Cancer is one of adult’s diseases that are most difficult to cure.

In order to treat various cancers, surgical operation, radiotherapy and chemotherapy (administration of anticancer agent) are generally used today. But in any case, their side effect is causing serious damage and suffering to patients. As an alternative to these treatment methods, immunotherapy is now gaining more attention than ever. Immunotherapy substantially reduces the sufferings of side effect and inherent pain of cancer, cures cancer in its last stage and prevents recidivation by promoting the power of immunity that is originally equipped to a human body.

“It is predicted that FOOD SCIENCE and IMMUNOLOGY will represent the medical treatment in the 21st century.”

1. Effect on the circulatory organ system Pulmonary cancer, severe pneumonia, leukemia, malignant lymphoma, lymphoma caused by Hodgkin’s disease, hemiplegia, septicemia, heart diseases, myocardial infarction, arteriosclerosis, angina pectoris, cardiac reinforcement, thrombotic diseases, vasoconstrictive diseases, hypertension/hypotension, restoration, stamina supply, etc.
2. Effect on the digestive system Ascitic cancer, colic cancer, rectal cancer, sigmoid cancer, intestinal cancer, pancreatic cancer, splenic cancer, Liver Cancer, hepatocarcinoma, hepatocirrhosis, hepatitis A/B/C, hepatomegaly, gastric cancer, gastric ulcer, chronic gastritis, gastroptosis, duodenal ulcer, viral enteritis, renal cancer, nephritis, renal failure, polyposis, pimples, chronic stomatitis, pyorrhea, constipation, lost appetite, hemorrhoids, etc.
3. Effect on the endocrine system Diabetes mellitus, hyperlipidemia (hyper cholesterol), allergy, edema, hepatopathy, etc.
4. Effect on the cranial nerve metabolism system multiple rheumarthritis, rheumatoid arthritis, autonomic imbalance, neurosis, Japanese encephalitis, etc.
5. Effect on the respiratory system chronic bronchitis, asthma, etc.
6. Effect on the genital system Breast cancer, ovary cancer, mammitis, gynopathy, monstrous irregularity, climacteric disorder, etc.
7. Effects on the urinary system Cystitis, prostatomegaly, nephrosis, etc.
8. Effect to the cutaneous system Athlete’s foot, eczema, atopic dermatitis, dermatomyositis, ciliary skin tumor, etc.
9. Other effects Pollinosis, chronic rhinitis, empyema, Basedow’s disease, collagen disease, roseolar arthritis, influenza, asthenia, lumbago, frozen shoulder, stiff shoulder, ophthalmopathy, blood purification, cold constitution, lost energy, hangover, acquired immunity deficiency syndrome (AIDS), etc.
It is predicted that Agaricus Blazei Murrill has effects to improve such symptoms as listed above

These effects are said to be the result of the activation of immunity function caused by b-glucan, a-glucan, b-galactoglucan, Ribonucleotide-protein complex (RNA), b-(1-6)-D-glucan protein complex, Xyloglucan, etc. that are abundantly included in Agaricus Blazei Murrill. In the experiment made by a research group led by Professors Hitoshi Ito (M.D./Pharmacologist) and Keishiro Shimura (Pharmacologist) at Mie (National) University of Japan, Agaricus Blazei Murrill was administered to mice with implanted cancer. Their complete recovery rate turned out to be 90.4% and arrest rate was 99.4%. The results were reported at general meetings at Japanese mycological, cancer, and pharmacological academies, etc.

Agaricus Max ™ is manufactured using a special technique from genuine Agaricus Blazei Murrill with confirmed efficacy through clinical tests at a national university hospital, which is specially grown in the region called Piedate in Brazil. Be sure to look for Agaricus Max ™, since there are other poor quality products on the market, carrying the name of Agaricus but no medical/scientific assurance of efficacy.

A U.S. Patent as an oral remedy for AIDS has been obtained. Patent Number: 6120772

Registration filing is also being made in Canada, Mexico and 15 European countries.
(The Brazilian country Ministry of Health and Welfare registration number: MS 001-002-004. 358-98)

Professor Inosuke Iwaide at Tokyo (National) University of Japan succeeded in artificial cultivation of Agaricus Blazei Murrill for the first time in the world. Its effect on various diseases was reported to general meetings of scientific academies by Professors Hitoshi Ito (M.D./Pharmacologist) and Keishiro Shimura (Pharmacologist) at Mie (National) University Japan. Agaricus Max ™ is made under supervision of these professors at Iwaide Fungology Research Corporation and distributed worldwide by U.S.-based MAX LIVE CORP.

Healed Cases

(1) Mr. Fujimoto’s case (overcame cancer with only 5% of possibility of healing)

Mr. Fujimoto’s testicular tumor was found in September 1988 and he had an operation immediately. However, the cancer had already metastasized to his lungs and peritoneum through his lymphoglandula. He was told that he could live for only 3 – 6 more months. Mr. Fujimori, who was diagnosed to have a cancer of 95% fatality rate, started to take Agaricus Blazei Murrill because one of his neighbors recommended it through his experience of overcoming a vesical cancer of the last stage. He was under intravenous feeding of anticancer when he started to take Agaricus Blazei Murrill. His doctor anticipated side effects, which turned out to be almost non-existent, and Mr. Fujimoto showed a recovery by walking around in the hospital. His cancer kept hanging in the balance, but one month later, the X-ray shadow in his lungs disappeared and many cancer cells in his peritoneum was eliminated. His blood test result demonstrated that he was a normal person. His doctor stopped further scheduled operation and it was confirmed through the last abdominal operation that all his cancer cells were gone. Mr. Fujimoto is alive and back to his job now.

(2) Mr. Miyoshi’s case (fine and sound 12 years after a big surgery)

Mr. Miyoshi was found to have stomach cancer and his remaining life was estimated as 3 months. The result of diagnosis was desperate even if he would go through a surgery, but Mr. Miyoshi insisted to have the operation. A big abdominal operation including extraction of the wholly undermined stomach and resection of esophageal, spleen and pancreas was performed. The operation was a success, but critical condition persisted due to his exhaustion. Mr. Miyoshi started to take Agaricus Blazei Murill because his family recommended it. After a certain period of progress and retrogress, he has survived well beyond the estimated 3 months – and now it is 12 years after the operation. Fine and sound in his 70’s he is vigorously working every day at a nearby farmland cultivating vegetables without using pesticides. No one would believe that he was expected to die 12 years ago.

(3) Ms. Kayoko T’s statement (overcame rheumatism with no hope of recovery)

Deformation of my fingers started when I was 32. My wrists got swollen and it was hard to bend them. Though I received continuous treatment at university and private hospitals, the situation only got worse. One day I asked my doctor if any surgery is possible for my disease. The doctor said no and passed me a large paper bag full of medicines as usual. So I had to continue taking the medicines, suffering from its side effects when a friend of mine recommended me to take Agaricus Blazei Murill. After a discussion about it with my husband, I started to take it, though a little dubiously. A month later, I felt less pain and fatigue. In two months, my swelling began to decrease in size. Once I could not even hold a glass, but I could hold a needle then. When I visited the hospital for checkup 3 months after I started to take Agaricus Blazei Murill, the doctor said that my rheumatic symptoms are gone. I am so happy now, since I recovered my health again with no side effects, thanks to Agaricus Blazei Murill.

(4) Ms. Kazuko O’s statement (overcame diabetes mellitus with decrease of sight)

One day I felt fatigue, lack of concentration and decreased energy. I consulted a doctor and learned that I had diabetes. The doctor gave me medicines and dietotheraphy instructions. Tasteless meals made me feel gloomy. What was worse, my diabetes was complicated by a second disease – I started to have a cerebral hemorrhage in the eyes – though I followed every instructions from my doctor.. The white of my right eye turned black and my left eye always carried some black spots. The doctor recommended an immediate operation which I accepted. After the operation, my eyes recovered whiteness. But I lost substantial sight, which I could not recover by wearing glasses. On the other hand, my blood sugar value did not drop while I lost my body weight. At this period my concerns reached the peak, worrying about possibility of side diseases and dystrophia even if I would follow all the instructions from my doctor. So I jumped when the idea of trying Agaricus Blazei Murrill was suggested by a friend of mine. During one month of trial of the recipe, my sight was regained without glasses, my blood sugar value fell down and the lost body weight was recovered. Now I am spending pleasant days again.

(5) Research on the treatment of acquired immune deficiency syndrome (AIDS)

The clinical research was performed in Thailand, where reportedly there are more AIDS patients than in any other Asian country. Agaricus Max ™ was administered to 50 AIDS patients for 3 months and the result was monitored.

Hospitalized patients had the lymphoglandula around their eyes, neck and vesica hypertrophied to the size of a clenched fist and their symptoms demonstrated that they were in the last stage of AIDS. One month after the administration of Agaricus Max was stated, its effect on their immunity revival was apparent. After three months of administration, the lymphoglandula of all 50 patients regained the original size. The patients were no longer in the last stage AIDS and released from the hospital, but they still have to visit the hospital since they have not been completely cured. Their clinical conditions are being studied further.

(6) Hypercholesterolemia was improved.
(7) Serious pollinosis was improved within one month of administration.
(8) Serious empyema with completely lost sense of smell was improved.
(9) Never-ceasing attack of asthma with hypotension was overcome.
(10) Acute pain in the lower half of body and cystitis were overcome.
(11) Hypertension that has long been distressing finally disappeared.
(12) Basedow’s disease was improved in 4^5 weeks after start of the recipe.
(13) Very severe pain unique to cancer patients was eased.
(14) There are too many other examples to be listed here.
Clinical Test Examples

1. Observation of Agaricus Blazei Murrill’s therapeutic effect against digestive tract tumor

20 patients with malignant digestive tract tumor were divided into experimental group that received administration of Agaricus Blazei Murrill and control group who did not receive it. These patients were observed for three months of treatment and all scheduled perspectives were recorded before and after administration.

Since it had been proven that Agaricus Blazei Murrill would not cause any side effects, all patients were also treated with anti-cancer agent, etc. during the same period.

Comparison between Basic Conditions of Patients in the Two Groups

Group # of Patients Age Cancer Location
Male Female Esophageal Stomach Colon
Experimental Group 8 2 40 – 77 3 5 2
Control Group 9 1 32 – 67 4 5 1
Result

(1) Symptoms and Signs: The symptoms such as fatigue, anorexia, nausea and vomiting of control group patients continued, and in some cases deteriorated. The symptoms of experimental group patients, including mental condition, anorexia and nausea were noticeably improved. No dysphoria or side effects caused by taking the recipe were observed.

(2) Changes in Blood Picture: Experimental group patients’ blood picture was improved. On the other hand, the blood picture of control group patients obviously deteriorated. Especially the depression of platelets was remarkable. Therefore, it is considered that Agaricus Blazei improves the peripheral blood picture.

(3) Conclusion: The inspection results of the kidney function, liver function and electrocardiogram of experimental group patients are all within normal ranges. Radiotherapy and chemotherapy, as well as malignant tumor itself, weakens the immunity function inside the human body. In addition to its hematopoietic promotion, Agaricus Blazei is also considered to promote the restoration of retarded myelopoiesis after radiotherapy and chemotherapy, while promoting the immunity function and inhibiting the tumor growth.

2. Observation of Agaricus Blazei Murrill’s therapeutic effect on liver function of chronic hepatitis patients

20 patients with chronic and active hepatitis B were divided into experimental group that received administration of Agaricus Blazei Murrill and control group who did not receive it. These patients were observed for three months of treatment and all scheduled perspectives were recorded before and after administration. All patients were presenting the general clinical expression of chronic hepatitis caused by virus. Since it had been proven that Agaricus Blazei Murrill would not cause any side effects, all patients were also treated with appropriate medicines during the same period.

