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1.
Xenobiotica ; 37(2): 180-93, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17484520

ABSTRACT

This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon, on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague Dawley rats. Rats were dosed with the conazoles at three dose levels by gavage for 14 days: myclobutanil (150, 75, and 10mgkg(-1) body weight day(-1); triadimefon (115, 50, and 10 mg kg(-1) body weight day-'), which included their maximum tolerated dose levels (MTD). Both myclobutanil and triadimefon significantly induced pentoxyresorufin O-depentylase activities at their MTD levels: myclobutanil, 8.1-fold at 150mgkg(-1) body weight day- ; and triadimefon, 18.5-fold at 115mgkg(-1) body weight day-'. Benzyloxyresorufin O-debenzylase activities were similarly increased: myclobutanil, 13.3-fold; triadimefon, 27.7-fold. Quantitative real-time reverse-transcription polymerase chain reaction assays were used to characterize the mRNA expression of specific CYP genes induced by these two conazoles. Myclobutanil and triadimefon treatment at their MTD levels significantly increased rat hepatic mRNA expression of CYP2B1 (14.3- and 54.6-fold), CYP3A23/3A1 (2.2- and 7.3-fold), and CYP3A2 (1.5- and 1.7-fold). Western immunoblots of rat hepatic microsomal proteins identified significantly increased levels of CYP isoforms after myclobutanil or triadimefon treatment at their MTD levels: CYP2BI/2 (4.8- and 5.3-fold), and CYP3A1 (2.2- and 2.9-fold). Triadimefon also increased CYP3A2 immunoreactive protein levels 1.8-fold. These results indicate that triadimefon and myclobutanil, like other triazole-containing conazoles, induced CYP2B and CYP3A families of cytochromes in rat liver.


Subject(s)
Antifungal Agents/pharmacology , Cytochrome P-450 Enzyme System/biosynthesis , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Nitriles/pharmacology , Triazoles/pharmacology , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Base Sequence , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP2B1/genetics , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , DNA Primers/genetics , Enzyme Induction/drug effects , Gene Expression/drug effects , Male , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Steroid Hydroxylases/biosynthesis , Steroid Hydroxylases/genetics
2.
Carcinogenesis ; 22(8): 1317-22, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11470764

ABSTRACT

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in mice when administered in drinking water. The mechanism of DCA carcinogenicity is not clear and we speculate that changes in gene expression may be important. In order to analyze early changes in gene expression induced by DCA treatment we used the differential display method. Mice were treated with 2 g/l DCA in drinking water for 4 weeks. Total RNAs were obtained from livers of both control and treated mice for analysis. Of approximately 48 000 bands on the differential display gels representing an estimated 96% of RNA species, 381 showed differences in intensity. After cloning and confirmation by both reverse-northern and northern analyses, six differentially expressed genes were found. The expression of five of these genes was suppressed in the DCA-treated mice while one was induced. After sequencing, four genes were identified and two were matched to expressed sequence tags through the BLAST program. These genes are alpha-1 protease inhibitor, cytochrome b5, stearoyl-CoA desaturase and carboxylesterase. Stearoyl-CoA desaturase was induced approximately 3-fold in the livers of DCA-treated mice and the other three genes were suppressed approximately 3-fold. Stearoyl-CoA desaturase, cytochrome b5 and carboxylesterase are endoplasmic reticulum membrane-bound enzymes involved in fatty acid metabolism. The expression pattern of four of these genes was similar in DCA-induced hepatocellular carcinomas and the 4 week DCA-treated mouse livers. The expression of stearoyl-CoA desaturase and one of the unidentified genes returned to control levels in the carcinomas. Understanding the roles and interactions between these genes may shed light on the mechanism of DCA carcinogenesis.


Subject(s)
Carcinogens/pharmacology , Dichloroacetic Acid/pharmacology , Gene Expression Profiling , Liver/drug effects , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA Primers , Liver/metabolism , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Male , Mice
3.
Matrix Biol ; 19(1): 37-46, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10686424

