Nanopore Analysis of the 5-Guanidinohydantoin to Iminoallantoin Isomerization in Duplex DNA.

In DNA, guanine oxidation yields diastereomers of 5-guanidinohydantoin (Gh) as one of the major products. In nucleosides and single-stranded DNA, Gh is in a pH-dependent equilibrium with its constitutional isomer iminoallantoin (Ia). Herein, the isomerization reaction between Gh and Ia was monitored in duplex DNA using a protein nanopore by measuring the ionic current when duplex DNA interacts with the pore under an electrophoretic force. Monitoring current levels in this single-molecule method proved to be superior for analysis of population distributions in an equilibrating mixture of four isomers in duplex DNA as a function of pH. The results identified Gh as a major isomer observed when base paired with A, C, or G at pH 6.4-8.4, and Ia was a minor isomer of the reaction mixture that was only observed when the pH was >7.4 in the duplex DNA context. The present results suggest that Gh will be the dominant isomer in duplex DNA under physiological conditions regardless of the base-pairing partner in the duplex.

Conformational stabilities of iminoallantoin and its base pairs in DNA: implications for mutagenicity.

The types of mutations induced by oxidatively damaged products of DNA are continuously in debate. For example, some biochemical studies have proposed that guanidinohydantoin (Gh) would induce exclusively G to C mutations, while other studies have predicted a mixture of various mutations including G to C, G to T and G to A. In addition to the nature of mutations, the exact reasons of these mutations are also not properly understood. It is suggested that Gh can easily isomerize to iminoallantoin (Ia) in a pH-dependent manner and the transition becomes complete at pH > 8. In order to understand Gh/Ia-induced mutations, we have here studied the role of the most stable tautomer of Ia in the R- and S-enantiomeric configurations in promoting mismatch base pair complexes in DNA by employing a density functional theoretical (DFT) approach. It is found that Ia can have 39 different possible tautomeric forms each in the R- and S-enantiomeric configurations, out of which the most stable tautomer would involve the deprotonation of the N1 atom and protonation of the N3 atom. The most stable tautomer of Ia can adopt three different rotameric conformations (Ia1, Ia2, and Ia3) of comparable stabilities. It is further revealed that these rotamers of Ia can interact with different bases of DNA in 88 different possible ways. However, the interaction of G with Ia3 in both the anti- and syn-conformations would be the most stable. It is further revealed that the base pairing patterns, binding energies and electronic environments of anti-Ia3:G and G:T complexes are similar. In addition to this, it is also found that the binding patterns and energies of Gh1:G and Ia3:G complexes are similar. Based on these results, it is proposed that under physiological conditions, Gh1 may be responsible for the observed G to C mutations in DNA, while in an acidic environment Ia3 may be responsible for the same mutations. This study has led to a solid foundation for further high resolution structural studies to completely unravel Ia-induced mutagenicity in DNA.

Aldioxa improves delayed gastric emptying and impaired gastric compliance, pathophysiologic mechanisms of functional dyspepsia.

Delayed gastric emptying and impaired gastric accommodation (decreased gastric compliance) play important roles in functional dyspepsia (FD). Here we screen for a clinically used drug with an ability to improve delayed gastric emptying in rats. Oral administration of aldioxa (dihydroxyaluminum allantoinate) partially improved clonidine- or restraint stress-induced delayed gastric emptying. Administration of allantoin, but not aluminium hydroxide, restored the gastric emptying. Both aldioxa and allantoin inhibited clonidine binding to the α-2 adrenergic receptor, suggesting that antagonistic activity of the allantoin moiety of aldioxa on this receptor is involved in the restoration of gastric emptying activity. Aldioxa or aluminium hydroxide but not allantoin restored gastric compliance with restraint stress, suggesting that aluminium hydroxide moiety is involved in this restoration. We propose that aldioxa is a candidate drug for FD, because its safety in humans has already been confirmed and its ameliorating effect on both of delayed gastric emptying and impaired gastric compliance are confirmed here.

Reaction of 3',5'-di-O-acetyl-2'-deoxyguansoine with hypobromous acid.

