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Lack of subchronic toxicity of an aqueous extract of Agaricus blazei Murrill in F344 rats
Food and Chemical Toxicology 43 (2005) 1047–1053 www.elsevier.com/locate/foodchemtox
Lack of subchronic toxicity of an aqueous extract of Agaricus blazei Murrill in F344 rats Y. Kuroiwa, A. Nishikawa *, T. Imazawa, K. Kanki, Y. Kitamura, T. Umemura, M. Hirose Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 24 November 2004; accepted 15 February 2005
Abstract Agaricus blazei Murrill, an edible mushroom, is widely used as a functional food due to its possible medicinal effects. Aqueous extracts are also used as food additive to provide an agreeable bitter taste. As a part of its safety assessment, the present 90-day subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), rats were fed powder diet containing A. blazei Murrill aqueous extract at dose levels of 0 (basal diet), 0.63, 1.25, 2.5 and 5% (maximum) for 90 days. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum blood urea nitrogen was slightly but significantly increased in males of the 2.5 and 5% groups, no related histopathological changes were observed in the kidney, and serum creatinine levels were rather reduced, suggesting the increase of blood urea nitrogen to be of little toxicological significance. Hematology, organ weight measurement and histopathological observation revealed no test compound-related toxicological changes. In conclusion, A. blazei Murrill extract even at 5% in the diet (2654 mg/kg b.w./day for male rats and 2965 mg/kg b.w./day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL was concluded to be 5% in the diet. 2005 Elsevier Ltd. All rights reserved. Keywords: Agaricus blazei; Murrill extract; Subchronic toxicity; F344 rat; Food additive
1. Introduction Agaricus blazei Murrill, an edible mushroom belonging to the Agaricaceae family, is native to southern Bra-
Abbreviations: AlB, albumin; AlP, alkaline phosphatase; AlT, alanine aminotransferase; AsT, aspartate aminotransferase; A/G, albumin/globulin ratio; BUN, blood urea nitrogen; Ca, calcium; Cl, chloride; CRN, creatinine; c-GT, c-glutamyl aminotransferase; Hb, hemoglobin; Ht, hematocrit; K, potassium; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; Na, sodium; NOAEL, no-observedadverse-effect level; P, inorganic phosphate; PLT, platelets; RBC, red blood cell count; T. Bil, total bilirubin; T. Cho, total cholesterol; TP, total protein; WBC, white blood cell count. * Corresponding author. Tel.: +81 3 3700 9819; fax: +81 3 3700 1425. E-mail address: [email protected] (A. Nishikawa). 0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.02.007
zil. It is popularly known as ‘‘Himematsutake’’ in Japan. At present, A. blazei Murrill is widely used as a medicinal and functional food rather than for nutrition proposes because of its potent medicinal properties. Although it was traditionally consumed as tea, various kinds of healthy foods containing A. blazei Murrill and/or its extracts are also produced in some countries such as Brazil, China and Japan and traded in the world. Aqueous extracts from the mycelium, fruit bodies or culture solution of A. blazei Murrill have been approved in Japan as food additive giving a bitter taste (MHLW, 1996a; JETRO, 2004). A. blazei Murrill has tested negative for mutagenicity in the Ames, micronucleus, chromosome aberration, sister chromatid exchange, and comet assays (Osaki et al., 1994; Menoli et al., 2001; Luiz et al., 2003;
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Guterrez et al., 2004), but a comprehensive toxicological evaluation of extracts has hitherto not been conducted. Therefore, a 90-day subchronic toxicity study in F344 rats was here performed as part of a safety assessment for food additive use.
2. Materials and methods 2.1. Experimental animals and housing conditions The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences. Specific pathogen-free F344/DuCrj male and female rats (5 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and acclimated for 1 week prior to commencement of the test. The animals were randomly allocated to five groups, each consisting of 10 males and 10 females, housed in a room with a barrier system, and maintained under the following conditions: temperature of 24 ± 1 C, relative humidity 55 ± 5%, ventilation frequency of 18 times/h and a 12 h light/dark cycle. The animals were housed in plastic cages (5 rats/ cage) on soft chip bedding (Sankyo Labo-service, Tokyo, Japan). Throughout the experiment, chips were renewed every 3 or 4 days and rats were given tap water ad libitum. 2.2. Test compound and administration dose levels A. blazei Murrill extract (ABM-EG3), a brown dry powder, was kindly provided by Iwade Mushroom Institute (Mie, Japan). The mycelium and fruit bodies of A. blazei Murrill (Iwade strain 101) harvested in Paraguay and Brazil were extracted with hot water and solvent, and concentrated in vacuum. The test material ABM-EG3 comprised 30% of the extract, and 70% of soluble dextrin and starch. By an enzymatic analysis, ABM-EG3 proved to include 4.7% of b-glucan as an indicator of mushroom constituents. On the basis of a preliminary 2-week feeding study (data not shown), the maximum dose level was set as 5% in compliance with guidelines for food additives (MHLW, 1996b). As the high, middle and low dose levels, 2.5, 1.25 and 0.63% were set with a common ratio of 2. The requisite amount of test compound for each dose was weighed and mixed with powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo) and the test compound/diet admixture was supplied ad libitum for 90 days. The animals in the control group received the basal diet only. Dry powder of A. blazei Murrill was found to hardly denature over 2 years under refrigeration. Therefore, diets were prepared at a forage supplier Oriental Yeast Co., Ltd., and preserved in the dark at 4 C prior to use.
