Journal of Oriental Medicine

 Vol.5, No.1, December, 2000

 

A study of the radioprotection effect of macro-glucan

(b-1-3glucan) on the fetuses of ICR mice

 

Gu Yeun-hwa*, Hasegawa Takeo*, Suzuki Ikukatsu**, Hayashi Ikuo**

Ahn Kyoo-Seok***, and Tawaraya Hitoshi****

 

*Dept. of Radiological Technology, Suzuka University of Medical Science,

1001-1 Kishiokacho, Suzuka City, Mie 510-0293, Japan

**Dept. of Clinical Nutrition, Suzuka University of Medical Science, 1001-1

Kishiokacho, Suzuka City, Mie 510-0293, Japan

***Dept. of Pathology, College of Oriental medicine, Institute of Oriental Medicine, Kyunghee University, Seoul, 130- 701, Korea.

****Japan Sunny Health Co.1m. 1-10-17 Hamatsucho, Minato-ku, Tokyo, 105-0013, Japan

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Abstract

 

In this study abundant malformations of ICR mice in the organogenesis stages were observed and the radioprotection effect of Macro-Glucan(b-1-3glucan) was shown. This research examined the radioprotection effect of Macro-Glucan(b-1-3glucan) and its teratogenic properties. The reason it has a preventive effect on a tall fetus was examined in the research. We examined the determination stage (organogenesis), and pointed to malformation toward the radiation in the environmental agent to the malformation. Moreover, we examined the cellularis level of the embryos toward the radiation and analyzed the mechanism of the protection effect of the Macro-Glucan(-1-3glucan) from malformation induction by the radiation. Therefore, the purpose of this research is to get a foundation of data of the radioprotection effect of Macro-Glucan(-1-3glucan). This study produced the following results: It was made clear that Macro-Glucan(-1-3glucan) had a radioprotection effect against embryonic death. Radioprotection effect was recognized as the teratogenesis rate by Macro-Glucan(-1-3glucan)'s administering it before the exposure. Therefore, the subject faced radiation exposure by the Macro-Glucan(-1-3glucan) administration, and radiological protecting effect was made clear.

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Key Words: Radioprotection effects · Organogenesis · Malformation ·  Macro- Glucan (ImmuDyne’s MacroForce sold by Sunny Health in Japan)

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Introduction

 

        The aims of the radioprotection are of a human and environmental concern. Radiation has many harmful points as well as positive ones for human life. First, nuclear the power plant industry has been contributing greatly to the use to the radiological peace. As for present clinical medicine, the medical techniques that have been developed as a result of radiation cannot be disputed. However, there are many problems, that a radioprotection plan at the nuclear power plant that haven't been solved. Moreover, the study of fetal effects by radiation for medical research is not only the concern of medical science but also has broader social implications as well. The mission of the nuclear power plant makes it critical to make radioprotection toward the people concerned engaged in the nuclear power plant and the periphery inhabitants a perfect thing. It is the aim of which radioprotection from the internal exposure of the human and the external exposure is the biggest.

  Recently, the individual, or the combination in the anti-cancer medicine is using much treatment of radiation that is one of radiation therapy for a cancer. On that occasion, the most serious problem is a side effect. Chemical material has been used as a radiation protection medicine until before. But, there is a side effect in it as well again. It was examined about effect on radiation protection of Macro-Glucan to malformation by using the ICR mouse being used for the teratogenesis experiment frequently in this research. It paid attention to malformation in the fetus whose sensitivity is high most to the radiation, and It was examined as a means. Moreover, it paid attention to it in this decision time, and effect on radiation protection of Macro-Glucan was examined in this research to the external malformation and skeletal malformation by radiation. As for effect on protection of the embryonic effect toward the radiation as well, effect on radiation protection of Macro-Glucan was studied by doing an examination. It effect on radiation protection to Macro-Glucan is explained, effect as a radiation protection medicine against the exposure of the amount of high line is clear. From now on, other oriental medicine and natural product combination will be done, and it will aim at obtaining information on the radiation protection medicine in the oriental medicine to do use to the radiation protection medicine of Macro- Glucan.

