The radioprotective effect of riboxine (inosine)

In experiments with mice subjected to whole-body X-irradiation a radioprotective effect of riboxine (inosine) was demonstrated. The observed effect may be attributed to the ability of the preparation to interfere with the cyclic nucleotide metabolism.     

Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice

The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to gamma rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80 degrees C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase-luminol-p-iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against gamma-radiation-induced death.     

Radioprotective effect of hydrogen in cultured cells and mice

Abstract

It has been demonstrated that hydrogen can selectively reduce hydroxyl and peroxynitrite in vitro. Since most of the ionizing radiation-induced cellular damage is caused by hydroxyl radicals, this study was designed to test the hypothesis that hydrogen may be an effective radioprotective agent. This paper demonstrates that treating cells with hydrogen before irradiation could significantly inhibit ionizing irradiation(IR)-induced Human Lymphocyte AHH-1 cells apoptosis and increase cells viability in vitro. This paper also shows that hydrogen can protect gastrointestinal endothelia from radiation-induced injury, decrease plasma malondialdehyde (MDA) intestinal 8-hydroxydeoxyguanosine (8-OHDG) levels and increase plasma endogenous antioxidants in vivo. It is suggested that hydrogen has a potential as an effective and safe radioprotective agent.     

Structure-function studies of a plant tyrosyl-DNA phosphodiesterase provide novel insights into DNA repair mechanisms of Arabidopsis thaliana

Our recent studies suggest that H2 (hydrogen) has a potential as a novel radioprotector without known toxic side effects. The present study was designed to examine the underlying radioprotective mechanism of H2 and its protective role on irradiated germ cells. Produced by the Fenton reaction and radiolysis of H2O, hydroxyl radicals (?OH) were identified as the free radical species that were reduced by H2. We used a H2 microelectrode to dynamically detect H2 concentration in vivo, and found H2 significantly reduced in situ fluorescence intensity of hydroxyphenyl fluorescein; however, as we treated the mice with H2 after irradiation, the decrease is not significant. We found that pre-treatment of H2 to IR (ionizing radiation) significantly suppressed the reaction of ?OH and the cellular macromolecules which caused lipid peroxidation, protein carbonyl and oxidatively damaged DNA. The radioprotective effect of H2 on male germ cells was supported by ameliorated apoptotic findings examined by morphological changes and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) in testicular tissue, and by preserved viability of stem spermatogonia examined for testicular histological parameters, daily sperm production and sperm quality; we used WR-2721 [S-2-(3-aminopropylamino)ethyl phosphorothioic acid] as a reference compound. Our results represent the first in vivo evidence in support of a radioprotective role of H2 by neutralizing ?OH in irradiated tissue with no side effects.     

The effect of riboxine on prophage induction and the survival of bacterial cultures exposed to gamma irradiation

In experiments with lysogenic cultures Pseudomonas aeruginosa and Escherichia coli K-12 proposed as a test for biological indication of relatively low doses of gamma-radiation, it was shown that when estimated by the criterion of prophage induction riboxine had a radioprotective action at nonlethal radiation doses. At the same time, when estimated by the criterion of the survival rate, riboxine produced the radioprotective effect at rather high radiation doses (survival rate of less than 10 per cent). It is suggested that riboxine is involved in the cell repair processes.     

Radioprotective effect of inosine and its enhancement by magnesium and global hypoxia.

The slight radioprotective action of inosine, when injected intraperitoneally to mice shortly before gamma-irradiation, can be enhanced by the administration of magnesium aspartate. This effect can be explained by the additivity of the vasodilatory actions of both agents. Inosine increases the radioprotective effectiveness of hypobaric hypoxia (10% O2), probably due to the additivity of the hypoxic effects in radiosensitive tissues. Acute hypoxic toxicity, however, is decreased by inosine administration. The cumulation of radioprotective effects of inosine and of its antihypoxic action in vitally important organs can have a favourable influence in hypoxic radiotherapy.     

The level of DNA damage in mouse hematopoietic cells and in frog and human blood cells,as induced by the action of reactive oxygen species in vitro

Comparative studies of the level of DNA damage induced in vitro by X-rays (0–8 Gy) or hydrogen peroxide (0–300 µM) in cells of blood, spleen, and bone marrow of mice and in blood cells of frogs and humans were performed using the alkaline comet assay. For both agents, the levels of induced DNA damage in leucocytes/splenocytes of mice were higher than those in blood cells of frogs and humans, while in human leucocytes, they were comparable with those in frog blood cells. The rate of DNA repair in frog blood cells was very slow. The results suggest that the levels of radiation-induced DNA damage are not in accordance with species radiosensitivity (according to LD_50/30) but rather with the intrinsic peculiarities of cells.     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.     

An experimental approach to determining the protective properties of drugs against ionizing radiation in low and sublethal doses]

In experiments with mice, the efficiency of radioprotective agents against acute gamma-irradiation with nonlethal and low doses (0.5-4 Gy) was estimated by nine parameters. The treatment with cystamine, cysteamine and riboxine prior to irradiation (< 2 Gy) did not influence the postirradiation values of the parameters under study. With a dose of up to 4 Gy, the effect of riboxine was noted. The determinations of the number of aberrant mitoses, cellularity and mitotic activity of the bone marrow have proved to be most informative. The data obtained are discussed with due regard for the specific effect of low-level radiation and mechanisms of the protective action of the agents under study.     

