DNA Distortion Caused by Uracil-Containing Intrastrand Cross-Links.

Four uracil-containing intrastrand cross-links have been detected in human cells upon UV irradiation of 5-bromouracil-containing DNA, namely 5'-G[8-5]U-3', 5'-U[5-8]G-3', 5'-A[8-5]U-3', and 5'-A[2-5]U-3'. These lesions feature unique composition and connectivity compared with other intrastrand cross-links reported in the literature. For the first time, structural information obtained using molecular dynamics (MD) simulations reveal that all four lesions distort the DNA helix, which can involve an extrahelical location of the cross-link, changes in the helical interactions of the complementary nucleotides, or disruption of hydrogen bonding in the flanking base pairs up to two positions from the cross-linked site; however, the degree of distortion varies between the cross-links, being affected by the sequence, nucleobase-nucleobase connectivity, and the purine involved. Most importantly, the relative distortion of the damaged DNA provides the first structural explanation for the observed abundances of the four uracil-containing cross-links. Furthermore, the highly distorted conformations suggest that these lesions will likely have severe implications for DNA replication and repair processes in cells.

5-bromodeoxyuridine induces transcription of repressed genes with disruption of nucleosome positioning.

5-Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of studies. Recently, we have demonstrated that BrdU disrupts nucleosome positioning on model plasmids mediated by specific AT-tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an α2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd-auxotrophic MATα cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT-tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabilized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associated with more marked disruption of nucleosome positioning on the promoter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5-bromouracil into the plasmid did not weaken the binding of the α2/Mcm1 repressor complex to its legitimate binding site, as revealed by an in vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters.

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

Mutagenic properties of 5-halogenuracils: correlated quantum chemical ab initio study.

The relative stability of all possible 5-bromouracil tautomers was studied theoretically in a gas phase, in a microhydrated environment (with one water molecule), and in bulk water. Tautomer structures were determined by gradient optimization at the correlated ab initio quantum chemical level with an extended basis set of atomic orbitals. The role of water was examined by using a self-consistent reaction field method. The relative stabilization and free energies in the gas phase, the microhydrated environment, and the bulk water clearly support the preference of the canonical keto form of 5-bromouracil in all mentioned environments. An increased abundance of enol tautomers when passing from uracil to 5-bromouracil is not supported by our calculations. Thus, the tautomeric model of the mutagenic activity of 5-bromouracil proposed previously [Hu et al. Biochemistry (2004) 43, 6361] can be refuted. The validity of other mutagenic models was also discussed, and finally a new mechanism for explaining the mutagenic activity of halogenuracils based on their different behaviors in triplet excited states was suggested.

Relationships among plasma [2-(13)C]uracil concentrations, breath (13)CO(2) expiration, and dihydropyrimidine dehydrogenase (DPD) activity in the liver in normal and dpd-deficient dogs.

Dihydropyrimidine dehydrogenase (DPD), the first enzyme in the sequential metabolism of pyrimidine, regulates blood concentrations of 5-fluorouracil and is deeply involved in its toxicity. This study was designed to examine the effects of a DPD inhibitor on blood concentrations of [2-(13)C]uracil ([(13)C]uracil) and (13)CO(2) concentration (Delta(13)C) expired in breath after oral or intravenous administration of [(13)C]uracil to DPD-suppressed dogs prepared by pretreatment with 5-(trans-2-bromovinyl)uracil (BVU), a DPD inhibitor. Area under the curve (AUC(t)) of [(13) C]uracil after oral administration at 20 micromol/kg to dogs pretreated with BVU at 2, 5, and 40 mmol/kg were 37-, 88- and 120-fold higher than those of the control dogs, respectively. In contrast, breath AUC(t) values of Delta(13)C were reduced to 0.88-, 0.47- and 0.13-fold the control values, respectively. Upon intravenous administration of [(13)C]uracil at 20 micromol/kg to dogs pretreated with BVU at 0.5, 2, and 40 micromol/kg, blood AUC(t) values of [(13)C]uracil were 1.4-, 4.2-, and 13-fold higher than those of the control group, respectively, whereas breath AUC(t) values were reduced to 1.0-, 0.83-, and 0.07-fold the respective control values. DPD activities in the liver cytosol of dogs pretreated with BVU at 0.5, 2, 5, and 40 micromol/kg were decreased to 0.71-, 0.12-, 0.06-, and 0.04-fold those of the control dogs, respectively. These findings demonstrate that breath output (Delta(13)C) is a good marker of hepatic DPD activity in vivo.

