Analysis of ß-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital.

In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for ß-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in ß-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated ß-catenin forms in just 4 male liver tumour samples, with the presence of the truncated ß-catenin forms being confirmed by ß-catenin exon 1-3 mutation analysis. Microarray gene expression analysis was performed with three of the NaPB-induced male mouse liver tumour samples where ß-catenin mutations had not been identified by Western immunoblotting and DNA/RNA analysis and with three liver samples from both NaPB-induced non-tumour tissue and control animals. Treatment with NaPB resulted in induction of Cyp2b subfamily gene expression in both NaPB-induced mouse liver tumours and in NaPB-treated non-tumour tissue. In addition, the gene expression analysis demonstrated that the ß-catenin and Hras pathways were not modified in NaPB-induced mouse liver tumours not exhibiting truncated ß-catenin forms. Overall, while chronic administration of the model CAR activator NaPB results in both hepatocellular adenoma and carcinoma in the low spontaneous liver tumour incidence C57BL/10J mouse strain, only 40 % of the liver tumours evaluated in this study had ß-catenin mutations. These results are in agreement with previous studies with the CAR activator oxazepam and demonstrate that mouse liver tumours induced by nongenotoxic CAR activators in the absence of initiation with a genotoxic agent are due to a number of mechanisms, including those largely independent of either the Wnt/ß-catenin signalling pathway or Hras oncogene mutations.

Effect of chronic phenobarbitone administration on liver tumour formation in the C57BL/10J mouse.

The hepatocarcinogenicity of sodium phenobarbitone (PB) was studied in male and female mice of the low spontaneous liver tumour incidence C57BL/10 J strain. Treatment with 200 and 1000 ppmPB for 1 month increased relative liver weight in both sexes, with 1000 ppmPB also producing a transient increase in replicative DNA synthesis. The treatment of male and female mice with 200 and 1000 ppm (the maximum tolerated dose) PB for 99 weeks resulted in centrilobular hypertrophy and a dose-dependent increase in relative liver weight. Altered hepatic foci were observed in both sexes given 1000 ppm PB. In male mice given 1000 ppm PB significant increases were observed in the incidence of hepatocellular adenoma and carcinoma, to 43% and 10% of the animals examined, respectively. No increase in liver tumours was observed in male mice given 200 ppm PB and in female mice given 200 and 1000 ppm PB. In summary, PB at a dose level which produces liver hypertrophy, a transient stimulation of replicative DNA synthesis and on chronic administration altered hepatic foci, three key events in the established mode of action for PB-induced rodent liver tumour formation, results in a significant increase in liver tumours in male C57BL/10 J mice.

Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat.

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multiomic data are considered.

Gene expression changes induced by estrogen and selective estrogen receptor modulators in primary-cultured human endometrial cells: signals that distinguish the human carcinogen tamoxifen.

Tamoxifen has long been the endocrine treatment of choice for women with breast cancer and is now employed for prophylactic use in women at high risk from breast cancer. Other selective estrogen receptor modulators (SERMs), such as raloxifene, mimic some of tamoxifen's beneficial effects and, like tamoxifen, exhibit a complex mixture of organ-specific estrogen agonist and antagonistic properties. However, accompanying the positive effects of tamoxifen has been the emergence of evidence for an increased risk of endometrial cancer associated with its use. A more complete understanding of the mechanism(s) of SERM carcinogenicity and endometrial effects is therefore required. We have sought to compare and characterise the transcript profile of tamoxifen, raloxifene and the agonist estradiol in human endometrial cells. Using primary cultures of human endometria, to best emulate the in vivo responses in a manageable in vitro system, we have shown 230 significant changes in gene expression for epithelial cultures and 83 in stromal cultures, either specific to 17beta-estradiol, tamoxifen or raloxifene, or changed across more than one of the treatments. Considering the transcriptome as a whole, the endometrial responses to raloxifene or tamoxifen were more similar than either drug was to 17beta-estradiol. Treatment of endometrial cultures with tamoxifen resulted in the largest number of gene changes relative to control cultures and a high proportion of genes associated with regulation of gene transcription, cell-cycle control and signal transduction. Tamoxifen-specific changes that might point towards mechanisms for its proliferative response in the endometrium included changes in retinoblastoma and c-myc binding proteins, the APCL, dihydrofolate reductase (DHFR) and E2F1 genes and other transcription factors. Tamoxifen was also found to give rise to the highest number of gene expression changes common to those that characterise malignant endometria. It is anticipated that this study will provide leads for further and more focused investigation into SERM carcinogenicity.

Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption.

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.

Biochemical and genetic characterization of a murine class Kappa glutathione S-transferase.

The class Kappa family of glutathione S-transferases (GSTs) currently comprises a single rat subunit (rGSTK1), originally isolated from the matrix of liver mitochondria [Harris, Meyer, Coles and Ketterer (1991) Biochem. J. 278, 137-141; Pemble, Wardle and Taylor (1996) Biochem. J. 319, 749-754]. In the present study, an expressed sequence tag (EST) clone has been identified which encodes a mouse class Kappa GST (designated mGSTK1). The EST clone contains an open reading frame of 678 bp, encoding a protein composed of 226 amino acid residues with 86% sequence identity with the rGSTK1 polypeptide. The mGSTK1 and rGSTK1 proteins have been heterologously expressed in Escherichia coli and purified by affinity chromatography. Both mouse and rat transferases were found to exhibit GSH-conjugating and GSH-peroxidase activities towards model substrates. Analysis of expression levels in a range of mouse and rat tissues revealed that the mRNA encoding these enzymes is expressed predominantly in heart, kidney, liver and skeletal muscle. Although other soluble GST isoenzymes are believed to reside primarily within the cytosol, subcellular fractionation of mouse liver demonstrates that this novel murine class Kappa GST is associated with mitochondrial fractions. Through the use of bioinformatics, the genes encoding the mouse and rat class Kappa GSTs have been identified. Both genes comprise eight exons, the protein coding region of which spans approx. 4.3 kb and 4.1 kb of DNA for mGSTK1 and rGSTK1 respectively. This conservation in primary structure, catalytic properties, tissue-specific expression, subcellular localization and gene structure between mouse and rat class Kappa GSTs indicates that they perform similar physiological functions. Furthermore, the association of these enzymes with mitochondrial fractions is consistent with them performing a specific conserved biological role within this organelle.

Tissue-specific inactivation of murine M6P/IGF2R.

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and T cell- mediated immunity. M6P/IGF2R is an imprinted gene in mice with expression only from the maternal allele. Complete knockout of this gene causes neonatal lethality, thus preventing analysis of its multifunctional role postnatally. To help elucidate the biological functions of M6P/IGF2R in adulthood, we generated both complete and tissue-specific M6P/IGF2R knockout mice using the Cre/loxP system. We confirm that complete M6P/IGF2R knockout results in fetal overgrowth and neonatal lethality. In contrast, tissue-specific inactivation of this gene in either the liver or skeletal and cardiac muscle gives rise to viable animals with no obvious phenotype. The successful creation of viable tissue-specific M6P/IGF2R knockout mouse models will now allow for detailed analysis of receptor function in a number of cellular processes including brain development, carcinogenesis, lysosomal trafficking, and T cell-mediated immunity.