Adaptive response and the influence of ageing: effects of low-dose irradiation on cell growth of cultured glial cells.

PURPOSE: To examine the molecular mechanism of radiation adaptive response (RAR) for the growth of cultured glial cells and to investigate the influence of ageing on the response. MATERIALS AND METHODS: Glial cells were cultured from young and older rats (1 and 24 months). RAR for the growth of glial cells conditioned with a low dose of X-rays and subsequently exposed to a high dose of X-rays was examined for cell number and BrdU incorporation. Involvement of the subcellular signalling pathway factors in RAR was investigated using their inhibitors, activators, and mutated and knockout glial cells. RESULTS: RAR was observed in cells cultured from young rats but was not in cells from older animals. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNA-PK) or phosphatidylinositol 3-kinase (PI3K) suppressed RAR. The activators of PKC instead of low-dose irradiation also caused RAR. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice (CB-17 scid) and ataxia-telangiectasia mutated (Atm) knockout mice showed no RAR. CONCLUSION: The results indicated that PKC, ATM, DNAPK and/or PI3K were involved in RAR for growth and BrdU incorporation of cultured glial cells and RAR decreased with ageing.

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation.

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 microM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.

Reduction of hydroxamic acids to the corresponding amides catalyzed by rabbit blood.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.

Debromination of (alpha-bromoiso-valeryl)urea catalysed by rat blood.

(Alpha-bromoiso-valeryl) urea, a sedative or hypnotic, is metabolized to (3-methylbutyryl)urea by reductive debromination. This study was designed to evaluate the role of blood in the debromination of (alpha-bromoiso-valeryl) urea. Rat blood containing an electron donor had significant debrominating activity toward (alpha-bromoiso-valeryl)urea. This debromination proceeded by enzymatic and non-enzymatic processes which required both NADH (or NADPH) and flavin mononucleotide (FMN), under anaerobic conditions. The debrominating activity was sensitive to inhibition by carbon monoxide, and the pH optimum was 8.5. When FMN was replaced by flavin adenine dinucleotide (FAD) or riboflavin, similar results were obtained. The optimum concentration of flavins was 10(-4) M. The reductive debromination was also mediated by rat erythrocytes, but not by plasma. When the blood or erythrocytes were boiled, the debrominating activity was not abolished, but was enhanced, suggesting that the activity arises from the haemoglobin in erythrocytes, and haemoglobin had debrominating activity when supplemented with both a reduced pyridine nucleotide and a flavin. Furthermore, haematin had significant debrominating activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FMN. The results imply that the debromination proceeds in two steps--enzymatic or non-enzymatic reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, then non-enzymatic reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea catalysed by the haem group of rat haemoglobin in the presence of the reduced flavin.

A unique tertiary amine N-oxide reduction system composed of quinone reductase and heme in rat liver preparations.

The results of this study show the quinone-dependent reduction of tertiary amine N-oxides to the corresponding tertiary amines by rat liver preparations. The reduction of imipramine N-oxide to imipramine mediated by liver mitochondria, microsomes, and cytosol proceeded in the presence of both NAD(P)H and menadione under anaerobic conditions. When menadione was replaced with 1, 4-naphthoquinone or 9,10-anthraquinone, similar results were obtained in the cytosolic reduction. The quinone-dependent reducing activity in liver cytosol was inhibited by dicumarol and carbon monoxide. This result suggested that the activity is caused by DT-diaphorase, a cytosolic quinone reductase, and hemoproteins in liver cytosol. In fact, catalase and hemoglobin showed the ability to reduce imipramine N-oxide when supplemented with DT-diaphorase. The hemoproteins also exhibited the N-oxide reductase activity with reduced menadione, menadiol. The N-oxide reductase activity of the hemoproteins was also exhibited with 1,4-dihydroxynaphthalene, 1,4,9, 10-tetrahydroxyanthracene, or 1,4-dihydroxy-9,10-anthraquinone. Furthermore, hematin revealed a significant N-oxide-reducing activity in the presence of menadiol. The reduction appears to proceed in two steps. The first step is reduction of menadione to menadiol by a quinone reductase with NADPH or NADH. The second step is nonenzymatic reduction of tertiary amine N-oxides to tertiary amines by menadiol, catalyzed by the heme group of hemoproteins. Cyclobenzaprine N-oxide and brucine N-oxide were also transformed similarly to the corresponding amine by the quinone-dependent reducing system.

Quinone-dependent tertiary amine N-oxide reduction in rat blood.

