Synthesis of some thiourea derivatives and their influence on biological and genetic effects of gamma rays

The synthesis of the new radioprotective compounds ATB (I, 2-allylthioureidobenzoic acid), PTB (II, 2-phenylthioureidobenzoic acid), A-2-PTU (III, N-allyl-N'-2-pyridylthiourea), and P-2-PTU (IV, N-phenyl-N'-2-pyridylthiourea) and their influence on biological and genetic effects of gamma rays was studied. In result of investigations it must be noted that PTB displayed radioprotective effect as a result of which more plants in M1 germination and survive in M2 of the induced mutations is increased. The cytological analysis reveals that the studied substance (PTB) decreases chromosome aberration in meristem cells of pea roots almost twice as a result of postirradiation treatment. The effect of A-2-PTU in the experiments with peas greatly depends on the dose of irradiation, i.e., on the degree of damaging of the processes of cell restoration and the possibility of their partial restoration after the treatment with the protector. The results obtained suggest that chemical compounds of N,N'-disubstituted thiourea group (A-2-PTU and P-2-PTU) exert strong radioprotective effect in the experiments with peas. This is of great importance to modern radiobiology and radiation mutagenesis and also to protect hereditary structures against radiation.     

Synthesis of Some New Thiourea Derivatives and Their Influence on Biological and Genetic Effects of Gamma Rays*

The synthesis of the new radioprotective compounds ATB (I, 2-allylthioureidobenzoic acid), PTB (II, 2-phenylthioureidobenzoic acid), A-2-PTU (III, N-allyl-N"-2-pyridylthiourea), and P-2-PTU (IV, N-phenyl-N"-2-pyridylthiourea) and their influence on biological and genetic effects of gamma rays was studied. In result of investigations it must be noted that PTB displayed radioprotective effect as a result of which more plants in M₁ germination and survive in M₂ of the induced mutations is increased. The cytological analysis reveals that the studied substance (PTB) decreases chromosome aberration in meristem cells of pea roots almost twice as a result of postirradiation treatment. The effect of A-2-PTU in the experiments with peas greatly depends on the dose of irradiation, i.e., on the degree of damaging of the processes of cell restoration and the possibility of their partial restoration after the treatment with the protector. The results obtained suggest that chemical compounds of N,N"-disubstituted thiourea group (A-2-PTU and P-2-PTU) exert strong radioprotective effect in the experiments with peas. This is of great importance to modern radiobiology and radiation mutagenesis and also to protect hereditary structures against radiation.     

Biochemical states of zinc incorporated in the liver of mice treated with a radioelement after irradiation with a sublethal dose of gamma-rays

Radioresistance has been observed in mice which have received an inorganic zinc 24 hours prior to gamma irradiation with a sublethal dose. In order to elucidate the mechanism of radioresistance, the biochemical states of the metal in filtrate extracted from the homogenated liver of the animal which have received an injection of zinc under the form of 65Zn as tracer 24 hours post irradiation, were analyzed and compared with those of non-irradiated (control) mice. Two peaks of radioactivity: P-1 and P-2, having molecular weight of about 45,000 and 20,000-25,000 respectively, were revealed by Sephadex G-75 separation. The radioactivity of P-2 (39%) from the irradiated mice is lower than that from the controls (46%). Chromatography of P-2 from control animals through DEAE-Sephadex A-50 revealed the existence of a large radioactive peak (P-U). Chromatography of P-2 from irradiated animals did not show any P-U peak. This P-U peak is composed principally from 2 to 3 proteins with a molecular weight of 12,000 to 26,000. Proteins of the P-U peak provoke a protection effect against the lethal effect of gamma-rays when they are injected into mice after irradiation.     

Induction by gamma-radiation of DNA synthesis in radicle cells of germinating seeds of Pisum sativum l.

