Carcinogenicity of triethanolamine in mice and its mutagenicity after reaction with sodium nitrite in bacteria.
Mice fed a diet containing 0.3 or 0.03% triethanolamine developed malignant tumors. Females showed a high incidence of tumors in lymphoid tissues, while this type was absent in males. Tumors in other tissues were produced at a considerable rate in both sexes, but no hepatoma was found. Triethanolamine was not mutagenic to Bacillus subtilis by itself, but it became mutagenic after reacting with sodium nitrite under acidic conditions or when the mixture was heated. Although N-nitrosodiethanolamine, a known carcinogen and mutagen, was detected in the reaction mixture by thin-layer chromatography, it may not be the main mutagenic product, because the product was a stable and direct mutagen and its mutagenic activity was destroyed by liver enzymes, unlike N-nitrosodiethanolamine. The lethal and mutagenic DNA damages produced by this unidentified product were susceptible to some extent to the repair functions of the bacteria. (+info)
Enantioselective inhibition of the biotransformation and pharmacological actions of isoidide dinitrate by diphenyleneiodonium sulphate.
1. We have shown previously that the D- and L- enantiomers of isoidide dinitrate (D-IIDN and L-IIDN) exhibit a potency difference for relaxation and cyclic GMP accumulation in isolated rat aorta and that this is related to preferential biotransformation of the more potent enantiomer (D-IIDN). The objective of the current study was to examine the effect of the flavoprotein inhibitor, diphenyleneiodonium sulphate (DPI), on the enantioselectivity of IIDN action. 2. In isolated rat aortic strip preparations, exposure to 0.3 microM DPI resulted in a 3.6 fold increase in the EC50 value for D-IIDN-induced relaxation, but had no effect on L-IIDN-induced relaxation. 3. Incubation of aortic strips with 2 microM D- or L-IIDN for 5 min resulted in significantly more D-isoidide mononitrate formed (5.0 +/- 1.5 pmol mg protein(-1)) than L-isoidide mononitrate (2.1 +/- 0.7 pmol mg protein(-1)) and this difference was abolished by pretreatment of tissues with 0.3 microM DPI. DPI had no effect on glutathione S-transferase (GST) activity or GSH-dependent biotransformation of D- or L-IIDN in the 105,000 x g supernatant fraction of rat aorta. 4. Consistent with both the relaxation and biotransformation data, treatment of tissues with 0.3 microM DPI significantly inhibited D-IIDN-induced cyclic GMP accumulation, but had no effect on L-IIDN-induced cyclic GMP accumulation. 5. In the intact animal, 2 mg kg(-1) DPI significantly inhibited the pharmacokinetic and haemodynamic properties of D-IIDN, but had no effect L-IIDN. 6. These data suggest that the basis for the potency difference for relaxation by the two enantiomers is preferential biotransformation of D-IIDN to NO, by an enzyme that is inhibited by DPI. Given that DPI binds to and inhibits NADPH-cytochrome P450 reductase, the data are consistent with a role for the cytochromes P450-NADPH-cytochrome P450 reductase system in this enantioselective biotransformation process. (+info)
Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas.
CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans. (+info)
Microbial desulfurization of organic sulfur compounds in petroleum.
Sulfur removal from petroleum is important from the standpoint of the global environment because the combustion of sulfur compounds leads to the production of sulfur oxides, which are the source of acid rain. As the regulations for sulfur in fuels become more stringent, the existing chemical desulfurizations are coming inadequate for the "deeper desulfurization" to produce lower-sulfur fuels without new and innovative processes. Biodesulfurization is rising as one of the candidates. Several microorganisms were found to desulfurize dibenzothiophene (DBT), a representative of the organic sulfur compounds in petroleum, forming a sulfur-free compound, 2-hydroxybiphenyl. They are promising as biocatalysts in the microbial desulfurization of petroleum because without assimilation of the carbon content, they remove only sulfur from the heterocyclic compounds which is refractory to conventional chemical desulfurization. Both enzymological and molecular genetic studies are now in progress for the purpose of obtaining improved desulfurization activity of organisms. The genes involved in the sulfur-specific DBT desulfurization were identified and the corresponding enzymes have been investigated. From the practical point of view, it has been proved that the microbial desulfurization proceeds in the presence of high concentrations of hydrocarbons, and more complicated DBT analogs are also desulfurized by the microorganisms. This review outlines the progress in the studies of the microbial desulfurization from the basic and practical point of view. (+info)
Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.
The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury. (+info)
Oxidative bioactivation of the lactol prodrug of a lactone cyclooxygenase-2 inhibitor.
