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Development of Enhanced Radioprotectors

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Development of Enhanced Radioprotectors KAERI/RR -2000/99 KR0000199 M Radiation Application for Development of Bioscience Development of Enhanced Radioprotectors if Sf 7| KAERI/RR-2000/99 M H M Radiation Application for Development of Bioscience Development of Enhanced Radioprotectors Please be aware that all of the Missing Pages in this document were originally blank pages 2000. 4. 71 71 -1- II III Ljf-g- in vitro 9| in w'ro IV. TSH In vivo U in vitro -§-*H -3- ^ NIH MCF 7]^ ^ 4s &#<>] vitro 31 in vivo<>\}*\$] apoptosis^] v. 71 £ 7H1M -4- SUMMARY I . Project Title Development Enhanced Radioprotectors II. Objective and Importance of the Project For the development of enhanced radioprotectors, it's required to establish methodologies for screening radioprotective effects of natural products. Such an effort should be made from the point of hoisting national competitiveness. The object of the project was to set up key technology to develop enhanced candidates of radioprotective agents and to elucidate the action mechanisms of those candidates in order to protect the human resources from various radiation damages. III. Scope and Contents of Project In order to develop radioprotective or radiosensitizing agents from natural products, experimental works have been carried out. • Elaboration of biological model systems for assessing radio- protective or sensitizing activities of natural products • In vivo and/or in vitro studies of the protecting effects of candidates compounds on radiation damage •Assessment of radiosensitizing activities of farnesyltransferase inhibitors • Molecular biochemical studies on radioprotective mechanism and genes related to radiation damage • Study on the induction of radioprotection using antioxidant -5- combinations IV. Result of Project Biological model systems such as TSH mutations, animals and their reproductive organs, cultured cell line, and human lymphocytes have been set up for screening radioprotective or radiosensitizing activities of natural products. About 100 natural plants were in vivo and in vitro tested. Among them, Sal via miltiorrhiza, Areca catechu, Cornus officinalis, and Portulaca oleracea showed 20% of radioprotective activities. On the other hand, Acanthopanax sessiliflous, Melia japonica, and Agastache rugosa revealed radiosensitizing activities. Experimental studies on the farnesyltransferase inhibitors showed the results that some of the inhibitors lead to higher levels of cell death after /-irradiation and increased radiosensitivity in H-ras-transformed NIH3T3 cells and MCF-10A human tumor cells. Molecular biochemical studies on the mechanism of radioprotection was carried out using cultured cells. The treatment of DTT increased cell survival after gamma-irradiation, decreased in the frequencies of micronucleus, and reduction in DNA fragmentation and apoptotic cells. Induction of apoptosis after irradiation was revealed by the changes in the relative cell death, increase in the relative amount of apoptotic cells, and the induction of DNA fragmentation. The radioprotective effects of antioxidants in combination was studied in the bone-marrow cells of experimental animals. V. Proposal for Applications The experimental model systems established in this study will make a key role for assessing radioprotective activities in the next stage of the project. Samples shown excellent radioprotective or radiosensitizing activities will be subjected to purification to develop radioprotectants and radiosensitizers applicable to human beings. The experimental results on farnesyltransferase inhibitors will supply with fundamental data for the following studies. The result of mechanism studies will- be applied to elucidate the radioprotection mechanism and to develop enhanced radioprotectors. Since the use of antioxidants in combination can efficiently modulate the radiation-induced cell damage like apoptosis, it can be applied to alleviate the cellular damage in tumor patients during radiotherapy. -7- CONTENTS Chapter 1. Introduction 19 Chapter 2. States of the Art 27 Section 1. Current States 27 Section 2. Assessment of the States •• 37 Chapter 3. Scope and Contents of the Project 39 Section 1. Extracts of Natural Medicinal Plants 39 Section 2. Model Systems for Screening Radioprotective Activities of Natural Products 40 Section 3. Assessment of Biological Damage Protection 59 Section 4. Studies on Radiosensitizing Activities of FTI's 118 Section 5. Biochemical and Molecular Genetical Approaches on the Radioprotective Mechanisms of Natural Products 145 Section 6. Study on Induction of Radioprotection Using Antioxidant Combinations 162 -9- Chapter 4. Achievements and Contributions of the Project 189 Chapter 5. Proposal for Applications 191 Chapter 6. References -10- Content of Tables Table 1. Experimental code for the group or material 43 Table 2. Change of body weights of control, FSH- injected, irradiated, and irradiated and FSH-injected immature female mice 76 Table 3. Changes of ovarian weights (mg) of control, FSH-injected, irradiated, and irradiated and FSH-injected immature female mice 