like variants among HCCs induced by carcinogens of different categories. Protein profiles of mouse HCCs induced by two different drugs of the same non-genotoxic category were distinct, demonstrating the great heterogeneity of pathways activated in HCC (Iida et al, 2003). Differentially expressed proteins in animal models can possibly be used as potential biomarkers for HCC diagnosis or targets for HCC chemotherapy as demonstrated in Zeindl-Eberhart's study; variants of aldose reductase-like proteins, which were found differentially expressed in rat HCC, were further validated as potential biomarkers for human HCC by immunohistology (Zeindl-Eberhart et al, 2004).…”