Molecular hydrogen and radiation protection

Molecular hydrogen (dihydrogen, H(2)) acts as a therapeutic antioxidant by selectively reducing hydroxyl radicals (?OH) and peroxynitrite (ONOO-). It has been well-known that ionising radiation (IR) causes oxidative damage and consequent apoptosis mainly due to the production of ?OH that follows radiolysis of H(2)O. Our department reported the protective effect of H(2) in irradiated cells and mice for the first time, and this effect is well repeated by us and another laboratory in different experimental animal models. A randomised, placebo-controlled investigation also showed consumption of H(2) can improve the quality of life of patients treated with radiotherapy for liver tumours. These encouraging results suggested that H(2) has a potential as a radioprotective agent with efficacy and non-toxicity.     

Hydrogen peroxide-induced base damage in deoxyribonucleic acid

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.     

The heart-protective mechanism of nitronyl nitroxide radicals on murine viral myocarditis induced by CVB3

Our previous researches showed that nitronyl nitroxyl derivatives, NNP and NNVP were good anti-oxidants and provided radioprotective effects in C6 cells. The objective of the present study is to investigate the possible antiviral effects and underlying pharmacological of the two nitronyl nitroxide radicals against CVB3 in vitro and in vivo. The results showed that NNP and NNVP were some of the most potent compounds in terms of their antiviral effects by protecting myocardial cells against oxidative damage of free radicals. Treatment with NNP or NNVP could decrease the intracellular ROS level in vitro. They could lead to a significant decrease in activities of biochemical markers AST, CK and LDH in infected murine serum and could increase SOD and CAT activities and decreased MDA activities compared with infected control in vivo. NNP and NNVP could reduce NO production in infected mice by reacting with NO to produce the imino nitroxides which was confirmed by ESR spectrometry. In addition, NNP and NNVP could both decrease the mRNA expression of proinflammatory cytokines, TNF-α, IL-2 and IL-6. In conclusion, nitronyl nitroxide radicals NNP and NNVP were shown to have antiviral activities against CVB3 and they may represent potential therapeutic agents for viral myocarditis.     

Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.     

The production of hydroxyl radicals by adriamycin in red blood cells

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.     

Protective effect of hydrogen‐rich saline against radiation‐induced immune dysfunction

Recent studies showed that hydrogen can be used as an effective radioprotective agent through scavenging free radicals. This study was undertaken to evaluate the radioprotective effects of hydrogen on immune system in mice. H₂ was dissolved in physiological saline using an apparatus produced by our department. Spleen index and histological analysis were used to evaluate the splenic structural damage. Spleen superoxide dismutase, GSH, MDA were measured to appraise the antioxidant capacity and a DCF assay for the measurement of radical oxygen species. Cell apoptosis was evaluated by an Annexin V‐FITC and propidium iodide staining method as well as the apoptotic proteins such as Bcl‐2, Bax, caspase‐3 and c‐caspase‐3. CD4+ and CD8+ T cells subtypes were detected by flow cytometry with FITC‐labelled antimouse CD4 and PE antimouse CD8 staining. Real‐time PCR was utilized to determine the CD4+ T cell subtypes and related cytokines. Our study demonstrated that pre‐treatment with H₂ could increase the spleen index and attenuate the radiation damage on splenic structure. Radical oxygen species level was also reduced by H₂ treatment. H₂ also inhibited radiation‐induced apoptosis in splenocytes and down‐regulated pro‐apoptotic proteins in living mice. Radiation‐induced imbalance of T cells was attenuated by H₂. Finally, we found that H₂ could regulate the polarization of CD4+ T cells and the level of related cytokines. This study suggests H₂ as an effective radioprotective agent on immune system by scavenging reactive oxygen species.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.     

Evaluation, in vitro, of the radioprotection of DNA from gamma-rays by naphazoline

Design of compounds that can protect efficiently against gamma-rays irradiation is a great challenge. An ionizing event can cause variety of DNA damage scenarios leading to mutagenesis, cell death. 2-(1-Naphthylmethyl)-2-imidazoline (naphazoline, NP) is a drug belonging to the vasoregulator class, which was shown to be a very interesting compound in radioprotection. In order to highlight the NP radioprotective activity, a comparison of its ability to protect DNA against either gamma-irradiation or radicals generated by Fenton's reaction was made. Results show that NP inhibits efficiently the generation of DNA single-strand breaks and that NP is a potent radioprotector and also an hydroxyl radical scavenger.     

DNA damage in thymocytes of mice under combined acute whole body exposure to cadmium ions and gamma-radiation

The effect of the combined acute whole body exposure to cadmium chloride (0.5 mg Cd2+ per kg body weight of animals) and gamma-radiation (1 Gy) on the DNA damage induction in thymocytes and thymic cellularity of mice was studied. It has been shown that CdCl2 solution injection 0.5 h before irradiation reduces the quantity of single-strand DNA breaks and alkali-labile sites in thymocytes 48 h after injection compared to gamma-radiation action only. The observed effect is accompanied by a sharp decrease of the thymic cellularity compared with the separate effects of both cadmium ions and irradiation, which masks the overall genotoxic effect of combined exposure and gives an illusion of cadmiumL ions radioprotective action. Cadmium chloride injection 24 h before irradiation leads to a significant additive increase in the single-strand DNA breaks and alkali-labile sites number as compared to the separate effects of cadmium ions and irradiation alone. At the same time the decrease in the percentage of DNA tightly bound to proteins (DNA-protein cross-links) was noted in comparison with the action of gamma-radiation only. Statistically significant changes in thymic cellularity compared with separate effects of cadmium ions and irradiation were not found. Thus, our research has shown that under a combined action of cadmium ions and gamma-radiation on thymocytes in mice at the applied doses and exposure schemes the additive effects, rather than antagonism or radioprotective effects are observed.     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.