Reductive metabolism In vivo of trans-4-phenyl-3-buten-2-one in rats and dogs.

The reductive metabolism in vivo of a flavoring additive, trans-4-phenyl-3-buten-2-one (PBO; trans-methyl styryl ketone) was investigated in rats and dogs. In both species, the double bond-reduced product, 4-phenyl-2-butanone (PBA), was detected by HPLC as the predominant species in blood after i.v. administration of PBO. PBA detected in rat blood was identified by comparison to the authentic sample. In contrast, the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) was also detected as a minor metabolite of PBO in both species. The area under the curve of PBOL in rat blood was only 3% of that of PBA. PBO was mutagenic in the Ames test using Salmonella typhimurium TA 100 when S-9 mix was added, but PBA and PBOL were not. It appears that PBO is mainly metabolized to PBA in vivo in rats and dogs as a detoxification pathway.     

Reductive metabolism of the sanguinarine iminium bond by rat liver preparations

BackgroundSanguinarine (SA) is a quaternary benzo[c]phenanthridine alkaloid that is mainly present in the Papaveraceae family. SA has been extensively studied because of its antimicrobial, anti-inflammatory, antitumor, antihypertensive, antiproliferative and antiplatelet activities. Metabolic studies demonstrated that SAbioavailability is apparently low, and the main pathway of SAmetabolism is iminium bond reduction resulting in dihydrosanguinarine (DHSA) formation. Nevertheless, the metabolic enzymes involved in SA reduction are still not known in detail. Thus, the aim of this study was to investigate the rat liver microsomes and cytosolinduced SA iminium bond reduction, and to examine the effects of cytosol reductase inhibitors on the reductive activity.MethodsDHSAformation was quantified by HPLC. The possible enzymes responsible for DHSAformation were examined using selective individual metabolic enzyme inhibitors.ResultsWhen SA was incubated with liver microsomes and cytosol in the absence of NAD(P)H, DHSA, the iminium bond reductive metabolite was formed. The reductase activity of the liver microsomes and cytosol was also enhanced significantly in the presence of NADH. The amount of DHSAformed in the liver cytosol was 4.6-fold higher than in the liver microsomes in the presence of NADH. The reductase activity in the liver cytosol was inhibited by the addition of flavin mononucleotide and/or riboflavin. Inhibition studies indicated that menadione, dicoumarol, quercetin and 7-hydroxycoumarin inhibited rat liver cytosol-mediated DHSA formation in the absence of NADH. However, only menadione and quercetin inhibited rat liver cytosol-mediated DHSA formation in the presence of NADH.ConclusionsThese results suggest that the SAiminium bond reduction proceeds via two routes in the liver cytosol. One route is direct non-enzymatic reduction by NAD(P)H, and the other is enzymatic reduction by possible carbonyl and/or quinone reductases in the liver cytosol.     

Metabolism of the alpha,beta-unsaturated ketones, chalcone and trans-4-phenyl-3-buten-2-one, by rat liver microsomes and estrogenic activity of the metabolites.

When chalcone and trans-4-phenyl-3-buten-2-one (PBO) were incubated with liver microsomes of untreated rats in the presence of NADPH, 4-hydroxychalcone and trans-4-(4-hydroxyphenyl)-3-buten-2-one (4-OH-PBO), respectively, were formed as major metabolites. Two minor metabolites of chalcone, 4'-hydroxychalcone and 2-hydroxychalcone, were also observed. The oxidase activity affording 4-hydroxychalcone was inhibited by SKF 525-A, disulfiram, ketoconazole, and alpha-naphthoflavone. The oxidase activities leading to 4-hydroxychalcone and 4'-hydroxychalcone were enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats, respectively. The activity generating 2-hydroxychalcone was enhanced in liver microsomes of 3-methylcholanthrene- and dexamethasone-treated rats. The oxidation of PBO to 4-OH-PBO was inhibited by SKF 525-A, ketoconazole, disulfiram, and sulfaphenazole. This activity was enhanced in liver microsomes of 3-methylcholanthrene-, acetone- and phenobarbital-treated rats. 4-Hydroxylation, 4'-hydroxylation, and 2-hydroxylation of chalcone were catalyzed by rat recombinant cytochrome P450 1A1, 1A2, and 2C6; by 1A1 and 2C6; and by 1A1 and 3A1, respectively. PBO was oxidized by cytochrome P450 1A1, 1A2, 2C6, and 2E1. Chalcone and PBO were negative in an estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, 4-hydroxychalcone, 2-hydroxychalcone, 4'-hydroxychalcone, and 4-OH-PBO exhibited estrogenic activity.     

Reductive metabolism of p,p'-DDT and o,p'-DDT by rat liver cytochrome P450.