In radicle meristem cells of germinating seeds of the pea (Pisum sativum L) before the onset of replicative synthesis of DNA, irradiation with 2-3 krad of gamma-rays induced the incorporation of 3H-thymidine (3H-TdR). Maximum isotope incorporation was noted during the first 2 hours after irradiation. Higher doses of radiation suppressed 3H-TdR incorporation. It was not seen after gamma-irradiation of air-dried seeds, nor after fast-neutron irradiation. The replication inhibitors hydroxyurea and 5-aminouracil had no effect on the gamma-induced incorporation of 3H-TdR, Whereas caffeine and acriflavine inhibited it to some extent. It is suggested that the gamma-radiation-induced incorporation of 3H-TdR in meristem cells during the pre-replicative period may be connected with repair phenomena.     

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by gamma-rays or ultraviolet (UV) light in cultured Chinese hamster V79 cells were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10(-5) M. Cobaltous chloride at a concentration of 3 x 10(-6) M had a marked effect in reducing 8AG-resistant mutations induced by gamma-rays of 2-6 Gy, when cells were incubated for 6-7 days in the presence of cobaltous chloride after gamma-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before gamma-ray irradiation reduced 8AG-resistant mutations induced by gamma-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and mammalian cell systems is discussed.     

Antioxidant vitamins C, E and beta-carotene reduce DNA damage before as well as after gamma-ray irradiation of human lymphocytes in vitro

The protective effect of Vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in human lymphocytes in vitro was investigated. Cultured lymphocytes were exposed to increasing concentration of these vitamins either before or after irradiation with 2Gy of gamma-rays and DNA damage was estimated using micronucleus assay. A radioprotective effect was observed when antioxidant vitamins were added to cultured cells before as well after irradiation; the strongest effect was observed when they were added no later than 1h after irradiation. The radioprotective effect of vitamins also depended on their concentration; Vitamins C added at low concentration (1 microg/ml) before exposure of the cells to radiation prevented induction of micronuclei. Vitamin E at the concentration above 2 microg/ml decreased the level of radiation-induced micronuclei when compared to the cells irradiated without vitamin treatment. beta-Carotene was effective at all tested concentrations from 1 to 5 microg/ml and reduced the number of micronuclei in irradiated cells. The vitamins had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The radioprotective action of antioxidant Vitamins C, E and beta-carotene was dependent upon their concentration as well as time and sequence of application.     

Phytochrome-mediated regulation of a monooxygenase hydroxylating cinnamic acid in etiolated pea seedlings

Cinnamic acid is hydroxylated by the mixed-function oxidase trans-cinnamic acid 4-hydroxylase (CA4H). The hydroxylation reaction involves the transfer of electrons from reduced pyridine nucleotides via the enzyme NADPH cytochrome P-450 reductase to the terminal oxidase cytochrome P-450. This multi-enzyme complex is localized in the microsomal fraction. Isopycnic and velocity gradient centrifugation suggest that in the apical bud of etiolated pea seedlings this complex is restricted to the endoplasmic reticulum membranes. CA4H activity which develops in dark germinating pea seedlings was found to be stimulated by light, an effect mediated by phytochrome. CA4H and NADPH cytochrome c reductase activities, cytochromes P-450 and b 5 contents were measured in seedlings submitted to either short pulses of red and far-red light, or to continuous far-red or blue irradiation. The results are discussed in terms of a specific effect of phytochrome on the different parts of the multi-enzyme complex.     

Tests which distinguish induced crossing-over and aneuploidy from secondary segregation in Aspergillus treated with chloral hydrate or gamma-rays