Rat blood exhibited a significant quinone-dependent N-oxide reductase activity towards imipramine N-oxide. The reduction mediated by the blood proceeded in the presence of both NAD(P)H and menadione under anaerobic conditions. When menadione was replaced with 1,4-naphthoquinone or 9,10-phenanthrenequinone, similar results were obtained. The reduction was also mediated by the combination of rat erythrocytes and plasma. The reducing activity was inhibited by dicumarol and carbon monoxide. When boiled plasma was combined with untreated erythrocytes, the N-oxide reducing activity was abolished. In contrast, when boiled erythrocytes were combined with untreated plasma, the activity was unchanged. These results suggest that the activity is caused by the heme of hemoglobin in erythrocytes and quinone reductase in plasma. In fact, erythrocytes and hemoglobin have the ability to reduce the N-oxide when supplemented with DT-diaphorase purified from rat liver in the presence of both NAD(P)H and menadione. Hemoglobin also exhibits N-oxide reductase activity with reduced menadione (menadiol). Furthermore, hematin exhibits a significant reducing activity in the presence of menadiol. The reduction appears to proceed in two steps. The first step is enzymatic reduction of quinones to dihydroquinones by quinone reductase(s) with NADPH or NADH in plasma. The second step is nonenzymatic reduction of imipramine N-oxide to imipramine by the dihydroquinones, catalyzed by the heme group of hemoglobin in erythrocytes. Cyclobenzaprine N-oxide and brucine N-oxide are similarly transformed to the corresponding amines by the above reducing system in blood. These results suggest that blood plays an important role in the reduction of tertiary amine N-oxides to tertiary amines.

Reductase activity of aldehyde oxidase toward the carcinogen N-hydroxy-2-acetylaminofluorene and the related hydroxamic acids.

Liver aldehyde oxidase (EC 1.2.3.1) was capable of reducing N-arylacetohydroxamic acids, N-hydroxy-2-acetyl-aminofluorene, N-hydroxy-4-acetylaminobiphenyl and N-hydroxyphenacetin, to the corresponding amides in the presence of an electron donor of the enzyme under anaerobic conditions. When supplemented with an electron donor of the enzyme, a significant reduction of N-hydroxy-2-acetylaminofluorene occurred, which was sensitive to an inhibitor of the enzyme. These observations were made with cytosolic fractions prepared from the livers of rabbits, guinea pigs, rats and mice.

Monoallelic expression of normal mRNA in the PIT1 mutation heterozygotes with normal phenotype and biallelic expression in the abnormal phenotype.

The combined deficiency of thyrotropin, growth hormone and prolactin, caused by PIT1 abnormality manifests in the homozygous or heterozygous state. We studied a patient having an allele with Arg271Trp mutation, which produces clinical symptoms in heterozygotes by a dominant-negative effect. However, in the family, her father, grandmother and aunts had the same mutation without clinical symptoms, although the proband had typical phenotypic expression. We analyzed the PIT1 transcript in peripheral lymphocytes by reverse transcription-polymerase chain reaction and found monoallelic expression of normal allele in the father and grandmother and skewed pattern of biallelic expression in the proband. The phenotypic expression of PIT1 abnormality may depend on different transcription of the PIT1 gene.

Metabolism of drugs in the eye. Menadione-dependent reduction of tertiary amine N-oxide by preparations from bovine ocular tissues.

As described previously, the microsomes and cytosol from bovine ciliary body exhibited a significant reductase activity toward tertiary amine N-oxide such as imipramine N-oxide when supplemented with menadione. In the present study, the menadione-dependent N-oxide reduction was further examined with preparations of bovine ocular tissues. The reduction of imipramine N-oxide occurred much more significantly when the microsomes and cytosols from bovine ciliary body were supplemented with both menadione and NAD(P)H, compared with menadione alone. The cytosolic menadione-dependent reduction, but not the microsomal one, was markedly inhibited by dicumarol, suggesting the involvement of DT-diaphorase in the reaction. Localization of the menadione-dependent N-oxide reductase activity in bovine ocular tissues indicated that the highest activity resided in the ciliary body, followed by retinal pigment epithelium-choroid, iris, retina and cornea. When the cytosol from bovine ciliary body was fractionated with ammonium sulfate, the distribution of the menadione-dependent N-oxide reductase activity in the resultant fractions was parallel, but roughly, to that of DT-diaphorase activity, supporting the assumption that the flavoenzyme was involved in the cytosolic menadione-dependent N-oxide reduction. We proposed a new mechanism for the metabolic reduction of tertiary amine N-oxide in the eye: Menadione is reduced to the corresponding diol by quinone-reducing enzymes and then tertiary amine N-oxide is reduced by the diol to the corresponding amine nonenzymatically.