The in vitro metabolism of p,p'-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], an important environmental pollutant, was examined in rat liver, focusing on reductive dechlorination. When p,p'-DDT was incubated with liver microsomes of rats in the presence of NADPH or NADH, a dechlorinated metabolite, p,p'-DDD [1,1-dichloro-2,2-bis(4-chlorophenyl)ethane], was formed under anaerobic conditions together with a dehydrochlorinated metabolite, p,p'-DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene]. p,p'-DDE was also formed from p,p'-DDD by liver microsomes. The dechlorinating activity was inhibited by carbon monoxide, metyrapone, and SKF 525-A (proadifen hydrochloride), but the dehydrochlorinating activity was unaffected. The reductase activity toward p,p'-DDT was induced by the pretreatment of rats with phenobarbital and dexamethasone. The dechlorination was catalyzed enzymatically by recombinant cytochrome P450 2B1, 3A1, 2B6, and 3A4. When p,p'-DDT was incubated with liver microsomes of rats in the presence of both a reduced pyridine nucleotide and FMN, p,p'-DDD was also formed under anaerobic conditions. In this case, the dechlorinating activity was not abolished when the microsomes were boiled. The reductase activities were inhibited by carbon monoxide. Hematin exhibited reductase activity toward p,p'-DDT in the presence of NADH and FMN. The activity of hematin was also supported by FMNH(2). The reductive dechlorination also seems to proceed nonenzymatically with the reduced flavin, catalyzed by the heme group of cytochrome P450. Similar enzymatic and nonenzymatic reducing activities were observed toward o,p'-DDT [1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane].     

Reductive metabolism of nitrofurantoin by rat lung and liver in vitro

In the present study, the metabolism of NF has been examined in detail in both rat lung and liver 9000 g supernatants using a specific radiometric HPLC assay. Over 92% of the total radioactivity chromatographed with authentic NF after incubations from either organ were carried out under oxygen for 60 min. Under anaerobic conditions, only 19% and 5% of the total unbound radioactivity corresponded to unchanged NF in lung and liver respectively. At least 4 metabolites were evident from the HPLC trace (M1, M2, M3, M4 according to increasing retention times). In the absence of oxygen, liver 9000 g supernatants generated 65% more M1 and 260% more M3 than did lung 9000 g supernatants, but the lung produced significantly more M4. Covalent binding to tissue macromolecules was similar in both tissues under oxygen but was 7 times greater in lung than in liver in the absence of oxygen (compared per unit protein). Neither piperonyl butoxide nor indomethacin affected NF metabolism. However, allopurinol almost completely inhibited the anaerobic and aerobic (superoxide generation measured by the rate of acetylated cytochrome c reduction) metabolism in the lung with little or no effect in the liver. The data indicate a quantitative difference in NF metabolism between the two tissues that may be related to the organ-selective toxicity of the drug.     

The metabolism of canrenone in vitro by rat liver preparations

1. The metabolism of [1-³H]canrenone, a primary metabolite of spironolactone and potassium canrenoate, by rat liver preparations in vitro has been investigated.

2. Canrenone was metabolized by 3-oxo-δ⁴-reduction to give 3α-hydroxy-5β-spirolactones, and also by a number of O₂ and NADPH-dependent microsomal hydroxylation reactions.

3. A major metabolic route requiring the presence of a microsomal fraction, but apparently independent of oxygen and NADPH, led to the formation of a number of compounds tentatively identified as trihydroxy-spirolactones.     

Reductive metabolism of furazolidone by Escherichia coli and rat liver in vitro

The metabolism of furazolidone by rat liver and Escherichia coli was characterized in vitro under aerobic and anaerobic incubation conditions. Rat liver 9000g supernatant rapidly metabolized 14C-furazo-lidone to more polar metabolites in the presence or absence of oxygen when NADPH was provided as a cofactor. At least five polar radiolabeled metabolites were detected in these incubations by high pressure liquid chromatography. Moreover, a significant (30-40%) proportion of the total radiolabeled metabolites remained tightly associated with liver protein despite repeated organic solvent extractions of the tissue. The major solvent-extractable metabolites produced under aerobic and anaerobic incubation conditions were isolated and analyzed by mass spectrometry. The mass spectra indicated that these derivatives possessed the same chemical structure. Subsequently, this metabolite was unequivocally identified as 3-(4-cyano-2-oxobutylideneamino)-2-oxazolidinone, an end product of reductive metabolism of the nitro group of furazolidone. The formation of the reduced metabolite under aerobic conditions indicated that this metabolic pathway was markedly less sensitive to oxygen than many previously studied nitroreduction reactions catalyzed by mammalian enzymes. This NADPH-dependent, oxygen-insensitive nitroreductase activity was further localized to the microsomal fraction of rat liver. E. coli also rapidly metabolized furazolidone (FZN) to a complex series of metabolites, including the reduced cyano metabolite, under both aerobic and anaerobic conditions. Sonic lysis of the bacteria released an NADPH-dependent, oxygen-insensitive nitroreductase which converted FZN to the cyano metabolite and other unidentified derivatives. The complete reduction of FZN by the solubilized bacterial enzyme was strongly inhibited by the addition of the thiol nucleophile glutathione to the incubation medium.     