  The fetus is the most sensitive issue when the issue of radiation exposure to humans is talked about 1,2,3,4,5,6,7. Therefore, if it is grasped precisely and protection criterion and protection resource are established, children and adults won't have a problem have a problem from the viewpoint of radioprotection. The aims of the radioprotection are a human and environmental safety concern. The malformation that research shows in the animals, and relations between the fetal death and the radiation are now being made distinct. There is even stage when expectant mother doesn't notice pregnancy until the organogenesis, the stage when a malformation is induced. Because it is the decisive stage which rational symptom is not like this, the potential that an effect is taken from the radiation is high. In this determination stage, the malformation toward the radiation was observed by using the ICR mice. These mice have often been used in Japan for this type of research. We examined the fetal effect toward the radiation. Previous research clearly shows TMG to be similar to the mechanism of the vitamin E and to have the protecting effect that faces free radical by the simulation of In vitro8. So, the reasons it is as a radioprotective agent of the effect on the fetus toward the radiation was examined by using the Macro-Glucan (b-1-3glucan) in this research. Therefore, it is a purpose to obtain information as a medicament of the radioprotection.

 

Materials and Methods

 

1. Experimental animals and mating procedure

  A closed colony of ICR (Crj:CD-l.) mice were purchased from Charles River Japan, Inc. They were housed in a room at a temperature of 22-23 degree and a relative humidity of 60 to 70% with a l2-hour light- dark cycle (the light phases starting at 6:00 and 18:00). The mice were given free access to food (CA-I, CLEA Japan, Inc.) and tap water. One or two female mice, 10 to18 weeks old, were placed together with one male mouse of the same age range in the same cage for only three hours from 6:00 to 9:00. The female mice in which vaginal plugs were found were assumed to have become pregnant at 8:00, at which time day 0 of gestation was, designated9,10. As for the medication method of the Macro-Glucan (b-1-3glucan), internal use of 25µg/kg for no less than two weeks was administered to the pregnant mice11.

 

2. Irradiation with X-rays

 

The pregnant mice were placed in plastic cages for exposure, and were treated with a single whole body X- radiation at 2 Gy with dose rates of 0.35 Gy/min. The pregnant mouse of 192 hpc (hours post conception): (organogenesis stage) was made within the irradiation gauge after the fertilized, Phillips X-ray irradiation device(225kV) was used. The time of embryo exposure was 196hpc. The total number of irradiated dams observed in this study was 40, a total of 38 non- irradiated control and sham control dams was also prepared, and 659 non-irradiated live fetuses served as controls.

 

3. Observation of external malformation and other effects

 

After irradiation, the pregnant mice were sacrificed by cervical dislocation on day 18 of gestation and the total number of corpus luteum in the ovaries, implantation sites, and live and dead embryos and fetuses were counted. The live fetuses were removed from the uterus and examined for external malformations using a dissecting microscope. The body weight and sex of each fetus were also determined.

 

4. STATISTICAL ANALYSIS

 

For teratological effects, it is not appropriate to consider the fetus and embryo to be an experimental unit. The litters (pregnant mice.) were taken into account during the statistical analysis of the experimental data. In the per litter analysis, an average fetal response within a litter was calculated. For statistical tests, we used non-parametric tests that are the Wilcoxon tests12. And, as for fetal body weight, a t-test was done.

 

Results

1. Preimplantation death

 

Statistical significant difference was recognized between the preimplantation death rate of the 2.0Gy group and the control group (P<0.05). Statistical significant difference wasn't recognized in other groups toward the control group. There is no this causal association directly because preimplantation death is different from the radiation exposure time of this study. Moreover, it is absorbed by the metabolism of which implantation site is early, and it is thought that it appeared with the preimplantation death.

 

2. Embryonic death

 

Implantation site, placental remnant and absorption embryo were considered to be embryonic death. Embryonic death is the death which induced from 4.5 hpc (days post conception) to 13.5 hpc. The result of the embryonic death rate is shown in Fig.1. Statistical significant difference was recognized between the embryonic death rate of the 2.0Gy group and the 2.0 Gy +Macro-Glucan (b-1-3glucan.administrated group toward the embryonic death rate of control group and sham control group (P<0.001). But when comparing the embryonic death rate of the 2.0Gy group and the 2.0Gy + Macro-Glucan group, the embryonic death rate of the 2.0Gy + Macro-Glucan group was less. If Macro-Glucan (b-I-3glucan.) wasn't administered, the embryo beyond the haploid number that did implantation was found out in the exposure beyond 2.0Gy.