The lactol derivative of a lactone cyclooxygenase-2 inhibitor (DFU) was evaluated in vivo and in vitro for its potential suitability as a prodrug. DFU-lactol was found to be 10 to 20 times more soluble than DFU in a variety of aqueous vehicles. After administration of DFU-lactol at 20 mg kg-1 p.o. in rats, a Cmax of 7.5 microM DFU was reached in the plasma. After oral administration, the ED50s of DFU-lactol in the carrageenan-induced paw edema and lipopolysaccharide-induced pyresis assays in rats are comparable with the ED50s observed when dosing with DFU. Incubations of DFU-lactol with rat and human hepatocytes demonstrated that the oxidation of DFU-lactol can be mediated by liver enzymes and that a competing pathway is direct glucuronidation of the DFU-lactol hydroxyl group. Assays with subcellular fractions from rat liver indicated that most of the oxidation of DFU-lactol occurs in the cytosolic fraction and requires NAD(P)+. Human liver cytosol can also support the oxidation of DFU-lactol to DFU when NAD(P)+ is added to the incubations. Fractionation of human liver cytosolic proteins showed that at least three enzymes are capable of efficiently effecting the oxidation of DFU-lactol to DFU. Incubations with commercially available dehydrogenases suggest that alcohol and hydroxysteroid dehydrogenases are involved in this oxidative process. These data together suggest that lactols may represent useful prodrugs for lactone-containing drugs. (+info)
The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin: effect on human cytochrome P-450 and implications for metabolic drug interactions.
Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, was metabolized by human liver microsomes to 5-hydroxy-, 6-hydroxy-, and N-deisopropyl-fluvastatin. Total metabolite formation was biphasic with apparent Km values of 0.2 to 0.7 and 7.9 to 50 microM and intrinsic metabolic clearance rates of 1.4 to 4 and 0.3 to 1.5 ml/h/mg microsomal protein for the high and low Km components, respectively. Several enzymes, but mainly CYP2C9, catalyzed fluvastatin metabolism. Only CYP2C9 inhibitors such as sulfaphenazole inhibited the formation of both 6-hydroxy- and N-deisopropyl-fluvastatin. 5-Hydroxy-fluvastatin formation was reduced by compounds that are inhibitors of CYP2C9, CYP3A, or CYP2C8. Fluvastatin in turn inhibited CYP2C9-catalyzed tolbutamide and diclofenac hydroxylation with Ki values of 0.3 and 0.5 microM, respectively. For CYP2C8-catalyzed 6alpha-hydroxy-paclitaxel formation the IC50 was 20 microM and for CYP1A2, CYP2C19, and CYP3A catalyzed reactions, no IC50 could be determined up to 100 microM fluvastatin. All three fluvastatin metabolites were also formed by recombinant CYP2C9, whereas CYP1A1, CYP2C8, CYP2D6, and CYP3A4 produced only 5-hydroxy-fluvastatin. Km values were approximately 1, 2.8, and 7.1 microM for CYP2C9, CYP2C8, and CYP3A, respectively. No difference in fluvastatin metabolism was found between the CYP2C9R144 and CYP2C9C144 alleles, suggesting the absence of polymorphic fluvastatin metabolism by these alleles. CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A5 did not produce detectable amounts of any metabolite. This data indicates that several human cytochrome P-450 enzymes metabolize fluvastatin with CYP2C9 contributing 50-80%. Any coadministered drug would therefore only partially reduce the metabolic clearance of fluvastatin; therefore, the likelihood for serious metabolic drug interactions is expected to be minimal. (+info)
Rapid liquid chromatography with tandem mass spectrometry-based screening procedures for studies on the biotransformation of drug candidates.
The accelerated pace of contemporary drug discovery and development in the pharmaceutical industry has generated increasing demands for early information on the metabolic fate of candidate drugs to guide the selection of new compounds for clinical evaluation. In response to these demands, we have developed a procedure for the rapid analysis of complex biological mixtures for the presence of drug-related materials and have embarked on the development of novel computer-based approaches whereby such procedures can be automated. The goal of this work was to rapidly identify drug metabolites (derived either from a single substrate or from a mixture of substrates) formed in vivo or in vitro. The approach that we have developed relies on the use of generic chromatographic and mass spectrometric methods for analysis of mixtures of drugs and metabolites and on correlation analysis of tandem mass spectrometry spectra to distinguish drug-related components from endogenous materials. Cross-correlation of the spectra also is used to identify the relationship between each metabolite and its respective parent drug in the mixture. In this manner, metabolites of a mixture of several drugs may be analyzed in the time it normally would take to analyze the products from a single substrate. We show that this rapid analytical approach can, with only minor sacrifices in the completeness of the data, significantly increase the number of compounds whose metabolic fate can be elucidated in a given time. (+info)