Mutagenic mechanism of the A-T to G-C transition induced by 5-bromouracil: an ab Initio Study.

The tautomerisms of uracil, 5-bromouracil (BrU), G-U, G-BrU, A-U, and A-BrU have been studied theoretically in an effort to investigate the mutagenicity of BrU. The ab initio calculations have been performed using HF and B3LYP methods with various basis sets. The relative stability of all tautomers was established. The intermolecular interactions between U, BrU, U*, BrU* (asterisks denote enol forms), and water have been studied. It shows that the possibility of tautomerism from BrU to BrU* is much more likely than that from U to U*. Further research indicates that BrU* tends to pair with guanine more easily than U*. The proton transfer process has been investigated by potential energy surface (PES) scan and transition state analysis. The results show that the proton transfer between G-U* and G*-U is monodirectional barrier-free proton transfer (BFPT), which terminates the base mismatch induced by U*. On the other hand, the proton transfer between G-BrU* and G*-BrU is bidirectional BFPT, which makes the base mismatch induced by BrU* sustained. On the basis of all of these calculated results, a new mutagenic mechanism for the A-T to G-C transition induced by 5-bromouracil is described in detail for the first time. It might give a new insight into the origin of the mutagenicity of the 5-Br derivative.

[Molecular toxicological mechanism of the lethal interactions of the new antiviral drug, sorivudine, with 5-fluorouracil prodrugs and genetic deficiency of dihydropyrimidine dehydrogenase].

In 1993, there were 18 acute deaths in Japanese patients who had the viral disease herpes zoster and were treated with the new antiviral drug sorivudine (SRV, 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil). All the dead patients had received a 5-fluorouracil (5-FU) prodrug as anticancer chemotherapy concomitant with SRV administration. Studies on toxicokinetics in rats and on hepatic dihydropyrimidine dehydrogenase (DPD), a rate-limiting enzyme for 5-FU catabolism in rats and humans, strongly suggested that in the patients who received both SRV and the 5-FU prodrug, tissue levels of highly toxic 5-FU markedly increased as a result of irreversible inactivation of DPD in the presence of NADPH by 5-(2-bromovinyl)uracil (BVU), a metabolite formed from SRV by gut flora in rats and humans. Recombinant human (h) DPD was also irreversibly inactivated by [14C] BVU in the presence of NADPH. MALDI-TOF MS analysis of radioactive tryptic fragments from the radiolabeled and inactivated hDPD demonstrated that a Cys residue located at position 671 in the pyrimidine-binding domain of hDPD was modified with an allyl bromide type of reactive metabolite, dihydro-BVU. Thus artificial DPD deficiency caused by BVU from SRV led to patient deaths when coadministered with the 5-FU prodrug. Human population studies using healthy volunteers have demonstrated that there are poor and extensive 5-FU metabolizers who have very low and high DPD activities, respectively. Administration of a clinical dose of 5-FU or its prodrug to poor 5-FU metabolizers may cause death unless DPD activity is determined using their peripheral blood mononuclear cells prior to the administration of the anticancer drug.

Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells.

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A. The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds.

Lack of susceptibility of bicyclic nucleoside analogs, highly potent inhibitors of varicella-zoster virus, to the catabolic action of thymidine phosphorylase and dihydropyrimidine dehydrogenase.

The susceptibility of the bicyclic nucleoside analogs (BCNAs), highly potent and selective inhibitors of varicella-zoster virus (VZV), to the enzymes involved in nucleoside/nucleobase catabolism has been investigated in comparison with the established anti-VZV agent (E)-5-(2-bromovinyl)-2'-deoxyuridine [BVDU; brivudine (Zostex)]. Whereas human and bacterial thymidine phosphorylases (TPases) efficiently converted BVDU to its antivirally inactive free base (E)-5-(2-bromovinyl)uracil (BVU), BCNAs showed no evidence of conversion to the free base in the presence of these enzymes. The lack of substrate affinity of TPase for the BCNAs could be rationalized by computer-assisted molecular modeling of the BCNAs in the TPase active site. Moreover, in contrast with BVU, which is a potent and selective inhibitor of dihydropyrimidine dehydrogenase (DPD) (50% inhibitory concentration; 10 microM in the presence of a 25 microM concentration of the natural substrate thymine), the free base (Cf 1381; 6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) of BCNA (Cf 1368; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) and the free base Cf 2200 [6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one] of BCNA (Cf 1743; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) did not inhibit the DPD-catalyzed catabolic reaction of pyrimidine bases (i.e., thymine) and pyrimidine base analogs [i.e., 5-fluorouracil (FU)] at a concentration of 250 microM. Consequently, whereas BVU caused a dramatic rise of FU levels in FU-treated mice, the BCNAs did not affect FU levels in such mice. From our data it is evident that BCNAs represent highly stable anti-VZV compounds that are not susceptible to breakdown by nucleoside/nucleobase catabolic enzymes and are not expected to interfere with cellular catabolic processes such as those involved in FU catabolism.

Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase.

A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.

[Effect of the mutation process on formation of alkaloids in Penicillium roquefortii BKM F-141 and P. fellutanum BKM F-1073]. / Vliianie mutatsionnogo protsessa na obrazovanie alkaloidov u Penicillium roquefortii BKM F-141 i P. fellutanum BKM F-1073.

Mutant strains of Penicillium roquefortii VKM F-141 and P. Fellutanum VKM F-1073 were obtained by mutagenesis induced by ultraviolet irradiation, N-methyl-N-nitronitrosoguanidine and bromouracil. By the rates of alkaloid production, the mutant strains can be divided into three groups: 1) unable to synthesize alkaloids; 2) with a high rate of biosynthesis; 3) with changed alkaloid composition. Compounds not characteristic of wild-type cultures were found in alkaloid fractions of some mutant strains.

Mechanism-based inactivation of human dihydropyrimidine dehydrogenase by (E)-5-(2-bromovinyl)uracil in the presence of NADPH.

Purified recombinant human dihydropyrimidine dehydrogenase (hDPD) was incubated with (14)C-labeled (E)-5-(2-bromovinyl)uracil ([(14)C]BVU) in the presence of NADPH to investigate a possible mechanism in the 18 patient deaths caused by interactions of 5-fluorouracil prodrugs with the new oral antiviral drug, sorivudine. BVU is formed from sorivudine by gut flora and absorbed through intestinal membrane. hDPD, a rate-limiting enzyme for the catabolism of 5-fluorouracil and endogenous pyrimidines in the human, was NADPH dependently radiolabeled and inactivated by [(14)C]BVU. Two radioactive tryptic fragments, I and II, isolated from radiolabeled hDPD were found by complete amino acid sequencing to originate from a common regional amino acid sequence located at positions 656 (Lys) to 678 (Arg) for I and positions 657 (Ser) to 678 (Arg) for II. However, only Cys(671), which should be present in the peptides, was not identified by amino acid sequencing. Mass spectrometric analysis of the tryptic fragments indicated that the sulfhydryl group of Cys(671) in the hDPD was modified with 5, 6-dihydro-5-(2-bromoethylydenyl)uracil (BEDU), a putative allyl bromide type of reactive molecule, to form a sulfide bond with loss of hydrogen bromide. The Cys(671) sulfide bearing the debrominated BEDU had a 5,6-dihydrouracil ring highly strained by the exocyclic double bond at the 5-position, so that it underwent facile hydrolytic ring fission with alkali and heated acid treatments. A new proposal is also made for the amino acid sequence of the pyrimidine-binding domain, including Cys(671), of DPD in the human and other species.

Prediction of in vivo drug-drug interactions based on mechanism-based inhibition from in vitro data: inhibition of 5-fluorouracil metabolism by (E)-5-(2-Bromovinyl)uracil.

