Yuichi Kawazoe

Metabolism of furazolidone by milk xanthine oxidase and rat liver 9000g supernatant: Formation of a unique nitrofuran metabolite and an aminofuran derivative

Abstract In vitro metabolism of furazolidone ( N -(5-nitro-2-furfuryliden)-3-amino-2-oxazolidone) was investigated by using milk xanthine oxidase and rat liver 9000 g supernatant. As a result, a new type of reduction product was isolated as one of the main metabolites from the incubation mixture and it was tentatively identified as 2,3-dihydro-3-cyanomethyl-2-hydroxyl-5-nitro-1a, 2-di(2-oxo-oxazolidin-3-yl)iminomethyl-furo[2,3- b ]furan. In addition, the present study demonstrated the formation of N -(5-amino-2-furfurylidene)-3-amino-2-oxazolidone as a minor metabolite of nitrofuran in a milk xanthine oxidase system. The aminofuran derivative was easily degraded by milk xanthine oxidase under aerobic, but not anaerobic, conditions. The degradation appears to be due to superoxide anion radicals, hydroxyl radicals, and/or singlet oxygen, which are produced in this enzyme system.

Ammonium lon as a possible regulator of the commencement of rRNA synthesis in Xenopus laevis embryogenesis

Recently, we have shown that ammonium salts and amines at an external concentration of 3 mM selectively inhibit rRNA synthesis in Xenopus disaggregated neurula cells. We studied here the change in the amount of ammonia within the embryo and its inhibitory action on the commencement of rRNA synthesis which normally occurs at the blastula stage of development. Ammonia exists at ca. 50 ng/egg (or ca. 3.0 mM at an intra-egg concentration) in the unfertilized egg. This level was maintained during cleavage and then sharply decreased during the blastula stage to the level of ca. 20 ng/embryo (or 1.2 mM) in postblastular stages. Ammonia was extracted from cleavage embryos in a form of ammonium chloride and confirmed to selectively inhibit rRNA synthesis in neurula cells. With authentic ammonium chloride, ammonia was found to be promptly incorporated into cells and to inhibit rRNA synthesis within 1 hr after treatment. In blastula cells, ammonium salts reversibly inhibited the commencement of the synthesis of rRNA, but not hnRNA, 5 S RNA and U1, U2, and U5 snRNAs. The inhibition was at the step of transcription of 40 S pre-rRNA but not the processing or degradation of the processed rRNA. Ammonium salts did not inhibit DNA synthesis, protein synthesis, cell division, and cellular reaggregation. These observations suggest that ammonium ion may be involved in the regulation of the commencement of rRNA synthesis in Xenopus embryogenesis, although it is not yet clear if the ammonium ion exerts its effect directly upon the rDNA transcription system.

Effects of various ammonium salts, amines, polyamines and α-methylornithine on rRNA synthesis in neurula cells of Xenopus laevis and Xenopus borealis

Xenopus neurula cells were cultured in a medium that contained ammonium salts, amines, polyamines or alpha-methylornithine, and their rRNA synthesis was examined. All the ammonium salts and amines, but not polyamines, were strong and selective inhibitors of rRNA synthesis at 1.25-5.0 mM. alpha-Methylornithine did not inhibit rRNA synthesis, although it inhibited ornithine decarboxylase, an enzyme claimed to be a direct stimulator of rRNA synthesis. During the treatment ammonium ions and monomethylamines were accumulated within the treated cells. However, monomethylamines did not induce the accumulation of ammonium ions, and vice versa. Ammonium salts and amines also selectively inhibited rRNA synthesis in Xenopus borealis neurula cells.

The mutagenic activity of ethyl N-hydroxycarbamate and its related compounds in Salmonella typhimurium

Alkyl N-hydroxycarbamates exhibited weak but significant mutagenic activity for Salmonella typhimurium TA100. The mutagenic potencies of these N-hydroxycarbamates were ranked thus: ethyl N-hydroxycarbamate greater than propyl N-hydroxycarbamate greater than methyl N-hydroxycarbamate. Acylation of ethyl N-hydroxycarbamate markedly enhanced its mutagenic activity for TA100. The highest mutagenic activity was observed with ethyl N-benzoyloxycarbamate among these acyl derivatives. Almost all the compounds were mutagenic to all the strains TA1535, TA100, TA98, especially to TA100.

Demonstration that inhibitor of rRNA synthesis in “charcoal extracts” of Xenopus embryos is artifactually produced ammonium perchlorate☆

Abstract A perchloric acid extract of Xenopus cleavage embryos partially purified with a charcoal column selectively inhibits rRNA synthesis in Xenopus neurula cells. The active substance has long been assumed to be nucleotide-related material, because it appeared to bind to a charcoal column. However, the active substance was proved to be ammonium perchlorate that was produced artifactually when a perchloric acid extract of cleavage embryos was eluted from a charcoal column with ammonia-alcohol. Ammonium ion, but not perchlorate ion, was responsible for the inhibitory activity.

Studies on enzymatic reduction of aliphatic nitro compounds: Reductive dimerization of β-nitrostyrene and 1-nitro-4-phenylbutadiene

Abstract Enzymatic reduction of aliphatic nitro compounds, β-nitrostyrene (I), 1-nitro-4-phenylbutadiene (II), 1-nitro-4-phenyl-1-butene (III), 1-nitro-2-phenylethane (IV), and nitrophenylethane (V) was investigated in a xanthine oxidase-hypoxanthine system. I and II were easily reduced by the enzyme system under anaerobic conditions, but III, IV, and V resisted to the enzymatic reduction. The reduction products of I and II were isolated from the reaction mixtures and were identified as dimolecular compounds, 1,4-dinitro-2, 3-diphenylbutane and 1,4-dinitro-2,3-distyrylbutane, respectively, by mass, nuclear magnetic resonance, and infrared spectrometries, and by elementary analyses.

Formation of novel nitrofuran metabolites in xanthine oxidase-catalyzed reduction of methyl 5-nitro-2-furoate.

After methyl 5-nitro-2-furoate was incubated with milk xanthine oxidase, three reduction products were isolated from the incubation mixture. Among them, two reduction products were new types of nitrofuran metabolites, i.e., metabolites 1 and 2 were identified as the dihydroxyhydrazine derivative (1,2-dihydroxy-1,2-di(5-methoxycarbonyl-2-furyl)hydrazine) and the hydroxylaminofuran derivative (methyl 5-hydroxylamino-2-furoate), respectively. Metabolite 3 was also identified as the aminofuran derivative (methyl 5-amino-2-furoate) by comparison with a synthetic sample.