Comparison between Basic Conditions of Patients in the Two Groups

Group # of Patients Age Diagnosis
Male Female 11 patients with complexion of liver disease, 7 with spider-like nevus and liver palms, 6 with jaundice, 6 with swollen liver and 5 with positive ascites.
Experimental Group 9 1 27 – 55
Control Group 8 2 21 – 57
Result

(1) The symptoms of most of the experimental group patients were improved. For example, symptoms such as abdominal distension, fatigue and hepatodynia either disappeared or improved. Signs were also improved to some degree. The number of patients with retraction of the liver and spleen decreased to 5 and 3 respectively, and 2 patients with ascites retraction was improved. It was obvious that the improvement in the clinical symptoms of the experimental group exceeded that of the control group.

(2) After 3-month of treatment using Agaricus Blazei, the liver function was obviously improved, and there was a remarkable change in values including ALT, AST, bilirubin. It is obvious that Agaricus Blazei has a therapeutic effect against enzyme depression and jaundice.

(3) Agaricus Blazei influences to all correlative perspectives of the liver function of chronic hepatopathy. Serum connective cholic acid, serum ferritin, g-globulin, coagulation time after the treatment showed remarkable depression. It is conceived that Agaricus Blazei has various therapeutic effects to hepatopaths and presents a comprehensive control in the positive direction.

(4) The treatment was especially effective to 2 patients and effective to 8 patients of total 10 patients of experimental group. Through this experiment, it was found out that Agaricus Blazei Murrill is effective in promoting the recovery of hepatocytes of chronic hepatitis patients as well as in its immunological enhancement and anticancer activity.

3. Clinical observation of Agaricus Blazei Murrill’s therapeutic effect against acute non-lymphocytic leukemia

20 patients with acute non-lymphocytic leukemia were divided into experimental group that received administration of Agaricus Blazei Murrill and control group who did not receive it. These patients were observed for three months of treatment and all scheduled perspectives were recorded before and after administration. The patients were with acute non-lymphocytic leukemia and were receiving treatment at the Department of Hematology, The First Affiliated Hospital of Medical College. Chemotherapy was given 3 times during the period they took medicine made with Agaricus Blazei Murrill, with 21 days of intermittence period.

Comparison between Basic Conditions of Patients in the Two Groups

Group # of Patients Age Diagnosis
Male Female M1 M2 M3 M4 M5
Experimental Group 6 4 22 – 58 0 2 2 1 5
Control Group 8 2 23 – 55 1 1 1 4 3
Result

(1) Because of the influence of Agaricus Blazei Murrill to the blood picture, peripheral blood hemoglobin, platelets and leukocyte remarkably increased. Among the patients in experimental group, 8 patients reached complete remission (CR) and 2 patients remained with no remission (NR). The bone marrow pressure time after the chemotherapy was short. Bone marrow proliferation ratio between red, granule and large nucleus cells recovered the normal level after the chemotherapy.

(2) Mental condition and appetite of the experimental group patients were remarkably improved. Obvious recovery in symptoms including fatigue, dizziness, nausea and vomiting of 8 patients was reported. The experimental group suffered fewer side effects compared to the control group.

(3) Compared to the control group, far less number of experimental group patients were found to have pulmonary, pharyngolaryngeal, or intestinal infection and the level of infection was lower. No adverse effect was observed during the administration period.

Clinical Observation on Treatment of Acute Non Lymphocytic Leukemia with Agaricus Blazei Murrill

by:
Drs. Tian Xiao Hui/Lun Zheng Guo/Wang Jing – Department of Hematology, The First Affiliated Hospital of Lanzhou Medical College, Drs. Hitoshi Ito/Keishiro Shimura/Wang Jun Zhi – Mie University School of Medicine

Overview
Ten cases of acute non-lymphocytic leukemia (ANLL) were treated with chemotherapy using Agaricus Blazei. The results showed that Agaricus Blazei could promote the bone marrow hemopoiesis in vivo, improve bone marrow inhibition after chemotherapy and remarkably increase the concentration of Hb, the number of Wbc and Plt in peripheral blood (P<005-0.02). It also enhanced the concentration of lgM (P<0.05), albumin and A/G ratio and decreased the concentration of globulin in plasma (P<0.01-0.05). It indicated that Agaricus Blazei could promote liquid immune function and had some inhibitory effects on leukemia cells in ANLL patients. No side effects were found in the treatment of Agaricus Blazei. The method is safe and remains to be further studied and applied.
(Keywords: Agaricus Blazei, Acute Non-Lymphocytic Leukemia (ANLL), Clinical Treatment)
Agaricus Blazei is a fruit body of fungi plant. As shown in the experiments and researches by Japanese academics, its hot water extract (includes water-soluble polysaccharides as a main component) has effects such as immunological enhancement and tumor growth inhibition. In animal experiments conducted in China, it was found that the polysaccharides extracted from Agaricus Blazei (ABPS) promote the proliferation of CFU-S, CFU-GM, CFU-E and CFU-F of rats. In order to make a progress in its clinical research and application perspectives, we carried out a clinical observation of 10 examples of acute nonlymphocytic leukemia patients under treatment with Agaricus Blazei from July 1992 to May 1994, which we report as follows:
Material and Method
I. Selection of Patients
The subject 20 examples were the patients with acute nonlymphocytic leukemia who received initial treatment at the Department of Hematology, The First Affiliated Hospital of Lanzhou Medical College. All examples comply with the diagnosis standard for acute nonlymphocytic leukemia established by Tientsin Leukemia Classification Conference held in September 1986. The patients were randomly divided into experimental and control groups, each consisting of 10 patients. There is no obvious difference between the two groups as to their age, the length of period with disease, condition (symptoms and signs), and chemotherapy that they had been receiving.
The experimental group consisted of 6 males and 4 females (2 x M2, 2 x M3, 1 x M4 and 5 x M5). Their age ranged 22 – 58 and the average was 34.1.
The control group consisted of 8 males and 2 females (1 x M1, 1 x M2, 1 x M3, 4 x M4, 3 x M5). Their age ranged 23 – 55 and the average was 36.4.
II. Treatment Method
The experimental group was treat under chemotherapy of standard induction and remission method including “daunorubicin + cytarabine” method with 14 – 21 days of intermittence period. The chemotherapy was given 3 times during the period they took medicine made with Agaricus Blazei. After the first treatment using hot water extract of Agaricus Blazei (supplied from Iwakin Co., Ltd. Laboratory of Japan), the daily amount was increased to 20g (twice a day). The patients took the medicine for total 3 months.
The control group patients received the chemotherapy and conditional support, but did not take the medicine made from Agaricus Blazei.
III. Monitored Values
1. Clinical Symptoms and Signs:
Mental condition, appetite, nausea, blood pressure, etc.
2. Laboratory Tests
2.1 Changes in Blood Picture
2.2 Changes in Bone Marrow Picture
2.3 Various items of biochemical blood tests including plasmatic protein, immunoglobulin, serum iron metabolism, liver and renal functions.
3. Infection:
The number of infected patients and infected parts of body were monitored.
4. Side Effect:
Patients went through rigid inspection during and after treatment for any side effects.
5. Statistical Processing:
“T” inspection processing was applied to the data of each item.
Result
1. Influence of Agaricus Blazei to the Blood Picture of Acute Nonlymphocytic Leukemia Patients:
As shown in the experiment result, peripheral blood hemoglobin, platelets and leukocyte remarkably increase after treatment with Agaricus Blazei (see Table 1).
2. Influence of Agaricus Blazei to the Bone Marrow Picture of Acute Nonlymphocytic Leukemia Patients:
Table 1: Influence of Agaricus Blazei to the Blood Picture of Acute Nonlymphocytic Leukemia Patients (x±S)
Group Timing Hb (g/L) P value WBC (x10/L) P value Plt (x10/L) P value
Experimental Group (10) Before 72.50±17.32 <0.001* 1.31±0.72 <0.001* 24.38±8.38 <0.001*
After 109.38±15.45 <0.005** 5.63±2.47 <0.02** 123.25±31.37 <0.02**
Control Group (10) Before 70.88±15.27 <0.01* 1.34±0.57 <0.001* 25.88±7.90 <0.001*
After 91.25±7.91 3.81±0.26 86.38±16.57
Note: * Comparison within the group
** Comparison with the result of control group patients after treatment
Among the 10 patients of the experimental group, 8 patients reached complete remission (CR) and 2 patients remained with no remission (NR). The bone marrow pressure time after the chemotherapy was short. Bone marrow proliferation, ratio between red, granule and large nucleus cells recovered the normal level within 7 – 8 days from the end of chemotherapy.
On the other hand, among the 10 patients of the control group, 5 patients reached CR, 2 patients reached partial remission (PR) and 3 patients remained with NR. The bone marrow pressure time after the chemotherapy was long. 8 patients recovered the normal level 14 – 21 days after chemotherapy and 2 patients remained abnormal.
3. Influence of Agaricus Blazei to Total Protein, Globulin, Albumin and A/G ratio of Acute Nonlymphocytic Leukemia Patients:
Plasmatic albumin of the experimental group patients obviously increased and globulin decreased, resulting in increased A/G ratio. Plasmatic total protein, albumin and globulin of the control group patients showed no change (see Table 2).
Table 2: Influence of Agaricus Blazei to the Total Protein, Globulin, Albumin and A/G Ratio of Acute Nonlymphocytic Leukemia Patients (x±S)
Group Timing Total Protein (g/L) P value Globulin (g/L) P value Albumin (g/L) P value A/G ratio P value
Experimental Group (10) Before 62.33±3.98 >0.05* 32.43±4.20 <0.1* 29.90±2.68 <0.01* 1.09±0.20 <0.01*
After 66.00±4.75 >0.05** 41.55±6.04 <0.01** 24.45±6.72 <0.05** 1.78±0.91 <0.05**
Control Group (10) Before 61.82±3.14 >0.05* 33.60±2.86 >0.05* 28.22±4.42 >0.1* 1.18±0.13 >0.1*
After 63.81±5.62 36.01±5.80 27.80±3.80 1.29±0.46
Note: * Comparison within the group
** Comparison with the result of control group patients after treatment
4. Influence of a Agaricus Blazei to the Immunoglobulin of Acute Nonlymphocytic Leukemia Patient
As shown in the experiment result, lgM of the experimental group patients obviously increased, while lgG and lgA remained close to those of the control group patients (see Table 3).
Table 3: Influence of Agaricus Blazei to the Immunoglobulin of Acute Nonlymphocytic Leukemia Patients (x±S)
Group Timing lgG (g/L) P value lgM (x10/L) P value lgA (x10/L) P value
Experimental Group (10) Before 9.09±3.28 >0.05* 1.07±0.31 <0.05* 1.64±1.21 >0.05*
After 14.62±9.71 >0.05** 1.74±0.91 <0.05** 1.76±0.82 >0.5**
Control Group (10) Before 8.20±2.57 >0.05* 0.96±0.22 >0.1* 1.41±0.84 >0.05*
After 12.75±6.92 1.23±0.76 1.75±0.93
Note: * Comparison within the group
** Comparison with the result of control group patients after treatment
5. Symptoms and Signs
Mental condition and appetite of the experimental group patients were remarkably improved. Obvious recovery in symptoms including fatigue, dizziness, and nausea of 8 patients was reported. The experimental group suffered fewer side effects compared to the control group.
6. Infection
Compared to the control group, far less number of experimental patients was found to have been infected and the level of infection was lower. The number of patients with infections (pulmonary, pharyngolaryngeal, intestinal) totaled 6 in the experimental group and 13 in the control group.
7. Side Effect
No adverse effect was observed during the administration period of Agaricus Blazei.
Discussion
Scholars in Japan are making researches on Agaricus Blazei in its aspects such as pharmaceutical components, structural characteristics and active elements. Agaricus Blazei, as shown in their studies, has effects to enhance immunity system and inhibit tumor growth. Research in China discloses that the polysaccharides extracted from Agaricus Blazei promote the proliferation of bone marrow multifunction hematopoietic cells, simple granule cells, red cells and fibroblasts of a normal rat. However, no report on its clinical application to leukemia patients has been released yet.
With the understanding of the said effects and mechanism, we observed the influence of Agaricus Blazei to leukemia patients based on chemotherapy. As a result of clinical research, it was confirmed that Agaricus Blazei promotes the hematopoiesis by the bone marrow. Direct reactions in the bodies of acute nonlymphocytic leukemia patients include short bone marrow pressure period, early hematopoietic restoration by the bone marrow and fast recovery of granule, red and large nucleus cells to normal level. Indirect reactions include remarkable improvement in peripheral blood picture and obvious increase of total number of hemoglobin, platelets and leukocyte, which is remarkable compared to control group after therapy (P<0.005 – 0.02). Myelopoiesis promoted by Agaricus Blazei apparently improves the condition of myelopoiesis pressure and promotes the restoration of peripheral blood picture of leukemia patients under chemotherapy. This is achieved to the same degree as the result of “Observation on Treatment Effect of Agaricus Blazei against Alimentary Tract Tumor”.
Various vegetable polysaccharide components extracted from Ganoderma Lucidum, Polyporus Umbellatus, etc. are effective in immunological enhancement and promote antitumor activity. By comparing before and after the treatment period in our experiment, we found the lgM value of experimental group patients obviously increased (P<0.05) while lgG and lgA remained at the same normal level as those of control group (P<0.05 – 0.1). Hence, Agaricus Blazei is conceived to have an effect to improve the restoration of damaged immunity function and promotes the reinforcement of humoral immunity function. As shown in the research by Lee Hai Qian et al., Agaricus Blazei also promotes the reinforcement of internal cellar immunity function. This is considered to have been achieved by activation of immunity system through the antitumor effect of Agaricus Blazei.
The experimental group patients were checked after treatment and were found to have obviously increased plasmatic albumin and decreased globulin, resulting in obviously higher A/G ratio. On the other hand, these values of control group patients did not obviously change during the treatment (P>0.05-0.1). Since no damage in liver function was observed for both groups during the treatment, the change in plasmatic protein of the acute leukemia patients can be considered as a result of certain action by Agaricus Blazei against tumorous cells and not due to change in liver function. However, further study will be necessary to determine if the action was achieved directly by Agaricus Blazei or indirectly through reinforced internal immunity activation.
In short, Agaricus Blazei demonstrates its clinical effectiveness against acute nonlymphocytic leukemia through such effects as promotion of myelopoiesis, improvement of peripheral blood picture, reinforcement of internal immunity and inhibition of tumor proliferation. Study and utilization of Agaricus Blazei should be continued as one supplemental method of therapy for acute leukemia because it increases the desirable effect of chemotherapy through reducing the side effects and reinforcing the durability of the body. Our experiment this time has presented an important cause of study and clinical application of Agaricus Blazei and has contributed to its development and utilization.
(Dr. Wang Jun Zhi is a co-researcher of Lanzhou Medicine College, currently at Mie University, Japan.)
Bibliography:
1. T. Mizuno et al. “Studies on the Host-Mediated Antitumor Polysaccharides: Part IX. Fractionation and Characterization of Antitumor Polysaccharides from Maitake, Grifoia Frondosa.” Agric Bio Chem 1986.56.1679
2.Zhang Xiu Wen et al. “Agaricus Blazei’s Influence to Myelopoiesis Cells of Normal Rats” Lanzhou Medical College Academic Report 1993.19(3).143
3.Wang Zhi Jian “Corrective Proposal on the Classification of Acute Nonlymphocytic Leukemia at Leukemia Classification Conference” China Hematology Magazine 1987.3.181
4.Wang Jing et al. “Observation on Treatment Effect of Agaricus Blazei against Alimentary Tract Tumor” Gansu Medical Journal 199