The fatal drug-drug interaction between sorivudine, an antiviral drug, and 5-fluorouracil (5-FU) has been shown to be caused by a mechanism-based inhibition. In this interaction, sorivudine is converted by gut flora to (E)-5-(2-bromovinyl)uracil (BVU), which is metabolically activated by dihydropyrimidine dehydrogenase (DPD), and the activated BVU irreversibly binds to DPD itself, thereby inactivating it. In an attempt to predict this interaction in vivo from in vitro data, inhibition of 5-FU metabolism by BVU was investigated by using rat and human hepatic cytosol and human recombinant DPD. Whichever enzyme was used, increased inhibition was observed that depended on the preincubation time of BVU and enzyme in the presence of NADPH and BVU concentration. The kinetic parameters obtained for inactivation represented by k(inact) and K'(app) were 2.05 +/- 1.52 min(-1), 69.2 +/- 60.8 microM (rat hepatic cytosol), 2.39 +/- 0.13 min(-1), 48.6 +/- 11.8 microM (human hepatic cytosol), and 0.574 +/- 0.121 min(-1), 2.20 +/- 0.57 microM (human recombinant DPD). The drug-drug interaction in vivo was predicted quantitatively based on a physiologically based pharmacokinetic model, using pharmacokinetic parameters obtained from the literature and kinetic parameters for the enzyme inactivation obtained in the in vitro studies. In rats, DPD was predicted to be completely inactivated by administration of BVU and the area under the curve of 5-FU was predicted to increase 11-fold, which agreed well with the reported data. In humans, a 5-fold increase in the area under the curve of 5-FU was predicted after administration of sorivudine, 150 mg/day for 5 days. Mechanism-based inhibition of drug metabolism is supposed to be very dangerous. We propose that such in vitro studies should be carried out during the drug-developing phase so that in vivo drug-drug interactions can be predicted.

Biochemical modulation of the catabolism and tissue uptake of the anticancer drug 5-fluorouracil by 5-bromovinyluracil: assessment with metabolic (19)F MR imaging.

Using chemical shift-selective (19)F magnetic resonance (MR) imaging, we investigated the biomodulating action of 5-bromovinyluracil (BVU) on the degradation of the anticancer drug 5-fluorouracil (5-FU) to its major catabolite alpha-fluoro-beta-alanine (FBAL) and the tissue uptake of 5-FU in ACI rats with transplanted Morris hepatoma. Rats in the control group (n = 7) received 200 mg/kg body weight of 5-FU intravenously, whereas the rats in the BVU group (n = 7) additionally received 30 mg/kg body weight of BVU intraperitoneally about 45 min before 5-FU injection. In each animal examination, three selective (19)F MR images were acquired sequentially after 5-FU administration with an acquisition time of 32 min each: an early 5-FU image (dominant Fourier line, 8 min p.i.) that characterized the early uptake of the drug into the various tissues, an FBAL image (dominant Fourier line, 56 min p.i.) that reflected the catabolism of the drug, and a late 5-FU image (dominant Fourier line, 78 min p.i.) that assessed the retention ("trapping") of unmetabolized 5-FU and its MR-visible anabolites. Pretreatment with BVU resulted in a highly statistical significant decrease (P < 0.001) of the FBAL signal in the liver. The marked effect of BVU on 5-FU degradation, however, improved neither the early uptake nor the retention of 5-FU in skeletal muscle and tumor tissue (P > 0.7). Moreover, our results indicate that 5-FU tumor uptake is not only dependent on the plasma concentration of unmetabolized 5-FU but is also determined by tumor-specific factors, these showing considerable variations between individual neoplasms. Magn Reson Med 42:936-943, 1999.

New molecular complexes of heterocyclic bis-adducts with bacterial lectins: synthesis and structure-activity relationship studies.

A strongly antitumour effect has been discovered for lectins of Bacillus bacteria [Bacillus subtilis 668(1 + 2)IMV, Bacillus polymyxa 102(1 + 2) KGU] and for their molecular complexes with some heterocyclic bis-adducts of unsubstituted benzimidazole and 6-methyluracile for the first time. These were tested on the tumours: Lymphosarcoma Plissa, Sarcoma 45, Carcinosarcoma Yokera 256. A new convenient method for the preparation of the heterocyclic bisadducts of imidazole, benzimidazole, uraciles with 1,1,1-trifluoro-2-bromo-2-chloroethane is described. The reactions are catalysed by the 18-crown-6-complex.

7-Deazaxanthine, a novel prototype inhibitor of thymidine phosphorylase.

7-Deazaxanthine (7DX) was identified as a novel inhibitor of thymidine (dThd) phosphorylase (TPase). It inhibited the TPase reaction in a concentration-dependent manner. At 1 mM, it almost completely prevented the TPase-catalysed hydrolysis of dThd to thymine. The 50% inhibitory concentration (IC50 of 7DX was 40 microM in the presence of 100 microM of the natural substrate dThd. 7DX is also endowed with a marked inhibitory effect on angiogenesis. It significantly prevents neovascularisation in the chicken chorioallantoic membrane during development. 7DX is the first purine derivative shown to be a potent inhibitor of purified TPase and angiogenesis.

Lethal drug interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs.