A system of tests with the ascomycete Aspergillus nidulans was devised that can detect 3 primary effects of genotoxic agents: (1) increases in mitotic crossing-over; (2) induced aneuploidy; and (3) clastogenic effects which cause chromosomal imbalance. Conidia of a new diploid tester strain, heterozygous for 4 recessive markers which alter conidial color, are treated and plated onto nonselective media. In cases of induced crossing-over, large color segments are found in normal green colonies, frequently adjacent to reciprocal twin segments. In contrast, both malsegregation and chromosome breakage produce unbalanced types which grow poorly and segregate further. Cases with yellow segregants are replated and their secondary diploid sectors tested for markers which are located on both chromosome arms in coupling with yA. Induced aneuploidy can be distinguished from chromosome breakage by the pattern of marker segregation. Any aneuploid type will produce euploid sectors solely by segregation of whole chromosomes; trisomic colonies (yA / yA / +) will show 1:2 ratios for yellow (homozygous yA) to parental green (yA/+) sectors and have characteristic phenotypes. Other induced unbalanced types, if heterozygous for deletions or aberrations may produce yellow diploid sectors by secondary crossing-over as well as by nondisjunction and such cases show unique patterns of genetic segregation and non- predictable phenotypes. As a complementary test, haploid strains are treated and induced abnormally growing types are replated and classified by phenotype. Aneuploids are unstable and produce many normal sectors, and some of these disomic or trisomic types can be visually identified.In contrast, induced deletions are lethal, and duplications or 'morphological' mutants show much more stable abnormal phenotypes. This test system was used to characterize the primary effects of gamma-rays and chloral hydrate. Results and evidence were as follows: (1) A dose-dependent increase of color segments resulting from reciprocal crossing-over was found after treatment of dividing nuclei in germinating diploid conidia with gamma-rays, but not with chloral hydrate. (2) Highly aneuploid and polyploid types were induced in diploid and haploid germinating conidia by chloral hydrate but not to any significant extent by gamma-rays. (3) gamma-Rays caused a dose- dependent increase off abnormally growing colonies when dormant or germinating diploid conidia were treated. These colonies produced secondary euploid sectors by spontaneous nondisjunction and frequently also by crossing-over, which provided evidence for induced semidominant and recessive lethal mutations of many types.     

Enzyme histochemical, biochemical and metabolic effects of Solcoseryl in connection with its radioprotective capability

The lethal irradiation with gamma-rays (LD 100/30) of adult mice at ambient temperature of 32.5 degrees C heavily alterates the pattern of LDH in liver. In close correspondence with the radioprotective effect of Solcoseryl on the survival rate of irradiated mice -- which amounts to 100% as compared with 40% survival of solely heat adapted animals -- Solcoseryl normalizes the LDH distribution both histochemically and biochemically. Since Solcoseryl alone generally enhances enzyme activity the normalization observed is obviously attributable to an intrinsically regulatory property of this natural compound.     

Dose-rate effects of gamma-ray-induced mutations in cultured mammalian cells

The dose-rate dependency of three radiobiological parameters, cell killing and mutations resistant to 6-thioguanine (6-TG^r) and to methotrexate (MTX^r), were studied in populations of mouse L5178Y cells exposed to gamma-rays. when the dose rate was reduced from 50 rad/min to 0.8 rad/min, the shape of the dose—response curves changed from sigmoidal to exponential for cell killing, from upward concave to linear in 6-TG^r mutations and remained linear in MTX^r mutations. A linear quadratic model appears capable of explaining the cell killing and 6-TG^r mutations but not the MTX^r mutations.The declining patterns of induced mutation frequencies of 6-TG^r and MTX^r with decreasing dose rate seem to be similar. The addition of DMSO resulted in protection of cells from cell killing, 6-TG^r and MTX^r mutations with acute exposure, but had little effect with chronic exposure. The reduction of mutation frequency of the 6-TG^r marker with chronic exposure was eliminated by holding cells in ice-cold condition during irradiation. These results suggest that there may be two components of induced mutation. One results primarily from repairable damage induced by the indirect action of radiation and shows a clear dose-rate dependency. The other is mainly from non-repairable damage by the direct action of radiation and is only slightly dose rate-dependent. Under chronic exposure conditions, the latter may predominate.     