ABSTRACT

This study describes the relative contribution of the 10 cysteine residues in lysyl hydroxylase 1 (LH1) to enzyme activity. We have identified a novel mutation of a 15-bp deletion in exon 11 in one LH1 allele, that codes for amino acids 367-371 (DLCRQ), in two unrelated compound heterozygous patients with Ehlers-Danlos type VI. The mutations in their other alleles were a C1119T change (exon 10) and a predicted Q49X (exon 2). We confirmed that the loss of cysteine 369 in the deleted sequence contributed to the diminished enzyme activity by structure/function analysis of mutant LH1 constructs, in which C369 and the nine other cysteines were individually mutated to serine by site-directed mutagenesis of a normal pAcGP67/LH1cDNA construct. Following their expression in an Sf9 insect cell/baculovirus system, SDS-PAGE and Western analysis showed that equivalent levels of correctly-sized (85-kDa) products were secreted. The mutation of residues C369 and also C375, C552 and C687 virtually eliminated LH activity, whereas mutations of C267, C270, and C680 had an intermediate effect. In contrast, the C204S, C484S and C566S constructs had normal activity. Although disulfide bond formation may affect the relative contribution of each cysteine to LH activity, catalytic activity does not appear to be directly related to dimerization of the enzyme.


Subject(s)
Cysteine/metabolism , Ehlers-Danlos Syndrome/enzymology , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Sequence Deletion , Alleles , Cells, Cultured , Cysteine/genetics , Ehlers-Danlos Syndrome/genetics , Heterozygote , Humans , Mutagenesis, Site-Directed , Oxidation-Reduction , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Protein Denaturation
4.
Biochem Biophys Res Commun ; 245(1): 185-9, 1998 Apr 07.
Article in English | MEDLINE | ID: mdl-9535805

ABSTRACT

Maleylated-bovine serum albumin (maleyl-BSA) elicits transcription and secretion of a number of proinflammatory genes via ligation of the low-affinity scavenger receptor (SR) on macrophages. We now demonstrate that while neither maleyl-BSA, nor interferon-gamma (INF-gamma) alone induce nitric oxide (NO) production, when combined they promote release of NO from murine peritoneal macrophages. This effect was blocked by treatment with oxidized-low density lipoprotein. Maleyl-BSA activated NF-kappaB dimers capable of binding the NF-kappaBd sequence unique to the iNOS promoter, but this failed to induce significant new transcription or accumulation of iNOS mRNA. The combination of maleyl-BSA and IFN-gamma failed to demonstrate synergy at the transcriptional or mRNA levels, as these levels were comparable to those elicited by IFN-gamma alone. These studies suggest that the synergy in NO production between maleyl-BSA and IFN-gamma occurs after the accumulation of iNOS-specific mRNA, possibly at the translational or post-translational level.


Subject(s)
Macrophages, Peritoneal/metabolism , Nitric Oxide/metabolism , Serum Albumin, Bovine/pharmacology , Animals , DNA-Binding Proteins/analysis , Gene Expression Regulation, Enzymologic/genetics , Interferon-gamma/pharmacology , Lipoproteins, LDL/pharmacology , Mice , Mice, Inbred Strains , NF-kappa B/metabolism , Promoter Regions, Genetic/genetics , RNA, Messenger/metabolism , Transcription, Genetic/genetics
5.
Mol Pharmacol ; 52(3): 421-9, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9281604

ABSTRACT

Aspirin has been reported to inhibit the activation of nuclear factor-kappaB (NF-kappaB) through stabilization of inhibitor kappaB (IkappaB). This observation led us to investigate the role of aspirin in suppressing the activation of the NF-kappaB-regulated tumor necrosis factor-alpha (TNF-alpha) gene expression in primary macrophages. We now report that therapeutic doses of aspirin suppress lipopolysaccharide-inducible NF-kappaB binding to an NF-kappaB binding site in the TNF-alpha promoter, lipopolysaccharide-induced TNF-alpha mRNA accumulation, and protein secretion. IkappaB is also stabilized under these conditions. The aspirin-initiated stabilization of IkappaB, suppression of induced TNF-alpha mRNA, and NF-kappaB binding to the TNF-alpha promoter are blocked by pretreatment with pertussis toxin. These studies suggest that aspirin may exert significant anti-inflammatory effects by suppressing the production of macrophage-derived inflammatory mediators.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Transcription Factors , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Binding Sites , Cells, Cultured , Gene Expression/drug effects , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , NF-kappa B/drug effects , NF-kappa B/metabolism , Pertussis Toxin , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , RNA, Messenger/metabolism , Transcription Factor RelB , Virulence Factors, Bordetella/pharmacology
6.
J Leukoc Biol ; 60(6): 784-92, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8975883