Hypobromous acid (HOBr) is formed by eosinophil peroxidase and myeloperoxidase in the presence of H2O2, Cl(-), and Br(-) in the host defense system of humans, protecting against invading bacteria. However, the formed HOBr may cause damage to DNA and its components in the host. When a guanine nucleoside (3',5'-di-O-acetyl-2'-deoxyguansoine) was treated with HOBr at pH 7.4, spiroiminodihydantoin, guanidinohydantoin/iminoallantoin, dehydro-iminoallantoin, diimino-imidazole, amino-imidazolone, and diamino-oxazolone nucleosides were generated in addition to an 8-bromoguanine nucleoside. The major products were spiroiminodihydantoin under neutral conditions and guanidinohydantoin/iminoallantoin under mildly acidic conditions. All the products were formed in the reaction with HOCl in the presence of Br(-). These products were also produced by eosinophil peroxidase or myeloperoxidase in the presence of H2O2, Cl(-), and Br(-). The results suggest that the products other than 8-bromoguanine may also have importance for mutagenesis by the reaction of HOBr with guanine residues in nucleotides and DNA.

Final report of the safety assessment of allantoin and its related complexes.

Allantoin is a heterocyclic organic compound. Allantoin ascorbate, allantoin biotin, allantoin galacturonic acid, allantoin glycyrrhetinic acid, allantoin panthenol, and allantoin polygalacturonic acid are complexes of allantoin. All of the ingredients in this review act as skin-conditioning agents. Allantoin was reported to be used in 1376 cosmetic products at concentrations up to 2%. There are data gaps regarding use and concentration of the remaining allantoin complexes. Ascorbic acid, biotin, glycyrrhetinic acid, and panthenol have been determined by the CIR Expert Panel to be safe. Galacturonic acid and polygalacturonic acid have not been reviewed by the CIR Expert Panel, and substantial data on these chemicals were not available. The safety test data in this safety assessment and in previous safety assessments were considered sufficient to support the safety of allantoin and the allantoin complexes in product categories and at concentrations reviewed in this safety assessment.

A novel superfamily of transporters for allantoin and other oxo derivatives of nitrogen heterocyclic compounds in Arabidopsis.

A wide spectrum of soil heterocyclic nitrogen compounds are potential nutrients for plants. Here, it is shown that Arabidopsis plants are able to use allantoin as sole nitrogen source. By functional complementation of a yeast mutant defective in allantoin uptake, an Arabidopsis transporter, AtUPS1 (Arabidopsis thaliana ureide permease 1), was identified. AtUPS1 belongs to a novel superfamily of plant membrane proteins with five open reading frames in Arabidopsis (identity, 64 to 82%). UPS proteins have 10 putative transmembrane domains with a large cytosolic central domain containing a "Walker A" motif. Transport of (14)C-labeled allantoin by AtUPS1 in yeast exhibited saturation kinetics (K(m) approximately 52 microM), was dependent on Glc and a proton gradient, and was stimulated by acidic pH. AtUPS1 transports uric acid and xanthine, besides allantoin, but not adenine. Protons are cosubstrates in allantoin transport by AtUPS1, as demonstrated by expression in Xenopus laevis oocytes. In plants, AtUPS1 gene expression was dependent on the nitrogen source. Therefore, AtUPS1 presumably is involved in the uptake of allantoin and other purine degradation products when primary sources are limiting.

Characterization of hydantoin products from one-electron oxidation of 8-oxo-7,8-dihydroguanosine in a nucleoside model.

Use of one-electron oxidants such as Na(2)IrCl(6) to oxidize 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) residues in oligodeoxynucleotides was previously shown to lead to predominant formation of a base lesion of mass M - 10 compared to starting material [Duarte et al. (1999) Nucleic Acids Res. 27, 596-502]. To thoroughly characterize the structure of this lesion, the oxidation of the nucleoside 9-N-(2',3',5'-tri-O-acetyl-beta-D-erythro-pentanosyl)-8-oxo-7,8-dihydroguanine with one-electron oxidants at pH 2-4 was used as a model for duplex DNA oxidation of OG residues. (1)H NMR and H,H COSY NMR studies in CD(3)OD along with LC-ESI-MS/MS fragmentation analysis are consistent with the assignment of the M - 10 species as a mixture of two pH-dependent equilibrating isomers, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) nucleoside, both present as mixtures of epimers at the C5 position of the hydantoin ring, i.e., four total isomers are formed. The Gh/Ia mixture is formed from hydration and decarboxylation of the initially formed intermediate 5-hydroxy-8-oxo-7,8-dihydroguanosine, a species that is also produced by four-electron oxidation (e.g., singlet oxygen) of guanosine. The product mixture can be further oxidized to a species designated Ia(ox), a hydrolytically unstable material at pH 7 that has been characterized by ESI-MS and (1)H NMR. Competition studies with 8-oxo-7,8-dihydroadenosine placed the redox potential of Gh/Ia at about 1.0 V vs NHE. These studies provide important information concerning the structures of lesions obtained when OG, a "hot spot" for oxidative damage, serves as a "hole trap" in long-range electron-transfer studies.