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

Luís Fernando Barbisan1, Clarissa Scolastici1, Maristela Miyamoto1,
Daisy Maria Favero Salvadori2, Lúcia Regina Ribeiro2,3,
Augusto Ferreira da Eira4 and João Lauro Viana de Camargo2
1Departamento de Morfologia, Instituto de Biologia, UNESP, Botucatu, SP, Brasil
2Departamento de Patologia – TOXICAN, Faculdade de Medicina, UNESP, Botucatu, SP, Brasil
3Universidade Luterana do Brasil, Canoas, RS, Brasil
4Departamento de Produção Vegetal, Módulo de Cogumelos, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP, Brasil
Corresponding author: L.F. Barbisan
E-mail: barbisan@ibb.unesp.br
Genet. Mol. Res. 2 (3): 295-308 (2003)
Received January 30, 2003
Accepted August 15, 2003
Published September 30, 2003

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine
Luís Fernando Barbisan1, Clarissa Scolastici1, Maristela Miyamoto1,
Daisy Maria Favero Salvadori2, Lúcia Regina Ribeiro2,3,
Augusto Ferreira da Eira4 and João Lauro Viana de Camargo2
1Departamento de Morfologia, Instituto de Biologia, UNESP, Botucatu, SP, Brasil
2Departamento de Patologia – TOXICAN, Faculdade de Medicina, UNESP, Botucatu, SP, Brasil
3Universidade Luterana do Brasil, Canoas, RS, Brasil
4Departamento de Produção Vegetal, Módulo de Cogumelos, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP, Brasil
Corresponding author: L.F. Barbisan
E-mail: barbisan@ibb.unesp.br
Genet. Mol. Res. 2 (3): 295-308 (2003)
Received January 30, 2003
Accepted August 15, 2003
Published September 30, 2003
ABSTRACT. The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.
Key words: Agaricus blazei, DNA damage, GST-P-positive liver foci, Hepatocarcinogenesis
INTRODUCTION
Among the mushroom species of higher Basidiomicetes, Agaricus blazei Murrill, a species native to Brazil, where it is popularly known as “sun mushroom”, has recently received attention in folk medicine due to its use in the treatment of ailments. Since 1965, strains have been exported from Brazil to Japan, where this mushroom has become popularly known as “Himematsutake” or “Agarikusutake”. This edible mushroom is often consumed as food and tea in different parts of the world, especially because of its reported medicinal properties.
In Brazil, infusion of the dried fruiting bodies of the mushroom A. blazei has been popularly consumed both as a stimulant and for auxiliary treatment of various diseases, including cancer. Nevertheless, no epidemiological and few in vivo experimental data exist on the beneficial effects of the crude aqueous extract of this mushroom. Recently, we demonstrated that this extract provides significant protection against mutagenicity induced by cyclophosphamide and methyl methanesulfonate, both in vivo and in vitro (Delmanto et al., 2001; Menoli et al., 2001; Martins de Oliveira et al., 2002). Various polysaccharides and protein-bound polysaccharides isolated from mycelia and fruiting bodies of A. blazei have shown anti-tumor activity in tumor-bearing mice by host immune response activation (Kawagishi et al., 1989; Itoh et al., 1994, 1997; Fujimiya et al., 1998; Mizuno et al., 1998).
Dielthylnitrosamine (DEN) is a potent genotoxic carcinogen that has been used as initiating agent in some two-stage (initiation-promotion) alternative protocols for hepatocarcinogenesis (Ito et al., 1988; Dragan et al., 1991). It has been reported that after its metabolic biotransformation, DEN produces the promutagenic adducts O6-ethyldeoxyguanosine and O4- and O6-ethyldeoxythymidine that may initiate liver carcinogenesis (Dragan et al., 1994; Verna et al., 1996). Consequently, the analysis of DNA damage may be relevant to evaluate the modifying influences of chemopreventive agents on the initiation stage of cancer (Moore et al., 1999).
The comet assay or single cell gel electrophoresis assay is a rapid and sensitive procedure for quantifying DNA lesions in individualized cells, both in vitro and in vivo (Tice et al., 1991; Fairbairn et al., 1995; Gontijo et al., 2001). The alkaline comet assay version was specially developed for detection of the DNA single-strand breaks and alkali-labile sites (Singh et al., 1988) and is also indicated to evaluate in vivo genotoxicity induced by carcinogen exposure (Anderson et al., 1998; Tsuda et al., 2000).
The DEN-partial hepatectomy (PH) model has proven to be a consistent bioassay for the detection of chemical hepatocarcinogens and for the assessment of the beneficial potential of chemopreventive agents (Ito et al., 1988, 1996; Moore et al., 1999). This 8-week-long medium-term rat liver assay uses as endpoint marker the development of putative preneoplastic foci of altered hepatocytes that express the placental form of the enzyme glutathione S-transferase (GST-P) (Ito et al., 1988). It has been indicated as a practical approach for the assessment of the potential hazard or benefit of chemicals, when associated with other surrogate end-points (Moore et al., 1999).
The standard DEN-PH assay protocol uses 200 mg/kg DEN to establish the initiation of the hepatocarcinogenesis process (Ito et al., 1988). In order to determine the protective influence of the aqueous solutions of the mushroom A. blazei against liver damage and preneoplasia development induced by the DEN dose level used in the DEN-PH assay, we evaluated DNA damage and foci of altered hepatocyte development by the comet assay and by the immunohistochemical expression of the placental enzyme, glutathione S-transferase, respectively.
MATERIAL AND METHODS
Animals and treatment
Male 4-week-old Wistar rats were obtained from CEMIB (UNICAMP, Campinas, SP, Brazil). The animals were kept in polypropylene cages (five animals/cage) covered with metallic grids in a room maintained at 22 ± 2oC, 55 ± 10% humidity, and with a 12-h light-dark cycle. They were fed with commercial Purina chow (Labina, Paulínia, SP, Brazil) and water ad libitum during a 2-week acclimation period.
The animals were randomly allocated into six groups (Figure 1). For two weeks, animals of groups 3 to 6 were treated with aqueous solutions of the A. blazei, with a mean dry weight of water-extractable solids of 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively. After this period, groups 2 to 5 were given a single ip injection of 200 mg/kg DEN (Sigma, St. Louis, MO, USA), and groups 1 and 6 were treated with 0.9% NaCl only (DEN vehicle). All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The University Ethics Committee for Animal Research approved the protocols used in this study (Protocol No. 99/22).