77 Table 4. Immunohistochemistry of ovarian sectionates obtained from immature mice 78 Table 5. Effect of ascorbic acid, tocopherol and cysteine on the number of WBC, RBC and platelet of mice irradiated with 6.5 Gy five days after oral administration 86 Table 6. Effect of ascorbic acid, tocopherol and cysteine on body weight, spleen weight and testis weight of mice irradiated with 6.5 Gy five days after oral administration 87 Table 7. Effect of ascorbic acid, tocopherol and cysteine on the number of sperms and testis volume of mice irradiated with 6.5 Gy five days after oral administration 88 Table 8. Effect of ascorbic acid, tocopherol and cysteine on the antioxidant activity in livers of mice irradiated with 6.5 Gy five days after oral administration 89 Table 9. The FACS effects of ascorbic acid, tocopherol and cysteine on leucocytes of BALB/c mice irradiated with 6.5 Gy five days after oral administration 90 Table 10. HPLC analysis conditions • 97 Table 11. Amino acid concentration of urine collected from mice -11- irradiated with 6 Gy, and pretreated with purslane extract for 5 days and then irradiated with 6 Gy during 19 days after irradiation 98 Table 12. Survival rate of mice injected with saline or purslane extract for 5 days and then irradiated with 8 Gy 99 Table 13. Relative protective effects of methanol extracts of medicinal plants on H202-induced oxidative stress in V79-4 cells • 104 Table 14. Relative protective effect of methanol extracts of medicinal plants against 10 Gy of y -ray-induced oxidative stress- 106 Table 15. Protective effect of fractions of medicinal plant extracts against oxidative stress in V79-4 cells 107 Table 16. Cytotoxicity of natural medicinal plant extracts against carcinoma (KB) cells 114 Table 17. Oxidative cell damage-sensitizing effect of natural medicinal plant extracts 116 Table 18. Changes in IC50 values of natural medicinal plant extracts when combined with chemically induced-oxidative stress •••• 118 Table 19. Induction of cell cycle changes by a gamma-irradiati on •••• 133 Table 20. Effects of HCA and BCA on radiation-induced cell death of N1H cell lines 139 Table 21. Effects of FPTase inhibitor 111 treatment on radiation- induced cell death 140 Table 22. Change of cell cycle after the treatment either with /-irradiation or FPTase inhibitors plus irradiation 145 Table 23. Micronucleus frequencies of Chinese hamster (V79-4) cells after y-ray irradiation in the absence or presence of 0.5 mM or 1 mM of DTT 151 -12- Table 24. Effect of DTT on radiation-induced apoptosis in small intestinal crypts of y -irradiated mice 152 Table 25. Effect of sodium selenite and its combination with N-acetylcysteine on radiation-induced cell death 170 Table 26. Effect of sodium selenite and its combination with N-acetylcysteine on radiation-induced cell death 171 Table 27. Effect of sodium selenite and its combination with N-acetylcysteine on radiation-induced cell death 172 Table 28. Effect of DL-ff-lipoic acid and its combination with N-acetylcysteine on radi at ion-induced cell death 174 Table 29. Effect of aminoguanidine and its combination with N-acetylcysteine on radiation-induced cell death 176 Table 30. Effect of aminoguanidine and its combination with N-acetylcysteine on radiation-induced cell death •••••• 177 Table 31. Effect of aminoguanidine and its combination with N-acetylcysteine on radiation-induced cell death 178 -13- Content of Figures Fig. 1. The effects of ethanol-extracts from plants on somatic cell mutation frequencies in Tradescantia stamen hairs 44 Fig. 2. The effects of water-soluble extracts from various materials on somatic cell mutation frequencies in Tradescantia stamen hairs 45 Fig. 3. Pink mutation frequencies induced by radiation in Tradescantia 4430 pretreated with 10-fold diluted natural extracts 46 Fig. 4. Pink mutation frequencies induced by radiation in Tradescantia 4430 pretreated with 20-fold diluted natural extracts 47 Fig. 5. Appearance of typical DNA comets 56 Fig. 6. Effect of peach kernel extracts on tail moment in human lymphocytes exposed to y -ray doses from 0 to 2.0 Gy 57 Fig. 7. Reduction of tail moment in the human blood lymphocytes pretreated with peach kernel extracts and then irradiated with gamma-radiation (0—2.0 Gy) 58 Fig. 8. Changes of atretic follicles {%) in y -ray irradiated immature mouse ovary 65 Fig. 9. Normal and atretic follicles after irradiation 66 Fig. 10. Percent {%) of PCNA-stained follicles to HE-stained total follicles of irradiated mouse ovaries 72 Fig. 11. Percentage of in situ 3'-end labelled follicles to HE-stained total follicles of irradiated mouse ovaries 73 Fig. 12. DNA fragmentation analysis showing radiation-induced atresia were mediated by apoptosis 74 Fig. 13. DNA fragmentation analysis showing the delay effect of FSH on -14- radiation-induced apoptosis • • 75 Fig.14. Relative cell survival of V79-4 cells after y -ray irradiation. Two different concentrations of cells were seeded on 96 well plates and irradiated with either 5 or 10 Gy of y -rays 108 Fig. 15. Relative cell survival of V79-4 cells after UV-C irradiation 109 Fig. 16. Relative cell survival of V79-4 cells after hydrogen peroxide treatment • 110 Fig.
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