Absence of gastrointestinal absorption or urinary excretion of aluminium from an allantoinate complex contained in two antacid formulations in patients with normal renal function.

We studied the plasma and urinary excretion levels of aluminum (Al) on day 0, 10 and 30 in 79 patients with gastrointestinal symptoms and normal renal function who were receiving a complex based on Al allantoinates [C4H5N4 O3 Al (OH)2] and [C4H5N4 O3 Cl Al2 (OH)4]. We evaluated the extent of Al absorption after repeated administration of this complex in two antacid formulations, Ulfon Lyoc in lyophilised tablet form (group 1; n = 40) and Ulfon suspension (group 2; n = 39). The total Al load for each antacid and patient was 512 mg daily for a total of 15360 mg during the 30-day treatment. No significant rise in plasma Al concentration was noted with either formulation between day 0 and 10, day 0 and 30 or day 10 and 30, nor was there any significant increase in urinary excretion levels. Al absorption was not increased and no toxic effects were noted, indicating that such formulations are suitable for long-term therapy in patients with gastrointestinal symptoms.

[Acidified aspirin-induced gastric lesion in rats with hepatic cirrhosis produced by N-nitrosodiethylamine or carbon tetrachloride. Effect of aldioxa on gastric lesions].

Gastric lesion was induced by the oral administration of acidified aspirin in rats with hepatic cirrhosis produced by N-nitrosodiethylamine (NDA) or carbon tetrachloride (CCl4). Gastric lesion by acidified aspirin was aggravated in NDA-induced cirrhosis, but not in CCl4-cirrhotic rats. To clarify this difference in the susceptibility of the gastric mucosa, gastric mucosal blood flow and gastric emptying were measured by the hydrogen gas clearance method and beads method, respectively. Gastric mucosal blood flow was lower and gastric emptying was significantly delayed in NDA-induced cirrhotic rats as compared with the controls, but not in CCl4-induced cirrhotic rats. Gastric mucosal blood flow in NDA-induced cirrhotic rats was significantly decreased by the oral administration of acidified aspirin as compared with the controls. Aldioxa dose-dependently inhibited the gastric lesion formation by acidified aspirin and inhibited the decrease of gastric mucosal blood flow in NDA-induced cirrhotic rats. These results suggest that aggravation of gastric lesion induced by acidified aspirin in NDA-induced cirrhotic rats would be due to the decrease of gastric mucosal blood flow and delay of gastric emptying. In addition, aldioxa showed a protective effect against gastric lesions induced by acidified aspirin in NDA-induced cirrhotic rats, suggesting that this compound would have an inhibitory effect on gastric lesions that are accompanied by hepatic cirrhosis.

[The effect of aldioxa on the formation of gastritis induced with sodium hydroxide].

The effects of aldioxa and cetraxate hydrochloride on the formative process of gastritis induced with intragastric application of 2% sodium hydroxide were studied. Rats were given food including either aldioxa or cetraxate hydrochloride for 6 weeks after the sodium hydroxide application. After sacrifice, the stomachs were removed, and the gastric mucosa were observed macroscopically and histologically. Gastric mucosal injury was widely induced with sodium hydroxide, being histologically characterized by mucosal hypertrophy, cell infiltration and intestinal metaplasia. These lesions seem to be the early stage of chronic gastritis. The aldioxa group showed a decrease of mucosal hypertrophy and cell infiltration as compared with the control group. In the cetraxate hydrochloride group, the mucosal surface of white gray color and the mucosal bosselation were observed macroscopically. Histologically, these were found to be cell infiltration and cyst formation. Moreover, intestinal metaplasia occurred at high incidence in this group. These findings in the cetraxate hydrochloride group are recognized to be an aggravation of chronic gastritis. From the above results, it is suggested that aldioxa promotes good regeneration of mucosa and should be useful for the clinical therapy of chronic gastritis.