Metabolism of drugs in the eye. Drug-reducing activity of preparations from bovine ciliary body.

Drug-metabolizing activities, especially the reductase activities towards N-oxide, hydroxamic acid, sulfoxide and nitro compounds were comparatively examined with bovine ciliary body. As described previously, the cytosol from the ocular tissue exhibits the nicotinamide N-oxide reductase activity when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. When the cytosol was fractionated with ammonium sulfate, followed by assays of aldehyde oxidase and nicotinamide N-oxide reductase activities in each fraction, the distribution of aldehyde oxidase activity in the resultant ammonium sulfate fractions was nearly parallel to that of nicotinamide N-oxide reductase activity. Furthermore, reductase activities towards drugs such as sulfoxide, hydroxamic acid and nitro compounds were observed with the cytosol in the presence of 2-hydroxypyrimidine or N1-methylnicotinamide. In general, these reductase activities of the fraction were markedly inhibited by menadione, an inhibitor of aldehyde oxidase. These results suggest that aldehyde oxidase present in ciliary body plays an important role in the reduction of a variety of xenobiotics in mammalian eyes. However, in the case of imipramine N-oxide, its reduction in the ocular tissue appears to be more readily catalyzed by a menadione-linked enzyme different from aldehyde oxidase.

Nicotinamide N-oxide reductase activity in bovine and rabbit eyes.

The nicotinamide N-oxide reductase activity of a variety of ocular tissues was investigated. The 9,000g supernatant of ciliary body, retinal pigment epithelium-choroid, iris, retina and cornea, but not lens, exhibited reductase activity under anaerobic conditions when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. Among these tissues, the highest activity was observed with ciliary body. When the 9,000g supernatant of ciliary body was fractionated, the 2-hydroxypyrimidine-linked reductase activity was mainly associated with the cytosolic fraction and was markedly inhibited by menadione, an inhibitor of aldehyde oxidase. Similarly, in the presence of 2-hydroxypyrimidine, the cytosolic fraction of rabbit ciliary body exhibited nicotinamide N-oxide reductase activity which was susceptible to inhibition by menadione. These facts strongly suggest that aldehyde oxidase present in mammalian eyes is involved in the reduction of nicotinamide N-oxide to nicotinamide.

Sulfoxide reduction catalyzed by guinea pig liver aldehyde oxidase in combination with one-electron reducing flavoenzymes.

The mechanism of the enhancing effect of methyl viologen (MV) and flavin-adenine dinucleotide (FAD) on sulfoxide reduction which is mediated by a combination of aldehyde oxidase (AO) from guinea pig liver and one-electron reducing flavoenzymes, such as milk xanthine oxidase (XO), was examined. The activity of anaerobic reduction of diphenyl sulfoxide (DPSO) to diphenyl sulfide (DPS) was less than 1 nmol/min/mg protein of AO preparation in a system consisting of hypoxanthine, XO and AO. However, the sulfoxide reduction by this system was enhanced about 6- and 100-fold by the additions of FAD and MV, respectively. In the system containing MV or FAD, other one-electron reducing flavoenzymes such as nicotinamide adenine dinucleotide (reduced form) (NADH) dehydrogenase, lipoamide dehydrogenase and glutathione reductase with an appropriate electron donor, could replace XO. The ability of supplemented flavoenzymes to facilitate DPSO reduction correlated with their abilities to reduce MV and FAD. When AO was omitted from the combined system, no sulfoxide reduction was observed. Stoichiometric study revealed that MV semiquinone and FADH2 were oxidized at ratios of 2 and 1 mol, respectively, per mol of DPS formed. These results indicate that either MV or FAD serves as an electron carrier from the supplemented flavoenzymes to AO, a terminal reductase of sulfoxide.

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase.

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.

Involvement of liver aldehyde oxidase in the reduction of nicotinamide N-oxide.

The present paper describes that mammalian liver aldehyde oxidase is involved in the reduction of nicotinamide N-oxide to nicotinamide. Rabbit liver aldehyde oxidase supplemented with its electron donor exhibited a significant nicotinamide N-oxide reductase activity under anaerobic conditions. Liver cytosols from rabbits, hogs, guinea pigs, hamsters, rats and mice, all of them, similarly exhibited the N-oxide reductase activity in the presence of an electron donor of aldehyde oxidase, but not xanthine oxidase. The cytosolic N-oxide reductase activity was almost completely inhibited by menadione, an inhibitor of aldehyde oxidase.

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.