Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat.
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We eliminated type beta transforming growth factor (TGF-beta) signaling by adenovirus-mediated local expression of a dominant-negative type II TGF-beta receptor (AdCATbeta-TR) in the liver of rats treated with dimethylnitrosamine, a model of persistent liver fibrosis. In rats that received a single application of AdCATbeta-TR via the portal vein, liver fibrosis as assessed by histology and hydroxyproline content was markedly attenuated. All AdCATbeta-TR-treated rats remained alive, and their serum levels of hyaluronic acid and transaminases remained at low levels, whereas all the AdCATbeta-TR-untreated rats died of liver dysfunction. The results demonstrate that TGF-beta does play a central role in liver fibrogenesis and indicate clearly in a persistent fibrosis model that prevention of fibrosis by anti-TGF-beta intervention could be therapeutically useful. (+info)
Phospholipid requirement for dimethylnitrosamine demethylation by hamster hepatic microsomal cytochrome P-450 enzyme system.
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Extraction with butan-1-ol of freeze-dried microsomal fractions from livers of 3-methyl-cholarthrene-pre-treated hamsters removed about 90% of the total lipid content, but the lipid remaining proved sufficient for the cytochrome P-450 enzyme system to retain about 15-40% of its original catalytic activity for dimethylnitrosamine demethylation. Addition of butan-1-ol-extracted total phospholipid or phosphatidylcholine could not restore any activity, whereas the addition of the synthetic phospholipid dilauroyl phosphatidylcholine was able to restore almost complete activity. Synthetic dipalmitoyl or distearoyl phosphatidylcholine was ineffective in restoring the activity in this reconstituted system. (+info)
Tightly regulated and inducible expression of rabbit CYP2E1 using a tetracycline-controlled expression system.
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A tetracycline (Tc)-controlled gene expression system that quantitatively controls gene expression in eukaryotic cells () was used to express cytochrome P-450 2E1 (CYP2E1) in HeLa cells in culture. The rabbit CYP2E1 cDNA was subcloned into the Tc-controlled expression vector (pUHD10-3) and transfected into a HeLa cell line constitutively expressing the Tc-controlled transactivator, a positive regulator of expression in the absence of Tc. The expression of CYP2E1 was tightly regulated. There was a time-dependent induction of CYP2E1 after removal of Tc. In the absence of Tc, the enzyme was induced more than 100-fold and expressed about 18 pmol of CYP2E1/mg microsomal protein. At maximal levels of expression the enzyme catalyzed the formation of 158 pmol 6-hydroxychlorzoxazone/min/mg total cellular protein. In addition, the level of the enzyme could be modulated by the concentration of Tc in the media. In the absence of Tc, exposure of cells to N-nitrosodimethylamine caused a significant dose-dependent decrease in cell viability. In contrast, menadione, a redox cycling toxicant, was less toxic to the cells after induction of CYP2E1 when compared with noninduced cells. Pulse-chase studies conducted 72 h after removal of Tc indicated a rapid turnover of CYP2E1 with a half-life of 3.9 h. Addition of the ligand, 4-methylpyrazole, and the suicide substrate, 1-aminobenzotrizole, decreased the degradation of CYP2E1. This cell line offers a useful system to examine the role of CYP2E1 in the cytotoxicity of xenobiotics and to investigate post-translational regulation of the enzyme. (+info)
Analysis of the inhibition of N-nitroso-dimethylamine activation in the liver by N-nitro-dimethylamine using a new non-linear statistical method.
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N-nitro-dimethylamine (NTDMA) is carcinogenic to rats: it induces nasal cavity tumours. It can be demethylated to N-nitromethylamine and formaldehyde and reduced to N-nitroso-dimethylamine (NDMA): a potent liver carcinogen and also of the nasal cavity if activation in the liver is blocked. To explain the mechanism of NTDMA carcinogenicity we compared its demethylation with that of NDMA in liver microsomes from female and male rats, untreated, fasted or treated with ethanol to induce cytochrome P450 2E1 (CYP2E1). Kinetic parameters were analysed by nonlinear statistical methods, which yielded unbiased parameter estimates for the calculated Km and Vmax values. Km for both compounds was very similar in females (24-47 microM) whereas Vmax for NTDMA was consistently higher than for NDMA as substrate: 1.07-4.70 nmol formaldehyde/mg microsomal protein x min and 0.52-2.76 nmol, respectively. In liver microsomes from induced male rats NTDMA was found to be a much more effective inhibitor of NDMA activation (KEI 39.6-73.6 microM) than NDMA of NTDMA demethylation (KEI 224-286 microM). Nasal microsomes can demethylate both NDMA and NTDMA but the kinetics are vastly different. NTDMA is demethylated at a linear rate and approximately 10-fold more effectively than NDMA. The mechanism of carcinogenicity of ingested NTDMA, we propose, is a partial reduction to NDMA in the liver and inhibition of NDMA activation in the liver by residual NTDMA, which enables NDMA to reach the nasal mucosa where it is activated to DNA-alkylating species and the observed tumours are formed. (+info)
Expression of cytochrome P450 2A3 in rat esophagus: relevance to N-nitrosobenzylmethylamine.