Contribution of N-oxygenation to the metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) by various liver preparations

Liver microsomes from uninduced mice and rats catalyze NADPH- and oxygen-dependent N-oxygenation of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The N-oxide is the principal product and accounts for 95-96% of the total MPTP metabolized by microsomes. Demethylation of MPTP is detectable but the rate of nor-MPTP formation was never more than 4-6% of the rate of N-oxygenation. Studies on the biochemical mechanisms for N-oxygenation of MPTP suggest that this reaction is catalyzed exclusively by the flavin-containing monooxygenase. This conclusion is based on the effects of selective cytochrome P-450 inhibitors, positive effectors, and alternate substrates for the flavin-containing monooxygenase as well as on studies with the purified hog liver enzyme. MPTP is an excellent substrate for this monooxygenase with a Km of 30-33 microM. Limited studies with human liver whole homogenates suggest that N-oxygenation is also a major route for the metabolism of MPTP in man and the rate of N-oxide formation is approximately equal to the rate of mitochondrial monoamine oxidase-dependent MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium species) production.     

Photolysis of 3-methyl- and 3-phenyl-4-amino-2-methyl-1-phenyl-3-pyrazolin-5-one (author's transl)

Photolysis of 3-Methyl- and 3-Phenyl-4-amino-2-methyl-phenyl-3-pyrazolin-5-one During the irradiation of aqueous solutions of 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one ( 1d ) besides the compounds 2 and 3 characteristic photolysis products of pyrazolones (Type I), the compound 12 is obtained, which was formed from two molecules of 1d . 4-Amino-2-methyl-1,3-diphenyl-3-pyrazolin-5-one, however isomerises to 8, which upon further irradiation fragments into several components two of them being N-phenyloxamide and N,N′-diphenyloxamide.     

In vitro metabolism of 1-phenyl-2-(n-propylamino) propane (N-propylamphetamine) by rat liver homogenates.

In vitro incubation of (+/-)-N-(n-propyl)amphetamine (NPA) with the 12000 g supernatant fraction of rat liver hmogenate resulted in the formation of two N-oxygenated products identified as N-hydroxy-1-phenyl-2(n-propylamino)propane and N-[(1-methyl-2-phenyl) ethyl]-1-propanamine N-oxide by g.l.c., g.l.c.-m.s. and t.l.c. Amphetamine, phenylacetone,...     

5-氯-6-苯基-2H-哒嗪-3-酮的合成

6-苯基-2H-哒嗪-3-酮(2)经三氯化磷/氯气氯代制得3,5-二氯-6-苯基哒嗪,经氢氧化钠皂化,再用盐酸调至pH 8.5得到5-氯-6-苯基-2H-哒嗪-3-酮,总收率约24%(以2计).副产物3-氯-5-羟基-6-苯基哒嗪可作为氯代反应的原料回收使用.     

Reductive dechlorination of chloramphenicol by rat liver microsomes

When chloramphenicol was incubated with rat liver microsomes anaerobically, it was metabolized predominantly to deschloro-chloramphenicol and products that become irreversibly bound to microsomal protein. Cytochromes P-450 induced by phenobarbital appeared to catalyze these reactions most effectively. Glutathione increased the formation of deschloro-chloramphenicol by 13% and decreased the amount of the irreversibly bound product by 18%, respectively. Only a small amount of the nitroaromatic-reduced product, chloramphenicol amine, was detected by high pressure liquid chromatography. These results are consistent with chloramphenicol being biotransformed and activated by cytochromes P-450 anaerobically through predominantly reductive dechlorination.     