Preparation and administration of aqueous solutions of Agaricus blazei
A sample of A. blazei Murrill (lineage 99/26) was obtained from the Departamento de Produção Vegetal, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP, Brazil. Twenty-five grams of powdered dry fruiting bodies of A. blazei mushroom was added to 1000 ml of deionized water (2.5% w/w) and left for 2 h at room temperature. This preparation corresponds to the popular form of use of A. blazei for beneficial health effects. This solution, referred from here on as “the crude aqueous extract”, was then centrifuged (800 g for 10 min) and filtered (commercial non-sterile filter). The final solution was provided as 10% (Ab1), 50% (Ab2) and 100% (Ab3) of the full 2.5 (w/w) aqueous extract of A. blazei. The mean amount of water-extractable solids in Ab1, Ab2, and Ab3 solutions was 1.2, 5.6, and 11.5 mg/ml, respectively. The yields were 4.8, 22.4 and 46%, respectively. The solutions were prepared daily and offered to rats ad libitum, in aluminum foil-wrapped bottles to avoid light decomposition. They were the sole source of drinking fluid, starting two weeks before DEN initiation.
Comet assay
Four hours after DEN or NaCl treatments, five animals of each experimental group were sacrificed and a small piece of the left hepatic lobe was collected and placed onto a small Petri disc with ice-cold mincing solution (Ca2+- and Mg2+-free HBSS containing 20 mM EDTA and 10% DMSO). The liver samples were cut into smaller pieces, using a disposable microtome razor blade, and the solution was aspirated. Then, a fresh mincing solution was added and the liver samples were minced again to finer pieces. Resulting cell suspensions were collected and filtered (100 µm nylon mesh). All samples were stocked on ice in appropriate conditions to avoid light until the comet assay procedures. The quantity of liver cells in the cell suspensions was determined in Giemsa-stained smears.
The viability of the liver cells was indirectly determined by analyzing the comet images after electrophoresis (Vaghef and Hellman, 1998) and by spontaneous DNA diffusion without DNA denaturation and electrophoresis (Vasques and Tice, 1997). The comet image was considered to be from a nonviable cell when it presented a “cloudy” appearance or a very small head and a tail like a balloon (necrotic or apoptotic cells). The viability of the cell suspension was considered acceptable when the frequency of such images was less than 2% (Vaghef and Hellman, 1998). Higher values were considered as indicative of cytotoxicity due to the carcinogen treatment. The extent of spontaneous DNA diffusion was evaluated by measuring the diameter of the liver cell “nucleoids”, considering that cells containing extremely low molecular weight DNA associated with apoptosis or necrosis develop spontaneous DNA diffusion into the agarose gel and consequently have larger diameters (Vasques and Tice, 1997).
The comet assay was performed under alkaline conditions according to a previously described standard protocol (Speit and Hartmann, 1999). Briefly, an aliquot of 5 µl of each prepared hepatic cell suspension was mixed with 120 µl of 0.5% low melting point agarose at 37oC and layered onto conventional microscope slides, precoated with 1.5% normal melting point agarose. The slides were placed overnight in freshly prepared cold lysing solution (1% Triton X-100, 2.5 mM NaCl, 0.1 mM Na2EDTA, 10 mM Tris with 10% DMSO, pH 10.0) and then in a horizontal electrophoresis cube with alkaline electrophoresis solution (0.3 M NaOH, 1 mM Na2EDTA, pH >13) at 4oC for 20 min. The electrophoresis was performed at 25 V and 300 mA for 20 min. After electrophoresis, the slides were washed twice for 5 min in neutralizing buffer (0.4 M Tris-HCl, pH 7.5), fixed for 5 min in absolute alcohol, air-dried, and stored at room temperature. In order to evaluate extremely low molecular weight DNA diffusion, two slides from each animal were removed after lysis procedure, rinsed with neutralizing solution, fixed and air-dried, and stored until analysis.
Immediately before analysis, the DNA was stained with 50 µl of 20 µg/ml ethidium bromide. The slides were examined with a 40X objective lens with epi-illuminated fluorescence microscopy (Olympus-Bx60, excitation filter: 515-560 nm; barrier filter: 590 nm) attached to a color CCD video camera and connected to an image analysis system (Comet II, Perspective Instruments, UK). Coded slides were scored blindly and 100 hepatic cell images were randomly analyzed for each animal (50 images per slide).
The comets were analyzed by a visual scoring method and computerized image analysis. The comets were classified into five categories, defined as types 0, 1, 2, 3 and 4 – where 0 indicates no or very low damage, 1, 2 and 3 indicate low, medium and long DNA migration, respectively, and 4 indicates apoptotic or necrotic DNA migration (Speit and Hartmann, 1999). The metrics for comet analysis included spontaneous DNA migration (measure of diameters larger than 34 microns of the “nucleoids”, expressed in microns), tail migration (distance from the end of the head to the end of the tail, expressed in microns), and tail moment (product of DNA density in the tail and the mean distance of DNA migration in the tail, expressed in arbitrary units).
Histology and GST-P-positive foci analysis
After sacrifice at the end of the 10th week, samples of each of the liver lobes were weighed and fixed in 10% phosphate-buffered formalin for hematoxylin-eosin (HE) staining and immunohistochemical demonstration of GST-P-positive foci, using the avidin-biotin complex method (Hsu et al., 1981).
Paraffin-embedded liver samples were cut into 5-µm thick sections, placed on poly/D/lysine-coated slides, deparaffinized in xylene and rehydrated with graded alcohol to water. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min. Nonspecific protein binding was minimized by the use of 1% nonfat dried milk in PBS for 60 min at 4oC. Slides were incubated with rabbit anti-rat GST-P primary antibody (Medical and Biological Laboratories Co., Tokyo, Japan) diluted at 1:1000 in 1% bovine serum albumin (BSA; Sigma) overnight at 4oC. Then, the slides were successively incubated with a biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories Inc., CA, USA) diluted at 1:200 in 1% BSA for 60 min, followed by an avidin-biotin-horseradish peroxidase complex kit (Vector Laboratories Inc.) diluted at 1:100 in PBS for 45 min. Subsequent chromagen color development was made using 0.038% 3-3’-diaminobenzidine tetrahydrochloride (Sigma) and 0.025% hydrogen peroxide in 0.1 M Tris-HCl, pH 7.4, for 4 min. The sections were counterstained with Harris’s hematoxylin, dehydrated, cleared in xylene, and a cover slip fixed with a xylene-based mounting medium.
GST-P-positive foci larger than 0.15 mm2 in diameter were measured using a Nikon photomicroscope (Microphot-FXA) connected to a KS-300 apparatus (Kontron Elektronic, Germany). Data are reported as number and area (mm2) of preneoplastic GST-P-positive foci per liver section (cm2).
Statistical analysis
Comparisons of body and liver weights among groups were carried out using analysis of variance and Student t-test. The DNA migration data and the number and area of the GST-P-positive foci were analyzed by Mann-Whitney and Kruskal-Wallis tests (Zar, 1984). The contrast between groups was analyzed by the Tukey test. A significant difference between the groups was assumed when P < 0.05.
RESULTS
Food and liquid consumption and body and liver weights
The two-week A. blazei treatment had no influence on the mean body weight gain (Table 1), on food consumption (~25 g/rat/day for all the experimental groups) or on liver morphology, evaluated by HE (data not shown). Significantly increased ingestion of the aqueous extract of A. blazei was observed for 50 and 100% of the full aqueous extract (2.5 w/w) of A. blazei (Ab2 and Ab3 solutions) (group G1 vs group G6, P < 0.001; group G2 vs groups G4 and G5, P < 0.001) (Table 1). The average ingestion of A. blazei (i.e., water-extractable material) calculated from the mean ingestion of the aqueous solutions of A. blazei was approximately 0.18, 0.90 and 1.97 g of solids/kg/day (Table 1).

No differences were found in the mean body and liver weights between non-initiated (groups G1 and G2) and DEN-initiated animals (group G2 to group G5) at the end of the experimental period (data not shown).
DNA damage evaluation
In untreated animals (group G1), there was a low frequency of necrotic hepatocytes, indicated by type 4 comet images (Figure 2). DEN treatment induced a higher frequency of type 4 comet images (necrosis/apoptosis process) and increased DNA migration in the alkaline comet assay, when compared to the non-initiated animals (group G1 vs group G2) (Figures 2 and 3). Necrosis/apoptosis of liver cells in DEN-treated animals was also indicated by increased spontaneous DNA diffusion (Table 2) and by extensive centrilobular liver necrosis observed 48 h after DEN treatment (Figure 4).

Agaricus blazei treatment alone did not change the basal level of DNA damage in liver cells, evaluated by DNA migration in the comet assay (Table 2 and Figure 2). Significantly smaller values of the DNA migration parameters (tail migration and tail moment) and of spontaneous DNA diffusion analysis without electrophoresis were observed in rats that received the highest concentration of the mushroom A. blazei (Ab3 solution) before 200 mg/kg DEN, than in rats solely treated with this dose of carcinogen (group G2 vs group G5, P < 0.001, Table 2).
GST-P quantitative data
Non-DEN-treated control and A. blazei-treated animals (groups G1 and G6) did not develop preneoplastic GST-P-positive liver foci (Table 3, Figure 5). Higher values of the number of but not the area of GST-P-positive liver foci per cm2 of liver sections were observed in rats that received the highest concentration of A. blazei (Ab3 solution) previously to 200 mg/kg DEN than in rats solely treated with this dose of carcinogen (group G2 vs group G5, P < 0.001, Table 3).

DISCUSSION
The potential of aqueous extracts of the mushroom A. blazei to ameliorate DNA damage and GST-P foci development induced by DEN was investigated in male Wistar rats.
Natural products have been traditionally accepted as remedies due to the popular belief that they produce few adverse side effects. Therefore, understanding the potential beneficial or adverse influence of natural products extensively used by human population is very important to implement public health safety measures. We did not observe adverse effects of treatment with relatively high doses of a crude aqueous solution of the mushroom A. blazei, determined from body weight, liver morphology, DNA damage and GST-P foci data.
In the comet assay, cells with damaged DNA displayed increased migration of DNA fragments (comet tail) from the nucleus (comet head), which may also be a feature of DNA fragmentation associated with the necrotic/apoptotic death process (Olive et al., 1993; Fairbairn et al., 1996). In contrast with the conventional dye exclusion assay to evaluate cell viability (e.g., Trypan blue), we used two other approaches to evaluate concurrent liver cytotoxicity due to the DEN treatment. These alternative cytotoxicity assays included liver histological analysis for the detection of necrosis/apoptosis and the single-cell neutral diffusion assay to detect cells with low molecular weight DNA fragments, indicative of apoptosis or necrosis (Tice et al., 2000).
Treatment with 200 mg/kg DEN induced liver cytotoxicity, evidenced by increased spontaneous DNA diffusion and also by severe centrilobular liver necrosis. The increased DNA migration observed 4 h after DEN treatment was likely a sum of genotoxicity and cytotoxicity induced by the carcinogen (Hartmann and Speit, 1997). A protective effect of the treatment with the higher A. blazei aqueous extract concentrations against DEN cytotoxicity/genotoxicity can be postulated because there was a diminished tail extension and diminished DNA content in the tails of the hepatocyte comets in A. blazei-treated animals when compared to the animals treated only with the standard 200-mg/kg DEN dose. It was previously found that A. blazei extracts inhibit the mutagenicity of benzo(a)pyrene in the Ames Salmonella microsome assay (Osaki et al., 1994) as well as mutagenicity induced by cyclophosphamide in the mouse bone marrow micronucleus test (Delmanto et al., 2001). In general, crude extracts and polysaccharides isolated from mushrooms have protective activity against chemical liver injury (Yeung et al., 1995; Ooi, 1996). Treatment with crude extracts of Lentinus edodes, Grifola frondosa, Tricholoma lobayence and an isolated polysaccharide peptide of Coriolus versicolor provides significant protection against paracetamol-induced hepatotoxicity by preventing a decrease in hepatic reduced glutathione and by increasing the conjugation and excretion of the drug reactive metabolites (Yeung et al., 1995; Ooi, 1996). The protective effect of A. blazei against the cytotoxicity induced by DEN could result from a modification in DEN metabolism due to active principles in the A. blazei extracts.
Altered foci of hepatocytes are believed to be early markers of the rodent liver cancer development (Bannasch and Zerban, 1992). A variety of phenotypic abnormalities identify the altered foci of hepatocytes, including abnormal expression of enzymes such as GST-P (Sato, 1988). In the DEN-PH model, the necrogenic dose of 200 mg/kg DEN was adopted because it induces a large number of GST-P-positive foci, favoring the analysis of the anti-cancer promoting potential of chemicals (Hasegawa and Ito, 1992). In a parallel study on DEN-induced cancer initiation of Wistar rats, we found that a previous treatment with a moderate dose of A. blazei (5.6 mg/ml) can exert hepatoprotection against liver toxicity and against the development of single GST-P-positive hepatocytes induced by 100 mg/kg DEN (Barbisan et al., 2002). Therefore, it was assumed that higher doses of A. blazei would also exert a beneficial influence against the initiation step of liver carcinogenesis by the standard protocol for medium-term hepatocarcinogenesis assay, which uses 200 mg/kg DEN for initiation. However, an increased number of the GST-P-positive foci were observed in animals treated with the higher doses of A. blazei before DEN. This could be attributed to the toxic environment induced by the higher dose of the carcinogen, when compared to the previous study. Also, the protection against liver cytotoxicity by the crude mushroom extract in the animals treated with the necrogenic dose of DEN could increase the survival of initiated cells (putative preneoplastic cells) through the process of liver initiation, resulting in a significant enhancement of GST-P-positive foci.
Numerous efforts towards reducing the chemical initiation of the carcinogenesis process have been unfruitful (Schmitt et al., 1993; Tessitore et al., 1996, 1997; Hambly et al., 1997). Schmitt et al. (1993) and Tessitore et al. (1996, 1997) were unable to detect an influence of fasting on the initiation stage of rat liver and mammary carcinogenesis when relatively high doses of carcinogens were used. Hambly et al. (1997) showed that diets containing low risk factors for colorectal cancer (e.g., low in fat and high in fiber and calcium) reduced dimethylhydrazine-induced DNA damage, as assessed by the comet assay, but did not change the aberrant crypt multiplicity, a preneoplastic marker for colorectal cancer in carcinogen-induced rodent models. Based on these results it appears that DNA damage does not always correlate with the development of histologically detected preneoplasia. We also showed that DNA damage does not correlate with GST-P-positive foci development. It is possible that chemically induced models using a relatively high dose of carcinogen are not well suited to study factors relevant to the initiation events (Tessitore et al., 1996, 1997; Rijken et al., 1999).
Apparently the protective influence of aqueous extracts of A. blazei against DEN genotoxicity, cytotoxicity and carcinogenicity is due to different mechanisms and is dependent on both the dose of the chemopreventive agent and of the carcinogen used. The highest concentration of A. blazei extract (11.5 mg/ml) demonstrated pro-carcinogenic properties by reducing the elimination of damaged cells (there was less apoptosis/necrosis of liver cells after DEN injection in rats receiving the mushroom extract) leading to the formation of an increased number of preneoplastic lesions.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical support provided by Paulo Roberto Cardoso and Mara Luíza Falagueira Ardanaz. Research supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant No. 98/07726-5) and Fundação para o Desenvolvimento da UNESP (FUNDUNESP, grant No. 119/99-DFP). C. Scolastici (PIBIC/UNESP), D.M.F Salvadori, L.R. Ribeiro and J.L.V. de Camargo were recipients of fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Copyright © 2003 by FUNPEC