 

3. Fetal death

 

A maceration fetus observed 18 dpc (days post conception.) was considered to be fetal death. Fetal death is defined as death that induced 14 dpc. The result of the fetal death rate is shown in Fig. 2. As for the fetal death rate of the 2.0 Gy+MG (Macro-Glucan.) group, statistical significant difference wasn't recognized as the control group in comparison with the sham control group with the 2.0 Gy group. Therefore, though b-1- 3glucan was used, it was found differences weren't significant toward the control group in the 2.0Gy exposure. Because the incidence of the embryonic death was very high, it isn't thought that the difference for the fetal death to be significant.

 

4. The incidences of the malformation

 

The number of the incidences of the malformation is shown in Fig.3. The modality of the malformation that it induced in each exposure group is exencephaly, cleft palate, open eye, gastroschisis, and anomalies of tail. A statistical significant difference was recognized as the development frequency of the malformation of the 2.0 Gy group in comparison with the induction frequency of the control group (P<0.001). A statistical significant difference was recognized as the teratogenesis rate of the MG+2.0 Gy group as well in comparison with it of the control group. As for the malformation, statistical significant differences were recognized as the control group from the above result toward the sham control group in all the exposure groups. But, though it was the highest when it was seen from the teratogenic rate, a decrease was recognized with the 2.0 Gy group and the 2.0 Gy+MG group by the MG+2Gy group of less than 2/3. Because there are many subjects that died because it had a malformation in the embryonic death, the effect of the malformation and the effect of each death, are separate issues and are not relevant to this research.

 

5. Fetal body weight

 

After one weighed fetal body weight respectively and the average of every mother was taken, the fetus' average weight of each exposure group was a mean by the male female of each exposure group. The fetal body weight of each exposure group is shown in Fig.4. Both significant difference of the fetal body weight decrease were recognized in the 2.0Gy group in embryonic age I8-day fetal body weight toward the body weight of the control group and the sham control group of the male and female (P<0.0I). But some thought the differences weren't recognized in it of the MG+2.0Gy group in the fetal body weight decrement toward the body weight of the control group and the sham control group. And, when a male and female ratio was compared, there was little fetal body weight difference. The female weighed about the circa 0.05g more as for each group as well.

Conclusion

 

Active oxygen (free radical.) appears in the organism, and oxidization target injury lesion is given to lipid, a protein, and the nuclei by radiation exposure. It follows that various histrionic damages are caused13,154,15 There is little clinical or epidemiological observation though much observation of it in vitro and in vivo with the vitamin E as for the radiation. It is chisels for medical use with the clinical observation used experimentally a little because of the therapeutic side effects prophylactics toward the bone marrow transfusion and the brain tumor radiological16). Actually, it has been shown that the lipid hyperoxidation when X-rays and gamma rays were radiated, and DNA damage occurred in the catastaltic by a natural agent1,8,17. But, this observation lacks radioprotection effect in the natural agent in one case, too18,19,20. Recently, oxidization affection by the active oxygen, the free radical is being made clear in various sicknesses and in retrogradation21. Moreover, attention to, and research about the natural anti-oxide substantial, the synthesis anti-oxide substantial is done very much radioprotection by the anti-oxide22. Fonseca has shown that the extracts of Macro-Glucan (b-1-3glucan.) were genotoxic as assessed by lysogenic induction in Escherichia coli and they were also able to induce mutagenesis in Salmonella typhimurium. The genotoxic activity in the extracts was related to the presence of a molecular complex formed by caffeine and a flavonoid (catechin or epicatechin in the presence of potassium23. Therefore, as for this research as well, it is thought by Macro-Glucan(b-1-3glucan) that its part as a radical scavenger that erases the active oxygen that occurred in the physical case is played, too. A flavonoid has a radioprotection effect23. The exposure group of the organogenesis of the sensibility against the embryonic death is the highest24. When an exposure group increased more in comparison with control group as for the embryonic death, it has by using F 1 of the male CDNMK mice and the female dd/MK mice as a result of doing simulation. But, a significant difference wasn't recognized, it is being reported for the preimplantation stage25. Murakami and Kameyama irradiated 0.5Gy to the 8 dpc(days post conception.)of pregnant mice, and it observes embryonic death. And an embryonic death of 20-30% was recognized (being reported as for the exposure of IGy26. Jacobsen irradiated 0.05Gy-IGy to the 7 hpc of pregnant mice, and the dose effect of the embryonic death was examined, and made dose response clear27.  A statistical significant