Rats were orally co-administered sorivudine (SRV: 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil), a new oral antiviral drug for herpes zoster, with the oral anticancer drug tegafur (FT: 1-(2-tetrahydrofuryl)-5-fluorouracil) as a prodrug of 5-flourouracil (5-FU) once daily to investigate a toxicokinetic mechanism of 15 Japanese patients' deaths recently caused within a brief period by the drug interaction of these drugs. All the rats showed extremely elevated levels of 5-FU in plasma and tissues, including bone marrow and small intestine, and died within 10 days, whereas the animals given the same dose of SRV or FT alone were still alive over 20 days without any appreciable toxic symptom. Before their death, there was marked damage of bone marrow, marked atrophy of intestinal membrane mucosa, marked decreases in white blood cells and platelets, diarrhea with bloody flux, and severe anorexia as reported with the Japanese patients. Data obtained by in vivo and in vitro studies strongly suggested that (E)-5-(2-bromovinyl)uracil generated from SRV by gut flora was reduced in the presense of NADPH to a reactive form by hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme determining the tissue 5-FU levels, bound covalently to DPD as a suicide inhibitor, and markedly retarded the catabolism of 5-FU.

Development and survival of Drosophila melanogaster fed a diet containing pyrimidine analogs.

A single generation of Drosophila melanogaster was raised on different media. One of the media was unsupplemented (the control) and the others were supplemented with pyrimidine analog at 10.3 mmol/kg culture medium. The relative numbers of larvae, pupae, and F1 adults reproduced from parent flies on each medium served as an indication of the relative toxicity of the supplements. The relative decreasing order of toxicity of the pyrimidines was as follows: 5-bromouracil < thymine < uracil = orotic acid = control = cytosine, control < UMP. The toxic effects of 5-bromouracil and thymine seem to be associated with the addition of a bromine or methyl group to carbon 5 of the pyrimidine ring. The UMP supplementation increased the number of adult F1 flies above the control group indicating that UMP was not only non toxic but also that it was beneficial.

Effects of 1-amino-5-bromouracil on the benzodiazepine-GABAA receptor complex.

We investigated the effects of 1-amino-5-bromouracil on the benzodiazepine-gamma-aminobutyric acid (GABA)A receptor complex to elucidate its central action. 1-Amino-5-bromouracil neither displaced nor enhanced [3H]muscimol, [35S]t-butylbicyclophosphorothionate (TBPS), or [3H]dehydroepiandrosterone sulfate binding to the rat brain synaptosomal membranes. The anesthesia induced by 1-amino-5-bromouracil was potentiated by diazepam, pentobarbital, and muscimol, and was antagonized by picrotoxin but not by bicuculline. 1-Amino-5-bromouracil protected mice from picrotoxin-induced seizure and slightly ameliorated TBPS-induced seizure, but did not antagonize bicuculline-induced seizure. Diazepam antagonized both the bicuculline- and the picrotoxin-induced seizure, and pentobarbital antagonized the picrotoxin- and the TBPS-induced seizure. Our in vivo studies suggest that part of the central action of 1-amino-5-bromouracil is concerned with the benzodiazepine-GABAA receptor complex including the chloride channel.

Effect of 1-amino-5-bromouracil on brain monoamine metabolism in rats.

The effect of 1-amino-5-bromouracil (ABU), a novel central-acting agent, on monoaminergic neurotransmitter levels of rat brain was investigated. Under the nonstressed condition, ABU (20 and 30 mg/kg intraperitoneally [IP]) did not affect monoamine metabolism, whereas diazepam (5 mg/kg IP) increased the 3-methoxy-4-hydroxyphenylethylene glycol (MHPG)/noradrenaline (NA) ratio. One-hour immobilization stress increased the MHPG/NA ratio in various brain regions of drug-naive rats, but did not increase the homovanilic acid (HVA) plus 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine (DA) ratio or the 5-hydroxyindole acetic acid (5-HIAA)/serotonin (5-HT) ratio. Pretreatment with ABU or diazepam suppressed the activation of noradrenergic neurons induced by immobilization stress. By contrast, electric foot shock stress increased the MHPG/NA and HVA+DOPAC/DA ratios. Pretreatment with ABU or diazepam suppressed the activation of noradrenergic and dopaminergic cortical neurons by electric foot shock stress. These results indicate that these two physiologic stresses affected monoaminergic neurons differently and that their effects were suppressed by ABU and diazepam.