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

Luís Fernando Barbisan1, Clarissa Scolastici1, Maristela Miyamoto1,
Daisy Maria Favero Salvadori2, Lúcia Regina Ribeiro2,3,
Augusto Ferreira da Eira4 and João Lauro Viana de Camargo2
1Departamento de Morfologia, Instituto de Biologia, UNESP, Botucatu, SP, Brasil
2Departamento de Patologia – TOXICAN, Faculdade de Medicina, UNESP, Botucatu, SP, Brasil
3Universidade Luterana do Brasil, Canoas, RS, Brasil
4Departamento de Produção Vegetal, Módulo de Cogumelos, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP, Brasil
Corresponding author: L.F. Barbisan
E-mail: barbisan@ibb.unesp.br
Genet. Mol. Res. 2 (3): 295-308 (2003)
Received January 30, 2003
Accepted August 15, 2003
Published September 30, 2003

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine
Luís Fernando Barbisan1, Clarissa Scolastici1, Maristela Miyamoto1,
Daisy Maria Favero Salvadori2, Lúcia Regina Ribeiro2,3,
Augusto Ferreira da Eira4 and João Lauro Viana de Camargo2
1Departamento de Morfologia, Instituto de Biologia, UNESP, Botucatu, SP, Brasil
2Departamento de Patologia – TOXICAN, Faculdade de Medicina, UNESP, Botucatu, SP, Brasil
3Universidade Luterana do Brasil, Canoas, RS, Brasil
4Departamento de Produção Vegetal, Módulo de Cogumelos, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP, Brasil
Corresponding author: L.F. Barbisan
E-mail: barbisan@ibb.unesp.br
Genet. Mol. Res. 2 (3): 295-308 (2003)
Received January 30, 2003
Accepted August 15, 2003
Published September 30, 2003
ABSTRACT. The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.
Key words: Agaricus blazei, DNA damage, GST-P-positive liver foci, Hepatocarcinogenesis
INTRODUCTION
Among the mushroom species of higher Basidiomicetes, Agaricus blazei Murrill, a species native to Brazil, where it is popularly known as “sun mushroom”, has recently received attention in folk medicine due to its use in the treatment of ailments. Since 1965, strains have been exported from Brazil to Japan, where this mushroom has become popularly known as “Himematsutake” or “Agarikusutake”. This edible mushroom is often consumed as food and tea in different parts of the world, especially because of its reported medicinal properties.
In Brazil, infusion of the dried fruiting bodies of the mushroom A. blazei has been popularly consumed both as a stimulant and for auxiliary treatment of various diseases, including cancer. Nevertheless, no epidemiological and few in vivo experimental data exist on the beneficial effects of the crude aqueous extract of this mushroom. Recently, we demonstrated that this extract provides significant protection against mutagenicity induced by cyclophosphamide and methyl methanesulfonate, both in vivo and in vitro (Delmanto et al., 2001; Menoli et al., 2001; Martins de Oliveira et al., 2002). Various polysaccharides and protein-bound polysaccharides isolated from mycelia and fruiting bodies of A. blazei have shown anti-tumor activity in tumor-bearing mice by host immune response activation (Kawagishi et al., 1989; Itoh et al., 1994, 1997; Fujimiya et al., 1998; Mizuno et al., 1998).
Dielthylnitrosamine (DEN) is a potent genotoxic carcinogen that has been used as initiating agent in some two-stage (initiation-promotion) alternative protocols for hepatocarcinogenesis (Ito et al., 1988; Dragan et al., 1991). It has been reported that after its metabolic biotransformation, DEN produces the promutagenic adducts O6-ethyldeoxyguanosine and O4- and O6-ethyldeoxythymidine that may initiate liver carcinogenesis (Dragan et al., 1994; Verna et al., 1996). Consequently, the analysis of DNA damage may be relevant to evaluate the modifying influences of chemopreventive agents on the initiation stage of cancer (Moore et al., 1999).
The comet assay or single cell gel electrophoresis assay is a rapid and sensitive procedure for quantifying DNA lesions in individualized cells, both in vitro and in vivo (Tice et al., 1991; Fairbairn et al., 1995; Gontijo et al., 2001). The alkaline comet assay version was specially developed for detection of the DNA single-strand breaks and alkali-labile sites (Singh et al., 1988) and is also indicated to evaluate in vivo genotoxicity induced by carcinogen exposure (Anderson et al., 1998; Tsuda et al., 2000).
The DEN-partial hepatectomy (PH) model has proven to be a consistent bioassay for the detection of chemical hepatocarcinogens and for the assessment of the beneficial potential of chemopreventive agents (Ito et al., 1988, 1996; Moore et al., 1999). This 8-week-long medium-term rat liver assay uses as endpoint marker the development of putative preneoplastic foci of altered hepatocytes that express the placental form of the enzyme glutathione S-transferase (GST-P) (Ito et al., 1988). It has been indicated as a practical approach for the assessment of the potential hazard or benefit of chemicals, when associated with other surrogate end-points (Moore et al., 1999).
The standard DEN-PH assay protocol uses 200 mg/kg DEN to establish the initiation of the hepatocarcinogenesis process (Ito et al., 1988). In order to determine the protective influence of the aqueous solutions of the mushroom A. blazei against liver damage and preneoplasia development induced by the DEN dose level used in the DEN-PH assay, we evaluated DNA damage and foci of altered hepatocyte development by the comet assay and by the immunohistochemical expression of the placental enzyme, glutathione S-transferase, respectively.
MATERIAL AND METHODS
Animals and treatment
Male 4-week-old Wistar rats were obtained from CEMIB (UNICAMP, Campinas, SP, Brazil). The animals were kept in polypropylene cages (five animals/cage) covered with metallic grids in a room maintained at 22 ± 2oC, 55 ± 10% humidity, and with a 12-h light-dark cycle. They were fed with commercial Purina chow (Labina, Paulínia, SP, Brazil) and water ad libitum during a 2-week acclimation period.
The animals were randomly allocated into six groups (Figure 1). For two weeks, animals of groups 3 to 6 were treated with aqueous solutions of the A. blazei, with a mean dry weight of water-extractable solids of 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively. After this period, groups 2 to 5 were given a single ip injection of 200 mg/kg DEN (Sigma, St. Louis, MO, USA), and groups 1 and 6 were treated with 0.9% NaCl only (DEN vehicle). All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The University Ethics Committee for Animal Research approved the protocols used in this study (Protocol No. 99/22).