Uptake, metabolism, and secretion of 3'-methyl-N,N-dimethyl-4-aminoazobenzene by isolated perfused rat liver

Uptake, metabolism, and biliary secretion of 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) were studied in isolated rat liver which was perfused with protein-free fluorocarbon medium. [14C]3'-Me-DAB (5-10 nmol) was injected into the portal vein and allowed to recirculate. The recovery of radioactivity in bile was 7.5, 14, and 20% at 15, 30, and 45 min of injection, respectively. At 45 min, the liver contained an additional 17% of injected radioactivity. Azo dye metabolites in perfused liver differed from those in vivo; metabolites co-migrating with 3'-CHO-DAB and 3'-methyl-n,n-methyl aminoazobenzene (Me-MAB) (and the parent compound 3'-Me-DAB) were present, while metabolites co-migrating with 3'-Me-4'-OH-AB and 3'-CH2OH-MAB were increased and metabolites co-migrating with 3'-CH2OH-DAB were decreased. In bile from perfused liver, metabolites co-migrating with 3'-CHO-DAB and 3'-Me-MAB were undetectable. When proteins from normal rat bile were injected into portal vein 15 min after the administration of 3'-Me-DAB, the compounds co-migrating with 3'-Me-MAB, 3'-CHO-DAB, 3'-Me-4'-OH-AB, and 3'-CH2OH-MAB decreased, and compounds co-migrating with 3'-CH2OH-DAB increased in the liver; in bile, there was increase in 3'-Me-MAB, 3'-CHO-DAB, and 3'-Me-4'-OH-MAB, which there was a decrease in N-Ac-3'-Me-4'-OH-AB and 3'-COOH-DAB. Appearance of protein-metabolite adducts in bile was also observed after addition of normal bile proteins to the perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reductive metabolism of the carcinogen 4-(5-nitro-2-furyl)thiazole to 1-(4-thiazolyl)-3-cyano-1-propanone by rat liver subcellular fractions

The reductive metabolism of 4-(5-nitro-2-furyl)thiazole (NFT), a rat mammary gland and forestomach carcinogen, was examined in vitro using rat liver tissues. NFT was reduced by rat liver cytosol or microsomes on anaerobic incubation with NADPH. The stoichiometry of microsomal reduction revealed that about 3 moles of NADPH were used per mole of NFT. Gas chromatographic analysis of the reaction mixture showed a major peak with a retention time of about 4.0 min in contrast to NFT with a retention time of about 6.5 min. Catalytic hydrogenation of NFT with palladium and activated carbon yielded a product with a retention time of 4.0 min. The component corresponding with the above metabolite was isolated from chemically reduced NFT and identified as 1-(4-thiazolyl)-3-cyano-l-propanone. The metabolite derived from enzymatic reduction had chromatographic and spectral properties and a mass spectral fragmentation pattern similar to those obtained chemically. These data establish that the enzymatically derived product is identical to that obtained by chemical reduction and that it corresponds to 1(4thiazolyl)3cyano1propanone.     

Synthesis of N-aryl-N'-[2-phenyl-3-quinazoline(3H)-4-one] acylthiourea derivatives as anticonvulsants.

By the reaction of [2-phenyl-3-quinazolineo (3H)-4-one] acyl isothiocynates and appropriate aryl amines in acetone, 24 new compounds, having a substituted thiourea grouping at the 3-position of the quianozolone moiety, were prepared. All compounds, except two, showed different degree of protection against pentetrazol induced seizures test in albino mice. While studying the effect of structural variation no definite pattern could be observed due to variations in 1-aryl moiety, but it was noticed that generally the branching or lengthening of 3-acyl chain either diminishes or does not effect the activity.     

Differential induction of 3-ethyl-2,6-dimethyl-4H-pyrido (1,2-alpha)pyrimidin-4-one metabolism by phenobarbital and 3-methylcholanthrene in microsomes and isolated perfused rat liver

1. The in vitro metabolism of 3-ethyl-2,6-dimethyl-4H-[2-14C]pyrido(1,2-alpha)pyrimidin-4-one (PYPY) was studied in liver microsomes and isolated perfused liver of 3-methylcholanthrene (MC) or phenobarbital (PB)-treated, and untreated rats. 2. Hydroxylation of the alkyl substituents was the main metabolic pathway for PYPY in both in vitro systems of untreated, and MC-treated animals, but with different proportions of the metabolites. PB enhanced the rate of ring hydroxylation, especially in the microsomes, and the product of this reaction became the main metabolite of PYPY biotransformation. Ring hydroxylation reactions in the microsomes and in the isolated perfused liver led to different products. 3. Differences arose in the rate of some oxidative reactions measured in the two in vitro systems resulting in altered metabolic patterns. PB enhanced not only quantitative but qualitative differences in the two systems. 4. The altered metabolite profile observed with whole liver compared with the products of microsomes, and the enhanced amount of water-soluble metabolites due to PB treatment in experiments with perfused liver indicate the involvement of further metabolic processes, perhaps conjugation reactions, in PYPY metabolism in the perfused liver. 5. The differences observed in the inducibility of some oxidative reactions by MC and PB indicate the involvement of at least three distinct cytochrome P-450 isozymes in the metabolism of PYPY.