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N-nitrosobenzylmethylamine (NBzMA) must be metabolically activated to exert its carcinogenic potential and is a potent inducer of tumors in the rat esophagus. The activation is believed to occur in the esophagus. Although the pathways of NBzMA metabolism are well studied, the principal cytochrome P450 enzyme(s) (P450) responsible for catalyzing its activation is unknown. Several preliminary studies have suggested that this enzyme may belong to the P450 2A family. We report here that P450 2A3 expressed in a baculovirus system metabolizes NBzMA, predominantly by methylene hydroxylation. To determine whether or not P450 2A3 is present in the rat esophagus, the relative level of P450 2A3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The mRNA levels of P450 2A3 were compared with the levels of P450 2A1 and 2A2 mRNA in the esophagus, liver, lung and nasal mucosa. P450 2A3 mRNA was detected in rat nasal mucosa, lung and esophagus, but not in liver, whereas P450 2A1 and 2A2 mRNAs were detected only in the liver. To determine the relative expression of P450 2A3 in each tissue, quantitative RT-PCR with PCR-MIMICS used as internal standards was performed. The expression level in the nasal mucosa was by far the greatest. The expression in the lung and esophagus was 60- and 1600-fold less, respectively. Using antibodies to P450 2A4/5 and P450 2A10/11 a 50 kDa immunoreactive protein was detected in all three tissues by western blot analysis. This is consistent with the expression of P450 2A3 in these tissues. However, the amount of protein detected in the nasal mucosa was much greater than that in the esophagus or lung. The expression of P450 2A protein was similar in the lung and esophagus. The rate of coumarin 7-hydroxylation in cultured rat esophagus was very low. This is a reaction efficiently catalyzed by P450 2A5, 2A6 and 2A10. In summary, our results clearly demonstrate the presence of P450 2A3 protein and mRNA in the esophagus, but the amounts are low and may not be sufficient to account for NBzMA activation in this tissue. (+info)
Isothiocyanates and freeze-dried strawberries as inhibitors of esophageal cancer.
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A group of arylalkyl isothiocyanates were tested for their abilities to inhibit tumorigenicity and DNA methylation induced by the esophageal-specific carcinogen, N-nitrosomethylbenzylamine (NMBA) in the F344 rat esophagus. Phenylpropyl isothiocyanate (PPITC) was more potent than either phenylethyl isothiocyanate (PEITC) or benzyl isothiocyanate (BITC). Phenylbutyl isothiocyanate (PBITC), however, had a lesser inhibitory effect on esophageal tumorigenesis, and phenylhexyl isothiocyanate (PHITC) actually enhanced esophageal tumorigenesis. Thus, the two- and three-carbon isothiocyanates were more effective inhibitors of NMBA-esophageal carcinogenesis than the longer chain isothiocyanates. The effects of the isothiocyanates on tumorigenesis were well correlated as to their effects on DNA adduct formation. The most likely mechanism of inhibition of tumorigenesis by these isothiocyanates is via inhibition of the cytochrome P450 enzymes responsible for the metabolic activation of NMBA in rat esophagus. A freeze-dried strawberry preparation was also evaluated for its ability to inhibit NMBA-esophageal tumorigenesis. It proved to be an effective inhibitor, although not as potent as either PEITC or PPITC. The inhibitory effect of the berries could not be attributed solely to the content of the chemopreventive agent, ellagic acid, in the berries. (+info)
Tumor necrosis factor receptor knockout (TNFR KO) mice were used to examine the role of tumor necrosis factor-alpha (TNFalpha) signaling during acute hepatotoxicant exposure. Mice were exposed intraperitoneally (ip) to either vehicle, phosphate-buffered saline (PBS), or dimethylnitrosamine (DMN, 100 mg/kg) for 24 h. Histological evaluation showed that DMN-treated TNFR-2 KO mice had increased liver damage compared to wild type (WT), TNFR-1 KO, or TNFR double KO (DKO) mice. Also, 3 of 8 TNFR-2 KO mice died following DMN treatment, suggesting that hepatic TNFR-2 signaling produces protective responses that counteract TNFR-1-mediated damage. DMN-induced cellular infiltration was absent in TNFR-1-deficient mice, indicating that infiltrating cells do not exacerbate acute hepatotoxic events. In separate experiments, mice were exposed ip to either DMN (5.0 or 100 mg/kg), carbon tetrachloride (CCl4, 0.3 or 1.0 ml/kg), or corresponding PBS/corn oil controls for 6 or 24 h to compare the hepatic mRNA expression of cytokine- and apoptotic-associated genes. Following 24 h of DMN (100 mg/kg) or 6-24 h of CCl4 treatment, hepatic transcripts for TNFalpha, interferon (IFN)-gamma, IL (interleukin)-1RI, and transforming growth factor (TGF)-betaRII were induced. Hepatotoxicant-treated WT and TNFR DKO mice induced liver transcripts for the pro- and anti-apoptotic genes, Bax and Bcl-X(L), respectively, indicating TNF-independent gene activation. The anti-apoptotic gene, Bfl-1, was highly expressed in CCl4-treated, TNFR-positive strains, but minimally expressed in TNFR DKO mice, suggesting that hepatic Bfl-1 is TNF-regulated. Taken together, these data show that acute hepatotoxicant exposure is followed by upregulation of liver cytokine, cytokine receptor, and apoptotic transcripts, and that TNFalpha regulates various aspects of liver inflammation and injury in a TNFR-specific fashion. (+info)
Muir-Torre-like syndrome in Fhit-deficient mice.
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To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129/SvJ x C57BL/6J) F(1) mice with a Fhit allele inactivated (+/-). Fhit +/+ and +/- mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the +/+ mice developed adenoma or papilloma of the forestomach, whereas 100% of the +/- mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome. (+info)