Preparation and administration of aqueous solutions of Agaricus blazei
A sample of A. blazei Murrill (lineage 99/26) was obtained from the Departamento de Produção Vegetal, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP, Brazil. Twenty-five grams of powdered dry fruiting bodies of A. blazei mushroom was added to 1000 ml of deionized water (2.5% w/w) and left for 2 h at room temperature. This preparation corresponds to the popular form of use of A. blazei for beneficial health effects. This solution, referred from here on as “the crude aqueous extract”, was then centrifuged (800 g for 10 min) and filtered (commercial non-sterile filter). The final solution was provided as 10% (Ab1), 50% (Ab2) and 100% (Ab3) of the full 2.5 (w/w) aqueous extract of A. blazei. The mean amount of water-extractable solids in Ab1, Ab2, and Ab3 solutions was 1.2, 5.6, and 11.5 mg/ml, respectively. The yields were 4.8, 22.4 and 46%, respectively. The solutions were prepared daily and offered to rats ad libitum, in aluminum foil-wrapped bottles to avoid light decomposition. They were the sole source of drinking fluid, starting two weeks before DEN initiation.
Comet assay
Four hours after DEN or NaCl treatments, five animals of each experimental group were sacrificed and a small piece of the left hepatic lobe was collected and placed onto a small Petri disc with ice-cold mincing solution (Ca2+- and Mg2+-free HBSS containing 20 mM EDTA and 10% DMSO). The liver samples were cut into smaller pieces, using a disposable microtome razor blade, and the solution was aspirated. Then, a fresh mincing solution was added and the liver samples were minced again to finer pieces. Resulting cell suspensions were collected and filtered (100 µm nylon mesh). All samples were stocked on ice in appropriate conditions to avoid light until the comet assay procedures. The quantity of liver cells in the cell suspensions was determined in Giemsa-stained smears.
The viability of the liver cells was indirectly determined by analyzing the comet images after electrophoresis (Vaghef and Hellman, 1998) and by spontaneous DNA diffusion without DNA denaturation and electrophoresis (Vasques and Tice, 1997). The comet image was considered to be from a nonviable cell when it presented a “cloudy” appearance or a very small head and a tail like a balloon (necrotic or apoptotic cells). The viability of the cell suspension was considered acceptable when the frequency of such images was less than 2% (Vaghef and Hellman, 1998). Higher values were considered as indicative of cytotoxicity due to the carcinogen treatment. The extent of spontaneous DNA diffusion was evaluated by measuring the diameter of the liver cell “nucleoids”, considering that cells containing extremely low molecular weight DNA associated with apoptosis or necrosis develop spontaneous DNA diffusion into the agarose gel and consequently have larger diameters (Vasques and Tice, 1997).
The comet assay was performed under alkaline conditions according to a previously described standard protocol (Speit and Hartmann, 1999). Briefly, an aliquot of 5 µl of each prepared hepatic cell suspension was mixed with 120 µl of 0.5% low melting point agarose at 37oC and layered onto conventional microscope slides, precoated with 1.5% normal melting point agarose. The slides were placed overnight in freshly prepared cold lysing solution (1% Triton X-100, 2.5 mM NaCl, 0.1 mM Na2EDTA, 10 mM Tris with 10% DMSO, pH 10.0) and then in a horizontal electrophoresis cube with alkaline electrophoresis solution (0.3 M NaOH, 1 mM Na2EDTA, pH >13) at 4oC for 20 min. The electrophoresis was performed at 25 V and 300 mA for 20 min. After electrophoresis, the slides were washed twice for 5 min in neutralizing buffer (0.4 M Tris-HCl, pH 7.5), fixed for 5 min in absolute alcohol, air-dried, and stored at room temperature. In order to evaluate extremely low molecular weight DNA diffusion, two slides from each animal were removed after lysis procedure, rinsed with neutralizing solution, fixed and air-dried, and stored until analysis.
Immediately before analysis, the DNA was stained with 50 µl of 20 µg/ml ethidium bromide. The slides were examined with a 40X objective lens with epi-illuminated fluorescence microscopy (Olympus-Bx60, excitation filter: 515-560 nm; barrier filter: 590 nm) attached to a color CCD video camera and connected to an image analysis system (Comet II, Perspective Instruments, UK). Coded slides were scored blindly and 100 hepatic cell images were randomly analyzed for each animal (50 images per slide).
The comets were analyzed by a visual scoring method and computerized image analysis. The comets were classified into five categories, defined as types 0, 1, 2, 3 and 4 – where 0 indicates no or very low damage, 1, 2 and 3 indicate low, medium and long DNA migration, respectively, and 4 indicates apoptotic or necrotic DNA migration (Speit and Hartmann, 1999). The metrics for comet analysis included spontaneous DNA migration (measure of diameters larger than 34 microns of the “nucleoids”, expressed in microns), tail migration (distance from the end of the head to the end of the tail, expressed in microns), and tail moment (product of DNA density in the tail and the mean distance of DNA migration in the tail, expressed in arbitrary units).
Histology and GST-P-positive foci analysis
After sacrifice at the end of the 10th week, samples of each of the liver lobes were weighed and fixed in 10% phosphate-buffered formalin for hematoxylin-eosin (HE) staining and immunohistochemical demonstration of GST-P-positive foci, using the avidin-biotin complex method (Hsu et al., 1981).
Paraffin-embedded liver samples were cut into 5-µm thick sections, placed on poly/D/lysine-coated slides, deparaffinized in xylene and rehydrated with graded alcohol to water. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min. Nonspecific protein binding was minimized by the use of 1% nonfat dried milk in PBS for 60 min at 4oC. Slides were incubated with rabbit anti-rat GST-P primary antibody (Medical and Biological Laboratories Co., Tokyo, Japan) diluted at 1:1000 in 1% bovine serum albumin (BSA; Sigma) overnight at 4oC. Then, the slides were successively incubated with a biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories Inc., CA, USA) diluted at 1:200 in 1% BSA for 60 min, followed by an avidin-biotin-horseradish peroxidase complex kit (Vector Laboratories Inc.) diluted at 1:100 in PBS for 45 min. Subsequent chromagen color development was made using 0.038% 3-3’-diaminobenzidine tetrahydrochloride (Sigma) and 0.025% hydrogen peroxide in 0.1 M Tris-HCl, pH 7.4, for 4 min. The sections were counterstained with Harris’s hematoxylin, dehydrated, cleared in xylene, and a cover slip fixed with a xylene-based mounting medium.
GST-P-positive foci larger than 0.15 mm2 in diameter were measured using a Nikon photomicroscope (Microphot-FXA) connected to a KS-300 apparatus (Kontron Elektronic, Germany). Data are reported as number and area (mm2) of preneoplastic GST-P-positive foci per liver section (cm2).
Statistical analysis
Comparisons of body and liver weights among groups were carried out using analysis of variance and Student t-test. The DNA migration data and the number and area of the GST-P-positive foci were analyzed by Mann-Whitney and Kruskal-Wallis tests (Zar, 1984). The contrast between groups was analyzed by the Tukey test. A significant difference between the groups was assumed when P < 0.05.
RESULTS
Food and liquid consumption and body and liver weights
The two-week A. blazei treatment had no influence on the mean body weight gain (Table 1), on food consumption (~25 g/rat/day for all the experimental groups) or on liver morphology, evaluated by HE (data not shown). Significantly increased ingestion of the aqueous extract of A. blazei was observed for 50 and 100% of the full aqueous extract (2.5 w/w) of A. blazei (Ab2 and Ab3 solutions) (group G1 vs group G6, P < 0.001; group G2 vs groups G4 and G5, P < 0.001) (Table 1). The average ingestion of A. blazei (i.e., water-extractable material) calculated from the mean ingestion of the aqueous solutions of A. blazei was approximately 0.18, 0.90 and 1.97 g of solids/kg/day (Table 1).

No differences were found in the mean body and liver weights between non-initiated (groups G1 and G2) and DEN-initiated animals (group G2 to group G5) at the end of the experimental period (data not shown).
DNA damage evaluation
In untreated animals (group G1), there was a low frequency of necrotic hepatocytes, indicated by type 4 comet images (Figure 2). DEN treatment induced a higher frequency of type 4 comet images (necrosis/apoptosis process) and increased DNA migration in the alkaline comet assay, when compared to the non-initiated animals (group G1 vs group G2) (Figures 2 and 3). Necrosis/apoptosis of liver cells in DEN-treated animals was also indicated by increased spontaneous DNA diffusion (Table 2) and by extensive centrilobular liver necrosis observed 48 h after DEN treatment (Figure 4).

Agaricus blazei treatment alone did not change the basal level of DNA damage in liver cells, evaluated by DNA migration in the comet assay (Table 2 and Figure 2). Significantly smaller values of the DNA migration parameters (tail migration and tail moment) and of spontaneous DNA diffusion analysis without electrophoresis were observed in rats that received the highest concentration of the mushroom A. blazei (Ab3 solution) before 200 mg/kg DEN, than in rats solely treated with this dose of carcinogen (group G2 vs group G5, P < 0.001, Table 2).
GST-P quantitative data
Non-DEN-treated control and A. blazei-treated animals (groups G1 and G6) did not develop preneoplastic GST-P-positive liver foci (Table 3, Figure 5). Higher values of the number of but not the area of GST-P-positive liver foci per cm2 of liver sections were observed in rats that received the highest concentration of A. blazei (Ab3 solution) previously to 200 mg/kg DEN than in rats solely treated with this dose of carcinogen (group G2 vs group G5, P < 0.001, Table 3).

DISCUSSION
The potential of aqueous extracts of the mushroom A. blazei to ameliorate DNA damage and GST-P foci development induced by DEN was investigated in male Wistar rats.
Natural products have been traditionally accepted as remedies due to the popular belief that they produce few adverse side effects. Therefore, understanding the potential beneficial or adverse influence of natural products extensively used by human population is very important to implement public health safety measures. We did not observe adverse effects of treatment with relatively high doses of a crude aqueous solution of the mushroom A. blazei, determined from body weight, liver morphology, DNA damage and GST-P foci data.
In the comet assay, cells with damaged DNA displayed increased migration of DNA fragments (comet tail) from the nucleus (comet head), which may also be a feature of DNA fragmentation associated with the necrotic/apoptotic death process (Olive et al., 1993; Fairbairn et al., 1996). In contrast with the conventional dye exclusion assay to evaluate cell viability (e.g., Trypan blue), we used two other approaches to evaluate concurrent liver cytotoxicity due to the DEN treatment. These alternative cytotoxicity assays included liver histological analysis for the detection of necrosis/apoptosis and the single-cell neutral diffusion assay to detect cells with low molecular weight DNA fragments, indicative of apoptosis or necrosis (Tice et al., 2000).
Treatment with 200 mg/kg DEN induced liver cytotoxicity, evidenced by increased spontaneous DNA diffusion and also by severe centrilobular liver necrosis. The increased DNA migration observed 4 h after DEN treatment was likely a sum of genotoxicity and cytotoxicity induced by the carcinogen (Hartmann and Speit, 1997). A protective effect of the treatment with the higher A. blazei aqueous extract concentrations against DEN cytotoxicity/genotoxicity can be postulated because there was a diminished tail extension and diminished DNA content in the tails of the hepatocyte comets in A. blazei-treated animals when compared to the animals treated only with the standard 200-mg/kg DEN dose. It was previously found that A. blazei extracts inhibit the mutagenicity of benzo(a)pyrene in the Ames Salmonella microsome assay (Osaki et al., 1994) as well as mutagenicity induced by cyclophosphamide in the mouse bone marrow micronucleus test (Delmanto et al., 2001). In general, crude extracts and polysaccharides isolated from mushrooms have protective activity against chemical liver injury (Yeung et al., 1995; Ooi, 1996). Treatment with crude extracts of Lentinus edodes, Grifola frondosa, Tricholoma lobayence and an isolated polysaccharide peptide of Coriolus versicolor provides significant protection against paracetamol-induced hepatotoxicity by preventing a decrease in hepatic reduced glutathione and by increasing the conjugation and excretion of the drug reactive metabolites (Yeung et al., 1995; Ooi, 1996). The protective effect of A. blazei against the cytotoxicity induced by DEN could result from a modification in DEN metabolism due to active principles in the A. blazei extracts.
Altered foci of hepatocytes are believed to be early markers of the rodent liver cancer development (Bannasch and Zerban, 1992). A variety of phenotypic abnormalities identify the altered foci of hepatocytes, including abnormal expression of enzymes such as GST-P (Sato, 1988). In the DEN-PH model, the necrogenic dose of 200 mg/kg DEN was adopted because it induces a large number of GST-P-positive foci, favoring the analysis of the anti-cancer promoting potential of chemicals (Hasegawa and Ito, 1992). In a parallel study on DEN-induced cancer initiation of Wistar rats, we found that a previous treatment with a moderate dose of A. blazei (5.6 mg/ml) can exert hepatoprotection against liver toxicity and against the development of single GST-P-positive hepatocytes induced by 100 mg/kg DEN (Barbisan et al., 2002). Therefore, it was assumed that higher doses of A. blazei would also exert a beneficial influence against the initiation step of liver carcinogenesis by the standard protocol for medium-term hepatocarcinogenesis assay, which uses 200 mg/kg DEN for initiation. However, an increased number of the GST-P-positive foci were observed in animals treated with the higher doses of A. blazei before DEN. This could be attributed to the toxic environment induced by the higher dose of the carcinogen, when compared to the previous study. Also, the protection against liver cytotoxicity by the crude mushroom extract in the animals treated with the necrogenic dose of DEN could increase the survival of initiated cells (putative preneoplastic cells) through the process of liver initiation, resulting in a significant enhancement of GST-P-positive foci.
Numerous efforts towards reducing the chemical initiation of the carcinogenesis process have been unfruitful (Schmitt et al., 1993; Tessitore et al., 1996, 1997; Hambly et al., 1997). Schmitt et al. (1993) and Tessitore et al. (1996, 1997) were unable to detect an influence of fasting on the initiation stage of rat liver and mammary carcinogenesis when relatively high doses of carcinogens were used. Hambly et al. (1997) showed that diets containing low risk factors for colorectal cancer (e.g., low in fat and high in fiber and calcium) reduced dimethylhydrazine-induced DNA damage, as assessed by the comet assay, but did not change the aberrant crypt multiplicity, a preneoplastic marker for colorectal cancer in carcinogen-induced rodent models. Based on these results it appears that DNA damage does not always correlate with the development of histologically detected preneoplasia. We also showed that DNA damage does not correlate with GST-P-positive foci development. It is possible that chemically induced models using a relatively high dose of carcinogen are not well suited to study factors relevant to the initiation events (Tessitore et al., 1996, 1997; Rijken et al., 1999).
Apparently the protective influence of aqueous extracts of A. blazei against DEN genotoxicity, cytotoxicity and carcinogenicity is due to different mechanisms and is dependent on both the dose of the chemopreventive agent and of the carcinogen used. The highest concentration of A. blazei extract (11.5 mg/ml) demonstrated pro-carcinogenic properties by reducing the elimination of damaged cells (there was less apoptosis/necrosis of liver cells after DEN injection in rats receiving the mushroom extract) leading to the formation of an increased number of preneoplastic lesions.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical support provided by Paulo Roberto Cardoso and Mara Luíza Falagueira Ardanaz. Research supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant No. 98/07726-5) and Fundação para o Desenvolvimento da UNESP (FUNDUNESP, grant No. 119/99-DFP). C. Scolastici (PIBIC/UNESP), D.M.F Salvadori, L.R. Ribeiro and J.L.V. de Camargo were recipients of fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Copyright © 2003 by FUNPEC