difference was recognized as the control group and the sham control group in comparison with the 2Gy group (P<0.001). But, a statistical significant difference wasn't recognized with the control group in comparison with the Sham control group by the MG+2Gy group. Therefore, radioprotection effect by the Macro-Glucan (b-1-3glucan) administration was clearly shown toward the embryonic death rate. The radioprotective action by the administration of the Macro-Glucan (b -1-3glucan) is radical scavenger prevention function due to the radiological chemical action and oxidation prevention function, and it is here as for this research as well28,29,30. The oxidization effects of that organism component are thought a possible protection by anti-oxide substantial. Actually, it has been observed that the lipid hyperoxidation when X- rays and gamma rays were radiated, and DNA damage occurred in the catastaltic by the manipulation of the Macro-Glucan (b-1-3glucan)23. It could get the same result as for this research as well to the embryos. Therefore, it is thought that Macro-Glucan(b-l- 3glucan) has a radioprotection effect. Brent recommends all permanent defects that it living being isn't prepared to repair be defined as malformation31. Ohzus irradiated 0.05-0.25Gy in 10 hpc and 34 hpc, and observed exencephaly and hyperdactyly25. And, the effect of the malformation of the organogenesis by the radiation has stage differences in developmental disorders32). It was made clear that sensibility to the malformation induction was the highest between the viviparity 7.5-10.5 days of the mice that radiological depends by preceding research32). Jacoobsen irradiates 0.05 in the viviparity 7.5 days, and the malformation induction of the cephalic, the jaw, the aperture was observed32. Muramamis irradiates 2Gy in the viviparity the ninth day and a malformation such as exencephaly and cleft palates are recognized10. The threshold dose of the malformation(exencephaly.)in the case of the radiation exposure is between 0.5Gy-l.5Gy, and the threshold dose of the embryonic death is between 1.5Gy-2.5Gy32. A difference in the threshold dose is a difference in the sensibility of the radiation dosage that affects a malformation and embryonic death. Perhaps, the number of the critical cellularis which causes teratogenesis, a difference in the number of the critical cellularis in the case of the embryonic death, and so on effects it24. Radioprotectinon effect by the Macro-Glucan (b-l- 3glucan.) administration was made clear to the malformation incidence of this research. 2Gy were irradiated to the mice after the body weights 50 micrograms kg-l of flavonoid administration, and Divis made radioprotection effect clear34. As for the fetal body weight, it is pointed out that it is critical as a character to grasp an effect such as a radiation by many researchers. Decrease of weight was recognized as the effect of the fetal body weight decrement by this research in the only 2Gy-exposure group. Decrease of fetal body weight was recognized in the organogenesis by the preceding research in the exposure group beyond 1.5Gy35. It isn't histrionic with the specific organizes against the decrement of the fetal body weight against the exposure group of the organogenesis, and histrionic development delayed type hypersensitivity effects the organizes of the body as a whole. Moreover, it is in proportion to the histrionic with the normal organizes, and it is thought that loss of weight happened36. The effect of radiation of the fetal body weight decrement in the organogenesis is thought about that an effects by not only the direct effect but also the uterus environment or blastogenesis stage didn't function normally, either. On the other hand, Konermanns radiated by 0.4 to the mice of the preimplantation stage (0-5 days) and the decrement of the fetal body weight wasn't recognized37. But, as for the fetal body weight as well, radioprotection effect by the Macro-Glucan (b-1-3glucan) was made clear in the same way as the effect of the embryonic death and the malformation.

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*Beta 1,3 glucan referred to in this study is the same as Beta-1,3/1,6-D-glucan.