Changes in concentration of endogenous growth inhibitors during growth recovery of dwarf pea seedlings

In order to clarify the role of endogenous growth inhibitors A-2α and A-2β in a dwarf pea plant, red light (emission peak 657 nm) treated, 9-d-old seedlings of dwarf pea (Pisum sativum L. cv. Progress No. 9) were transferred to darkness, and the resulting changes in growth rate and concentrations of A-2α and A-2β were monitored. The growth rate of the epicotyls increased, and the concentration of the inhibitors in the epicotyls decreased, according to sigmoidal time courses. The relationship between the logarithms of the concentration of the inhibitors and the corresponding growth rate was linear. These results suggest that A-2α and A-2β, may play an important role in the growth recovery process of the dwarf pea cultivar after termination of red light irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Radioprotection by mangiferin in DBAxC57BL mice: a preliminary study

The radioprotective effects of various concentrations (0, 0.25, 0.5, 1, 2, 5, 10, 17.5, 25, 50, 75 and 100 mg/kg b.wt.) of mangiferin (MGN) was studied in the DBAxC57BL mice whole body exposed to 10 Gy of gamma-irradiation. Treatment of mice with different doses of MGN, one hour before irradiation reduced the symptoms of radiation sickness and delayed the onset of mortality when compared with the non-drug treated irradiated controls. The radioprotective action of MGN increased in a dose dependent manner up to 2mg/kg and declined thereafter. The highest radioprotective effect was observed at 2mg/kg MGN, where greatest number of animals survived against the radiation-induced mortality. The administration of 0.5, 1, 2, 5, 10 and 17.5 mg/kg MGN reduced the radiation-induced gastrointestinal death as evident by a greater number of survivors up to 10 days in this group when compared with the DDW + 10 Gy irradiation group. A similar effect of MGN was observed for the radiation-induced bone marrow deaths also. Our study demonstrates that mangiferin, a gluosylxanthone, present in the Mangifera indica protected mice against the radiation-induced sickness and mortality and the optimum protective dose of 2mg/kg was 1/200 of LD50 dose (400 mg/kg) of MGN. The administration of 400 mg/kg MGN induced 50% mortality, therefore LD50 of the drug was considered to be 400 mg/kg.     

The mutagenic effect of gamma rays on leaf protoplasts of haploid and dihaploid Nicotiana plumbaginifolia,estimated by valine resistance mutation frequencies

Summary Leaf protoplasts isolated from haploid and dihaploid Nicotiana plumbaginifolia plantlets were treated with different doses of gamma-rays and their survival was determined by scoring for plating efficiency at each irradiation dose. A fixed number of surviving protoplast-derived colonies was then plated in the presence of inhibitory concentrations of L-valine and incubated until growing resistant calli could be scored and mutation rates calculated. Though haploid protoplasts were found to be a little more sensitive than dihaploids to the lethal effect of radiation, the two dose-response curves of gamma-rays that induced mutagenesis were very similar. The irradiation dose capable of causing a ten-fold increase of spontaneous mutation frequencies was about 500 rads with both haploid and dihaploid protoplasts.Contribution No. 2173 of the Biology Radiation Protection and Medical Research Programme, Directorate General XII of the Commission of the European Communities     

Mutagenic action of cadmium on the sex cells of male mice

Possible mutagenic effect of cadmium chloride was studied by determining the frequency of dominant lethal mutations induced in germ cells of male mice. Water solution of CdCl2 was injected intraperitoneally to male mice at doses of 1.0, 2.0 and 4.0 mg/kg. The results obtained did not reveal any mutagenic effect of this compound. The dose of 4.0 mg/kg CdCl2 resulted in the death of spermatocytes and spermatogonia and the sterility of male mice. Cadmium chloride at a dose of 2.0 mg/kg did not affect the frequency of dominant lethal mutation induced by gamma-rays 60Co at a dose of 450 r in germ cells of male mice.     

NO-dependent pathway of the modifying of the cellular sensitivity to gamma- and neutron irradiation

The modifying effect of L-NAME, the NO-synthase inhibitor and D-NAME, the inactive enantiomer was investigated in human carcinoma cells (HeLa) and Chinese hamster fibroblasts (V-79) exposed to different doses of gamma-rays and 0.85 MeV neutrons. We estimated the level of the chromosome aberrations manifested as the bridges and fragments in anaphases. Radioprotective effect of L-NAME showed the inverse dependence on the exposure dose and at low doses (1 Gy) it was higher in the V-79 cells, than in the HeLa cells. However, at high doses (3, 4, 6 Gy) the efficiency of L-NAME for these cellular lines was almost equal (DFR = 2). The modifying effect of L-NAME was almost equal for gamma-irradiation and neutrons, although the exposure of V-79 cells to neutrons induced more the asymmetric chromosome aberrations (RBE = 4). The D-NAME had no effect on the level of the radiation-induced chromosome aberrations, although D-NAME treatment of cells increased the chromatin condensation, as well as L-NAME. The counteractive condensation does not play the major role in the radioprotective effect of L-NAME. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the inducible NO-synthase.     