Observation on the Treatment Effect of Agaricus Blazei Murrill to the Liver Function of Chronic Hepatitis Patients

by:
Drs. Wang Li Rong/Feng Qing Rong/Xu Xiao Yuan/Ma Huai Liang – Department of Infection, The 1st Affiliated Hospital of Lanzhou Medicine College, Drs. Wang Jun Zhi/Hitoshi Ito/Keishiro Shimura – Mie University School of Medicine.

by:
Drs. Wang Li Rong/Feng Qing Rong/Xu Xiao Yuan/Ma Huai Liang – Department of Infection, The 1st Affiliated Hospital of Lanzhou Medicine College, Drs. Wang Jun Zhi/Hitoshi Ito/Keishiro Shimura – Mie University School of Medicine.
Overview
Experimental group (10 cases) of chronic hepatitis B patients who received treatment using Agaricus Blazei grown in Japan was studied. Inspection of items such as liver function, g-globulin and hemocyte was carried out before and after the treatment. In the comparison with control group, there was a significant change (P<0.005-0.001) in the values such as connective cholic acid, serum ferritin, protein electrophoresis, coagulation time, ALT, AST, bilirubin, leukocyte and platelets of experiment group patients. It is conceivable that Agaricus Blazei improves the clinical symptom of chronic hepatitis B patients, promotes the restoration of liver function and reinforces hematopoietic cell.
(Keywords: Agaricus Blazei, Vegetable Polysaccharides, Chronic Hepatitis B, Therapeutic Effect)
As proven by experiments and researches, Agaricus Blazei, a fungous plant of “Shiitake” family is known to provide reinforcement of nutrition liver and kidney. In order to examine its protective function to hepatocytes, we conducted a treatment using Agaricus Blazei for patients with chronic hepatitis and confirmed a satisfactory therapeutic effect through clinical observation from May 1992 to November 1993, which we report as follows.
Data and Method
I. Clinical Data
1. Background of the Subject Patients
The subject patients (total 20 examples) were a part of chronic and active hepatitis B patient who were hospitalized at the Department of Hematology, The First Affiliated Hospital of Lanzhou Medical College during the period from May 1992 to November 1993. They were randomly divided into an experimental group and a control group, each consisting of 10 patients. The examples comply with the diagnosis standard established at Shanghai Hepatitis Conference held in May 1990.
2. General Data
The age of the patients of the experimental group ranged 24 – 55 and the average was 41. The experimental group consisted of 9 males and 1 female.
The age of the patients of the control group ranged 21 – 57 and the average was 39. The control group consisted of 8 males and 2 females.
3. Expression of the Clinical Symptom
All the 20 patients were presenting the general clinical expression of chronic hepatitis caused by virus, including fatigue, anorexia, dark urine color, dull pain in the liver and abdominal distension. Among them, 11 patients wore the color of liver disease on their faces, 7 patients carried spider-like nevus and liver palms, and 6 patients presented various degrees of jaundice (serum bilirubin of one
patient was as high as 335mmol/L). All the patients had their ALT exceeding the normal level. 6 patients were experiencing the increase of AST or ALT/AST ratio lower than 1. Liver swellings were felt up to 0.5 – 2.0cm under ribs or 2-4cm under xiphoid. 6 patients had such swellings and 5 patients were presenting positive ascites.
4. Diagnosis Standard
Our diagnosis complies with the clinical and pathological standards for chronic and active hepatitis B established at Shanghai Hepatitis Conference held in May 1990.
II. Treatment Method
1. Experimental Group (10 examples):
While they were given a standard treatment for liver disease, the experimental group patients took Agaricus Blazei (supplied by Japan Iwakin Co. Ltd. Laboratory) boiled in the water (daily amount of 20g, twice a day) for 3 consecutive months. After the 3-month treatment period, the conditions of patients were re-checked.
2. Control Group (10 examples):
The control group patients received the same standard treatment for liver disease and they also took vitamins and energy mixture including diuretics, digestives and antiinfectives. Albumin and complex amino acid were also given to patients in serious condition.
III. Monitored Values
1. Changes in the Liver Functions:
ALT, AST, bilirubin, serum ferritin (ST), connective cholic acid (CG), g-globulin and coagulation time were monitored at pre-treatment test and post-treatment biweekly tests to see their dynamic changes.
2. Changes in the Blood Picture:
Leukocyte and platelets were monitored before and after treatment.
IV. Rating of Therapeutic Effect
1. The rating of the therapeutic effect established at the Shanghai Hepatitis Conference held in May 1990 is as follows:
• Especially effective: (1) Disappearance of subjective symptom, (2) decrease or no change in swelling and other chronic symptom, (3) recovery of liver function ALT to normal level and improvement in A/G ratio, and (4) negative HBVM activity replisome.
• Effective: (1) Improvement or disappearance of subjective symptom, (2) no change in swelling and other chronic symptom, (3) recovery of liver function ALT to normal level and no change in A/G ratio, and (4) negative HBVM activity replisome.
• Ineffective: Any result short of the above standards.
V. Side Effect
Patients went through rigid inspection during and after treatment for any side effects.
VI. Statistical Processing
“T” inspection processing was applied to the data of each item.
Result
1. Symptoms and Signs:
The symptoms of most of the experimental group patients were improved. For example, symptoms such as abdominal distension, fatigue and hepatodynia either disappeared or improved. Signs were also improved to some degree. The number of patients with retraction of the liver and spleen decreased to 5/8 and 3/6 examples respectively, and 2 examples of ascites retraction disappeared. It was obvious that the improvement in the clinical symptoms of the experimental group far exceeded that of the control group.
2. After the 3-month treatment using Agaricus Blazei, the liver function was obviously improved, and there was a remarkable change in values including ALT, AST, bilirubin (P<0.05-0.001). It is obvious that Agaricus Blazei has a therapeutic effect against enzyme depression and jaundice (see Table 1).
Table 1: Influence of Agaricus Blazei to the Liver Function of Chronic Hepatopaths (x±S)
Group Timing ALT(m) P value AST(m) P value Bilirubin
(mmol/L) P value
Experimental Group (10) Before 83.1±33.21 <0.001* 83.1±33.21 <0.005* 114.14±127 <0.05*
After 24.12 ±8.97 <0.001** 139.3 ±4.7 <0.001** 17.83 ±4.02 <0.05**
Control Group (10) Before 88.3 ±35.26 <0.01* 137.7 ±3.4 >0.1* 103.55±50.96 <0.001*
After 64.6 ±28.48 128.4 ±5.72) 31.36 ±17.16
Note: * Comparison within the group
** Comparison with the result of control group patients after treatment
3. Agaricus Blazei influences to all correlative perspectives of the liver function of chronic hepatopath. Serum connective cholic acid, serum ferritin, g-globulin, coagulation time after the treatment showed remarkable depression (P<0.05-0.001). It is conceived that Agaricus Blazei has various therapeutic effects to hepatopaths and presents a comprehensive control in the positive direction.
Table 2: Influence of Agaricus Blazei to the Correlative Perspectives of Liver Function of Chronic Hepatopaths (x±S)
Group Timing Connective Cholic Acid (mg/dl) P value Serum Ferritin (Ng/ml) P value g-Globulin (g%) P value Coagulation Time (S) P value
Experimental Group (10) Before 2803 ± 1535.53 <0.001* 340.7 ± 136.5 <0.005* 27.78 ± 0.91 <0.025* 19.8 ±3.27 <0.001*
After 748 ± 951.36 <0.005** 198 ± 110.79 <0.025** 22.24 ± 5.68
Control Group (10) Before 2803 ± 1189.77 <0.01* 302.8 ± 41.84 >0.5* 26.37 ± 7.93 >0.01* 19.7 ±3.09 <0.005*
After 2456.1±835.6 293.1±28.75 27.56±4.82
Note: * Comparison within the group
** Comparison with the result of control group patients after treatment
4. Agaricus Blazei Influences the Peripheral Blood of Chronic Hepatopaths
As shown in the experiment result, Agaricus Blazei causes promotional and formational action to the hematopoietic cells of mice. It was also found in clinical studies that it controls the peripheral hemocyte of chronic hepatitis patients to the positive direction. As shown in Table 3, the values after treatment using Agaricus Blazei obviously increases and presents a remarkable statistical significance (P<0.025-0.001). It can be said that Agaricus Blazei has obvious stimulative and proliferative effects to hemocytes.
Table 3: Influence of Agaricus Blazei to the Peripheral hemocytes of Chronic Hepatopaths (x±S)
Group Timing Leukocytes (x109/L) P value Platelets (x1012/L) P value
Experimental Group (10) Before treatment 4.55±0.85 <0.01* 67.6±11.29 <0.001*
After treatment 5.06±1.41 <0.025** 96.6±1.73 <0.025**
Control Group (10) Before treatment 4.68±0.46 <0.05* 68.7±15.9 >0.5*
After treatment 4.79±0.51 71.6±16.09
Note: * Comparison within the group
** Comparison with the result of control group patients after treatment
5. Comparison of Total Therapeutic Effect
The treatment was especially effective to 2 patients (20%) and effective to 8 patients (80%) of total 10 patients of experimental group. For control group, the treatment was especially effective to 1 patient (10%), effective to 6 patients (60%) and ineffective to 3 patients (30%).
6. Side Effects
No obvious reaction was observed during the treatment period except minor nausea and thirst reported by individual patients. These reactions disappears as they continue the recipe. No one stopped the recipe due to these minor reactions.
Discussion
Agaricus Blazei belongs to Fungi plant and it originated from Brazil. Recently, it is widely grown in Japan and Indonesia. It is sometimes called “Shiitake fungi” because its appearance resembles Shiitake. Since the edible part of the mushroom produces elegant aroma and has soft and delicious, it has been regarded as a high-quality food material. 100g of dried Agaricus Blazei contains 45g of protein and 38-40g of carbohydrate as well as various elements such as vitamin B group and nicotinic acid. According to the researches in Japan, it is found that these elements have nutrient effects to liver and kidney. The water extract from Agaricus Blazei contains rich polysaccharides that features immunological enhancement anticancer activity. This time, Agaricus Blazei’s function to promote the recovery of hepatocytes of chronic hepatitis patients was found through our clinical research.
As it is shown in the result of the test treatment this time, the disappearance and improvement of symptoms and signs of experimental group patients far exceeded those of control group. The mushroom also has an effect to improvement in liver function including dramatic enzyme depression. After the patients stopped the treatment, so far there is no sign of rebound phenomenon. AST, ALT, serum bilirubin, g-globulin, CG and SF are all improved and the difference in these values before and after the treatment is remarkable (P<0.05-0.001). Liver functions of the patients checked after the treatment period and were maintaining the normal level. So the therapeutic effect is stable and long lasting. As seen in the completely improved situation of liver function, Agaricus Blazei provides a reliable protection to hepatocytes through its remarkable promotion of the recovery of liver function and enhancement of liver reserve function of chronic hepatitis.
As shown in number of result in research and experiment, vegetable polysaccharides remarkably promote the formation of hematopoietic cells. Although the mechanism is still unknown, the result is presumed to have been achieved by directly promoting the approach to proliferation and differentiation stage of hematopoietic cells or by effectively stimulating the control factor of these cells. As confirmed by out experiment result, it is certain that Agaricus Blazei, with its voluminous vegetable polysaccharide contents, has an effect to increase the number of peripheral hemocyte. Especially, the leukocyte value increased after the treatment to 6.06±1.40 (x109/L) from pre-treatment value of 4.55±0.85. The same result has been obtained through other academic experiments as well.
As observed in our experiment, Agaricus Blazei is, when compared to other medicines, an ideal medicine for chronic hepatitis. Considering its potential applications to human society and contribution to human health, Agaricus Blazei is worth further study and research not only as a new crude drug but also as a therapeutic medicine for the next healthcare era of the 21st century.
(Dr. Wang Jun Zhi is a co-researcher of Lanzhou Medicine College, currently at Mie University, Japan.)
Bibliography:
1.T. Mizuno et al. “Studies on the Host-Mediated Antitumor Polysaccharides” 1986.56.1679
2.“The 6th National Virus Hepatitis Conference Report” China Epidemic Disease Magazine 1991.1.52
3.Ma Lan Fang et al. “Influence of the Polysaccharides to Hematopoietic Cell Formation” China Hematology Magazine 1988.9.14
4.Lee Xing Yu et al. “Observation of the Effect of Seaweed Pent-Polysaccharides against Anti-radiation Damages” 1988.9.14
5.Zhou Zhi Wen et al. “Influence of Polysaccharides to Normal Hematopoietic and Colony Stimulation Factors of Rats” China Hematology Magazine 1991.12.409
6.Zhu Qi et al. “Influence of Vegetable Polysaccharides to the Formation of Lymphocytes and Hematopoietic Cells of Rats” Military Medical Science Institute Publication 1985.3.279