[Evaluation of the anti-ulcer drugs using image analysis technology: effect of aldioxa containing preparation on the experimental gastric ulcer in rats].

Direct measurements under a microscope have been employed to evaluate experimental gastric ulcers, but the following problems have been left unsettled: it takes too much time, and much experience is required to perform accurate, objective measurements. The present study demonstrates the usefulness of image analysis technology for evaluating anti-ulcer drugs. With this newly developed method, the region of gastric mucosal injury can be automatically and/or mechanically chosen on the basis of three different factors: intensity, hue and purity. Consequently, this method can be used to rapidly obtain analytical data such as the number, area and length of gastric ulcers. Effect of NNP, a preparation containing aldioxa, on three different types of experimental ulcer formation (stress-induced, ethanol-induced, and pylorus-ligation) was studied by image analysis technology. In all cases, NNP (2,562 mg/kg, p.o.) almost completely inhibited the formation of rat gastric ulcer. From the finding that image analysis technology is very useful for evaluating anti-ulcer drugs, we conclude that this method will help further the development of anti-ulcer drugs.

1,3,8-Trimethylallantoin: a major caffeine metabolite formed by rat liver.

The formation of 1,3,8-trimethylallantoin from caffeine is demonstrated for the first time in vitro in rat liver slices. This compound constitutes 15% of total in vitro metabolites. With this identification, all the metabolic pathways of caffeine shown in vivo (N-demethylation, oxidation to uric acids and formation of uracil and allantoin derivatives) are demonstrated in vitro.

High-performance liquid chromatographic method for the determination of plasma allantoin.

A new method has been devised for the determination of nanomole levels of allantoin in human plasma. Allantoin was converted into xanthylallantoin, which was chromatographed on a reversed-phase silica gel using a mixture of acetonitrile-water (27:73) as mobile phase. The eluted compound was measured using an ultraviolet detector. The detection limit of the assay for plasma was about 100 ng/ml. This method was applied successfully to the determination of allantoin in human plasma after oral administration of 100 mg of aldioxa.

[Experimental studies on gastric ulcer (5). Endoscopical evaluation of healing processes of acetic acid ulcer in rats (1). Ulcer reducing process].

The following results were obtained as the result of the sequential observations with an endoscope of the gastric ulcer produced by submucosal injection of acetic acid in rats. 1) The size of the ulcer produced by acetic acid injection observed on the 3rd day after ulcer induction depended on the concentration and volume of acetic acid. 2) The natural reducing rates of the size of ulcers in the region between the fundus and pylorus on the anterior wall (A) and in the glandular region on the greater curvature (B) were compared. The reducing rate of B ulcer was significantly faster than that of A ulcer. 3) No significant difference was observed in the reducing processes of untreated ulcers in 7 weeks old rats and 25 weeks old rats. 4) The ulcer reducing rate of female rats was found to be slightly faster than that of male rats. 5) The ulcer reducing rates of the aldioxa (ALD) and sucralfate (SUC) treated group were significantly faster than that of the control group. However, no difference in the ulcer reducing process between the cimetidine (CIM) treated group and the control group was observed. 6) The ulcer indices (UI) of both the combinations of ALD and SUC and of ALD and CIM were found to be significantly less than that of the control group on the 20th day. Treatment with drug combinations shortened the healing period of ulcers more than treatment with one drug alone. The percent healing by the combination of ALD and SUC was the highest among the groups treated. 7) An approximately linear relationship exists between the logarithm of the days after ulcer induction (x) and logarithm of UI (y), and the following equation was obtained: log y = a log x + b. Slope (a) indicates the ulcer reducing rate in evaluating the ulcer reducing process.

Determination of allantoin and chlorohydroxyaluminium allantoinate in cosmetic and pharmaceutical products by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for convenient and direct determination of allantoin derivatives, by which as little as 0.1 micrograms of the compounds can be determined without any conventional sample preparation required to eliminate interferences. Chromatographic separation on a resin-based strong cation exchanger (H+) using water as eluent is combined with sensitive and selective post-column reaction of the allantoin derivatives. After dissolution of the sample in hot water, the determinations take 12 min per sample. The relative standard deviation is less than 1.8% and quantitative recoveries are obtained. Several commercial cosmetic and pharmaceutical products were analysed successfully. The method can be applied to test the stability of the compounds in formulations.