ABSTRACT

The interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated-bovine serum albumin (maleyl-BSA) binds a low-affinity SR, initiating PIP2 hydrolysis, [Ca2+]i spikes, phospholipase A2 (PLA2) activation, nuclear factor-kappa(B) (NF-kappa(B)) binding to its cognate nucleotide and tumor necrosis factor alpha (TNF-alpha) gene transcription. We recently reported that oxidized low-density lipoprotein (ox-LDL), which binds another macrophage SR, induced pertussis-toxin-sensitive hydrolysis of PIP2 and elevations in [Ca2+]i [J. Biol. Chem. 270, 3475-3478, 1995]. By contrast, maleyl-BSA-initiated events were not pertussis toxin-sensitive and produced less [Ca2+]i spiking than ox-LDL. Furthermore, maleyl-BSA led to binding of NF-kappa(B) to its cognate nucleotide and TNF-alpha gene transcription, whereas ox-LDL suppressed these events. Collectively, this data suggests that maleyl-BSA and ox-LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects.


Subject(s)
Albumins/pharmacology , Calcium/metabolism , Macrophages/drug effects , Membrane Proteins , NF-kappa B/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Receptors, Immunologic/physiology , Receptors, Lipoprotein , Serum Albumin, Bovine , Tumor Necrosis Factor-alpha/genetics , Animals , Diglycerides/metabolism , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/physiology , Hydrogen-Ion Concentration , Inositol 1,4,5-Trisphosphate/metabolism , Lipoproteins, LDL/pharmacology , Mice , Mice, Inbred C57BL , Pertussis Toxin , Phospholipases A/metabolism , Phospholipases A2 , Receptors, Scavenger , Scavenger Receptors, Class B , Second Messenger Systems/physiology , Signal Transduction , Sodium/metabolism , Sodium-Hydrogen Exchangers/metabolism , Transcription, Genetic/drug effects , Virulence Factors, Bordetella/pharmacology
7.
J Leukoc Biol ; 57(3): 427-33, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7884314

ABSTRACT

A critical step in development of atherosclerosis is the interaction of oxidized low-density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl-LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP-1, TNF-alpha, IL-1 alpha, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)-stimulated macrophages. By contrast, acetyl-LDL does not inhibit these genes at the doses at which oxidized-LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages.


Subject(s)
Chemotactic Factors/genetics , Cytokines/genetics , Interleukin-1/genetics , Lipoproteins, LDL/chemistry , Macrophages/drug effects , Nitric Oxide/metabolism , Tumor Necrosis Factor-alpha/genetics , Animals , Cells, Cultured , Chemokine CCL2 , Chemokine CXCL1 , Chemokines , Chemokines, CXC , Cytotoxicity, Immunologic , Gene Expression , Humans , Immunity, Cellular , In Vitro Techniques , Lipopolysaccharides/pharmacology , Lipoproteins, LDL/pharmacology , Macrophage Activation/drug effects , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Oxidation-Reduction , RNA, Messenger/genetics , Tumor Cells, Cultured
8.
J Biol Chem ; 270(8): 3475-8, 1995 Feb 24.
Article in English | MEDLINE | ID: mdl-7876078

ABSTRACT

The interaction of oxidized low density lipoprotein (ox-LDL) and macrophages is generally believed to be a significant inductive step in atherogenesis. Endocytosis of ox-LDL by scavenger receptors (SR) on macrophages is one result of this interaction, as is suppressed expression of several lipopolysaccharide (LPS)-stimulated, inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha). Events subsequent to SR ligation, including intracellular signaling events if any, have not been established. We report here that ox-LDL initiates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate 2 (PIP2) and intracellular fluxes of Ca2+ in macrophages, both of which are sensitive to pertussis toxin. ox-LDL also suppresses the LPS-induced binding of macrophage extracts to an NF kappa B sequence oligonucleotide and the LPS-initiated accumulation of RNA specific for TNF-alpha. These latter two effects are pertussis toxin-sensitive. Ligation of SR by ox-LDL thus initiates a pertussis toxin-sensitive signaling pathway in macrophages, which involves hydrolysis of PIP2 and which can suppress expression of the TNF-alpha gene by modulating activation of NF kappa B.


Subject(s)
Lipoproteins, LDL/pharmacology , Macrophages/drug effects , NF-kappa B/metabolism , Pertussis Toxin , Signal Transduction , Virulence Factors, Bordetella/pharmacology , Animals , Base Sequence , Cells, Cultured , DNA , Hydrolysis , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oxidation-Reduction , Protein Binding
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