Observation on Treatment Effect of Agaricus Blazei Murrill against Alimentary Tract Tumor

By:
Drs. Wang Jing/Mao Xin Min – Lanzhou Medical College, China
Drs. Cheng Ru Zheng – Gansu Tumor Hospital, China
Drs. Wang Jun Zhi/Hitoshi Ito/Keishiro Shimura – Mie University, Japan

Overview

Through clinical study of 10 patients with malignant tumor in their alimentary tracts who took Agaricus Blazei for 3 months, it was found that their clinical symptoms and signs were improved to a certain degree, their peripheral blood picture (hemoglobin, leukocyte and platelets) obviously rose, the ratio between plasma albumin and globulin was maintained within normal level and serum immunoglobulin (lgG, lgM and lgE) obviously rose. Hence, Agaricus Blazei is considered to have comparatively high medicinal value and fine development potential through its effect of improvement in clinical expression, promotion of normal hematopoiesis and enhancement of internal immunity function.

(Keywords: Agaricus Blazei, Alimentary Tract Malignant Tumor, Immunomodulator.)

The terrestrial stem of Fungi plant called Agaricus Blazei was originally grown in Brazil and has later come to be grown popularly in Japan and Indonesia. The mushroom looks close to “Shiitake (Lentinula Edodes)” mushroom and is regarded as a high-quality food material with soft and delicious body. The mushroom is recently conceived to have nutrient function as well. As it has been proven by basic medicinal research, the mushroom is worth further study and development since the polysaccharides extracted from it in the water reinforces internal immunity function and antitumor activity. The following report is based on a clinical therapy observation that was carried out at Mid-West Medical Department of Gansu Tumor Hospital from October 1992 to October 1993 on 10 patients with last stage malignant tumors in their alimentary tracts.

1. The Subject and Experiment Method

1.1 Clinical Data:

20 patients with malignant alimentary tract tumor who had all received various correlative inspections or post-operation diagnosis was chosen as the subject. The 20 patients were randomly divided into experimental group (to take Agaricus Blazei) and control group. There was no obvious difference between the two groups as to the type and condition of disease. See Table 1.
Table 1: Comparison between the Basic Condition of the Patients in the Two Groups

Group # of Patients Average Age Cancer Location
Male Female (Range) Esophageal Stomach Colon
Experimental Group 8 2 58.8 (40 – 77) 3 5
Control Group 9 1 53.7 (32 – 67) 4 5 2
1.2 Monitored Values

1.2.1 Symptoms and Signs

Mental condition, appetite, nausea, pulses, blood pressure, respiration, etc.

1.2.2 Laboratory Inspections

General blood composition, plasma total protein, albumin – globulin ratio, blood creatinin, blood urea nitrogen, liver function, serum lgG/lgM/lgE, urobilinogen, urine protein, etc.

1.2.3 Special Inspections

Endoscope, renogram and electrocardiogram.

1.3 Treatment Method

Normal treatment methods of western medicine (including radiotherapy, chemotherapy and surgery) were applied to both groups, while the experimental group took, twice a day, extract of daily amount (20g) of Agaricus Blazei using boiled water. Every value was monitored again 3 months after the treatment was started.

2. Result

2.1 Symptoms and Signs

The symptoms such as a fatigue, anorexia, nausea of control group patients continued, and in some cases deteriorated. The symptoms of experimental group patients, including mental condition, anorexia and nausea were gradually improved. No dysphoria or side effects caused by taking the extract were observed at this point.

2.2 Changes in Blood Picture

3 months after the experimental group patients started taking Agaricus Blazei extract, their blood picture was improved (though it is within a normal range). On the other hand, the blood picture of control group patients obviously deteriorated. Especially the depression of platelets was remarkable. Therefore, it is considered that Agaricus Blazei improves the peripheral blood picture. See Table 2.

Table 2: Comparison between the Changes in Blood Pictures of the Two Groups (x±S)

Group
Hb/(g/L)
Wbc/(x109/L)
Plt/(x109/L)

Experimental Group
Before treatment
135.5±6.3
6.6±0.8
60.8±4.3

After treatment
139.3±4.7
7.6±0.71)
72.5±3.73)

Control Group
Before treatment
137.7±3.4
6.9±0.5
67.0±5.4

After treatment 128.4±5.72) 5.1±0.62) 48.1±4.63)
Note: 1) P<0.05 when compared to before treatment
2) P<0.05 when compared to before observation
3) P<0.01 when compared to before observation
2.3 Proportional Changes in Plasma Total Protein, Albumin and Globulin of the Two Groups

Table 3: Proportional Changes in Plasma Total Protein, Albumin and Globulin of the Two Groups
(x±S)

Group Total Protein Albumin Globulin A/G
Experimental Group Before treatment 57.0±5 36.0±4 21.0±2 1.7±0.3
After treatment 58.0±3 36.0±3 22.0±3 1.7±0.4
Control Group Before treatment 64.0±5 42.0±7 22.0±3 1.9±0.2
After treatment 53.0±4 28.0±5 25.0±4 1.0±0.41)
Note: 1) P<0.05 when compared to before observation

While the albumin/globulin ratio of the experimental group stayed within the normal level, that of the control group became almost inversus. Therefore, it is considered that Agaricus Blazei inhibits cancer by controlling the immunity.

2.4 Comparison of Changes in Immunoglobulins of the Two Groups after Taking Agaricus Blazei

The lgG value did not obviously change, while lgM and lgE values obviously increased after taking Agaricus Blazei. See Table 4.

Table 4: Changes in Immunoglobulins after Taking Agaricus Blazei (x±S)

Group lgG lgM LgE
Experimental Group Before treatment 6.13±0.74 0.61±0.08 0.32±0.04
After treatment 6.94±0.68 0.77±0.061) 0.46±0.063)
Control Group Before treatment 6.04±0.53 0.62±0.06 0.30±0.03
After treatment 4.29±0.492) 0.45±0.052) 0.16±0.043)
Note:

1) P<0.05 when compared to before treatment
2) P<0.01 when compared to before observation
3) P<0.001 when compared to before observation
The inspection results including kidney and liver functions and electrocardiogram of the patients in the two groups are all within normal ranges.

3. Discussion

So far, the effect of Agaricus Blazei is not widely reported in China. The scholars in Japan are making various research on its pharmaceutical and pharmacological aspects. As seen in its pharmaceutical analysis, Agaricus Blazei contains protein, sugar, and small quantity of various elements and vitamins. Its pharmacological study reveals that neutral and acidic polysaccharides separated from water extract of mushrooms such as Agaricus Blazei, Ganoderma Lucidum or Polyporos Umbellatus have both immunological enhancement and tumor inhibition effects. This time, the lgG, lgM and lgE values of the experimental group were all increased from the values before treatment. The increase of lgM and lgE values were significant (P<0.05). On the other hand, as a result of monitoring the control group more than 3 months, all the three values were obviously decreased (P<0.01 – 0.001). Therefore, Agaricus Blazei demonstrated its internal immunity function. The decrease in the three values of control group is caused by the deterioration of internal immunity function from the radiotherapy and chemotherapy, as well as from the malignant tumor itself.

There is a difference between the two groups of patients in the change of ratio between plasma albumin and globulin. The A/G ratio of experimental group after the treatment was unchanged from the ratio before the treatment (the ratio was maintained within a normal range). On the other hand, the A/G ratio of the control group was almost inverted (P<0.05). This decrease in A/G ratio is considered to have been caused by the increase of globulin and not by decrease of albumin, since no obvious damage was found in liver function. Accordingly, the condition of cancer will be worsened. The fact that the A/G level remains within a normal level means that Agaricus Blazei has a cancer inhibition effect. As proven in the past experiments and clinical studies, many plant polysaccharides play the roll of an immunomodulator and are called antitumor polysaccharides. While such polysaccharides do not have an effect to kill cancer cells, they discharge certain cell factors effective in cancer inhibition through activation of internal immunity system and reinforce the anticancer function of natural killer cells and LAK cells. Therefore, Agaricus can be conceived to have the same immunomodulation activity, since it contains such polysaccharides voluminously.

Although patients in both groups experienced excessively low peripheral blood picture derived from retarded hematopoiesis caused by radiotherapy and chemotherapy, the blood pictures of the experimental group patients were maintained in a normal range and could obviously rise from an already low level. On the contrary, the blood picture of the control group, without the help of Agaricus Blazei kept dropping all the way. Hence, in addition to its hematopoietic promotion, Agaricus Blazei is also considered to promote the restoration of retarded myelopoiesis after radiotherapy and chemotherapy. This stands as the ratification of the result obtained in our animal experiment. We had found through our experiment (to be reported) using mice that Agaricus Blazei promoted the restoration of myelopoiesis damaged by chemotherapeutical drug and radiation (99Co-g), while it also promoted the growth of CFU-S, GM-CFU and CFU-F and reinforced serum hematopoietic factor. As shown in the result of the animal experiment, the cause of the increase of peripheral blood cells of the subject patients is related to the promotional effect of Agaricus Blazei for proliferation of internal hematopoietic, ancestor and fibroblastic cells.

Because of its effects in promotion of hematopoietic action, reinforcement of immunity function and inhibition of tumor growth, there lies a substantial value in the future development and application of Agaricus Blazei.

(Dr. Wang Jun Zhi is a co-researcher of Lanzhou Medicine College, currently at Mie University, Japan.)

Bibliography:

1.T. Mizuno et al. “Studies on the Host-Mediated Antitumor Polysaccharides: Part IX. Fractionation and Characterization of Antitumor Polysaccharides from Maitake, Grifoia Frondosa.” Agric Bio Chem 1986.56.1679

2.T. Mizuno et al. “Studies on Carcinostatic Polysaccharides (The second report): The Antitumor Activity of b-D-Glycan and Chitin Obtained from a Few Medicinal Fungi (Shizuoka University faculty of agriculture research report)” 1988.38.39

3.Liu Rui Hai et al. “Overview of the Present and the Future of Carcinostatics from the Viewpoint of Crude Drug Pharmacology (Foreign and Chinese Medicine and Pharmaceutical Sciences/Separate Volume)” 1989.11.16

4.Haranaka “Antitumor Activities and Tumor Necrosis Factor Producibility of Traditional Chinese Medicine and Crude Drugs.” Cancer Immunol Immunother 1985.20.1