Bio-antimutagenic effects of tannic acid on UV and chemically induced mutagenesis in Escherichia coli B/r

Tannic acid suppressed the mutagenesis in E. coli B/r WP2 trp- induced by UV or 4-nitroquinoline 1-oxide (4NQO), but not that induced by gamma-rays or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The depression of mutations induced by UV was most remarkable in the DNA-repair-proficient strain (WP2). Tannic acid, however, showed no bio-antimutagenic effect in the excision repair-deficient strain (WP2s uvrA- or ZA159 uvrB-) under the test conditions where no cellular toxicity was observed. The effect ceased within 30 min after UV irradiation. The inhibition of the expression of Trp+ phenotype and the delay of the first cell division after UV irradiation were not observed in the presence of tannic acid. From these results we conclude that tannic acid may enhance the excision-repair system probably by activating the repair enzymes or by interacting with DNA.     

An inhibitor of potentially lethal damage (PLD) repair reduces the frequency of gamma-ray-induced mutations in cultured Chinese hamster V79 cells.

Cordycepin (3'-deoxyadenosine, 3'-dA) is an RNA antimetabolite and a radiosensitizer in cultured mammalian cells. In the present paper, the effects of 3'-dA on gamma-ray-induced lethality and 6-thioguanine (6TG)-resistant mutations in cultured Chinese hamster V79 cells were examined. 3'-dA had the effect of sensitizing the lethality induced by gamma-rays. The potentially lethal damage (PLD) repair produced by post-incubation of cells in Hanks' solution after gamma-irradiation was almost completely suppressed by 5 x 10(-5) M 3'-dA. When cells were irradiated with 10 Gy gamma-rays and incubated with 3'-dA for 5 h, the frequency of 6TG-resistant mutations induced by gamma-rays decreased to one-sixth of that of irradiated cells incubated without 3'-dA. The decrease in the frequency of gamma-ray-induced mutations was dependent on the length of incubation time with 3'-dA. It is suggested that the inhibition of PLD repair by 3'-dA may be that of error-prone repair.     

Effect of copper ions on chromosome structural mutations caused by Cs1 37 gamma rays in moist seed cells of Crepis capillaris L]

When seeds of Crepis capillaris were germinated in solutions of Cu(NO3)2 (at 0.55-10(-5) M and 1.1-10(-5) M concentrations) the frequency of structural chromosome mutations induced by gamma-rays (137Cs) at 200 and 400 r doses was higher as compared to that induced in the absence of copper ions.     

Radioresistance of kohlrabi (Brassica oleracea L,var.gongylodes L.) seeds in relation to the metabolism of indoles,auxins and gibberellins

Irradiation of the seeds of radioresistant kohlrabi with gamma-rays causes at doses SO to 300 kR a decrease in growth and a drop in tryptophan (Try) level in seven-day-old plants. The level of glucobrassicin (GLUBR), 3-indolylacetonitrile (IAN) and gibberellic acid (GA3) in these plants increases up to a maximum with a dose of about 150–200 kR, afterwards it decreases. We assume that the specific system of auxin synthesis inBrassicaceae plants takes an important part in the reparation processes and thus in the radioresistance of these plants as well. In contrast to the divergent systems of auxin biosynthesis in other plants which are damaged by lower doses of irradiation, the specific system of auxin formation via GLUBR synthesis, under formation of IAN intermediate, is widely not. attacked by radiation. Thus, these irradiated plants are supplied with auxins and with gibberellins too, the both hormones having a radioprotective effect.