2.3. Observation and examination methods Clinical signs and general appearance were noted once daily, and body weights and food consumptions were measured once a week. Before the day of necropsy, the animals were deprived of food overnight. They were necropsied after blood samples were collected from the abdominal aorta under ether anesthesia. Hematological parameters, measured with an automated hematology analyzer (Sysmex M-2000, TOA Medical Electronics Co., Ltd., Hyogo, Japan) were: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelets (PLT). The distributions of white blood cells were assessed with a blood cell automatic analyzer (MICROX HEG-120A, Omron Co., Tokyo). Serum biochemical parameters, measured at SRL Inc. (Tokyo, Japan) using sera frozen after centrifugation of blood, were: total protein (TP), albumin/globulin ratio (A/G), albumin (AlB), total bilirubin (T. Bil), total cholesterol (T. Cho), blood urea nitrogen (BUN), creatinine (CRN), calcium (Ca), inorganic phosphate (P), sodium (Na), potassium (K), chloride (Cl), aspartate aminotransferase (AsT), alanine aminotransferase (AlT), alkaline phosphatase (AlP) and c -glutamyl aminotransferase (c-GT). At necropsy, all the organs were examined macroscopically, and the brain, heart, lungs, liver, spleen, adrenals, kidneys and testes were weighed. In addition to the organs mentioned above, the cranium with nasal cavity, pituitary, eyeballs, Harderian glands, spinal cord, salivary glands, thymus, stomach, small and large intestine, caecum, pancreas, urinary bladder, skin, mammary gland, lymph nodes, sternum, trachea, esophagus, thyroid gland, tongue, femoral muscle and bone, trigeminal and ischiatic nerves, epididymis, seminal vesicles, prostate and coagulating glands, uterus, ovary and vagina were fixed in 10% buffered formalin solution. Histopathological examination was carried out only on the 5% and basal diet (control) groups for both sexes. The organs of those groups were routinely processed for embedding in paraffin, sectioned and stained with hematoxylin and eosin. 2.4. Statistical analysis The data for relative organ weights, hematology and serum biochemistry were analyzed statistically as follows. The BartlettÕs test was applied to test homogeneity of variance between groups. If no significant heterogeneity was detected, one-way analysis of variance was applied. If significant heterogeneity of variance was detected, the Kruskal–WallisÕs test was conducted. If parameters were found to significantly differ between
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groups, a Bonferroni/DunnÕs multiple comparison was conducted.
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males, significantly increased BUN was noted in the 1.25% group without change of other serum parameters. 3.3. Organ weights
3. Results 3.1. General signs, body weight and food consumption No deaths occurred and no remarkable changes in general appearance were observed. Changes of body weight during the experiment are shown in Fig. 1. There were no differences between control and any of the dose groups in either sex. Data for food consumption are shown in Table 1. No suppression was evident during the experiment, and average intakes per day were approximately 14 g in male rats and 9 g in female rats. The average daily A. blazei Murrill extract intake in the 0.63, 1.25, 2.5 and 5% groups was 340, 669, 1346 and 2654 mg/kg b.w./day for males and 376, 745, 1491 and 2965 mg/kg b.w./day for females, respectively. The total intakes of A. blazei Murrill extract during the 90 days were respectively 8.0, 15.5, 31.9 and 64.0 g for males and 5.2, 10.2, 20.1 and 40.5 g for females. Thus, the total intake of A. blazei Murrill extract roughly depended on the dose level. 3.2. Hematological and serum biochemical data The results of hematological examinations are summarized in Tables 2 and 3. Except for MCV, which was significantly increased in females of the 5% group, no significant differences were observed for any parameter in either sex. Serum biochemical data are summarized in Tables 4 and 5. Significant increase of AlB was observed in 1.25% males and T. Cho was significantly increased in 0.63 and 1.25% males. BUN was significantly increased in 2.5 and 5% males and Cl in 1.25 and 2.5% males. In contrast, CRN was significantly decreased of 0.63% and higher doses in males. In fe-
350
0% 0.63% 1.25% 2.5% 5%
300
Body weight (g)
250
Males
200 150
Females
100 50 0 0
1
2
3
4
5
6
7
8
9
10
11
12
13
Fig. 1. Body weight curves for male and female F344 rats treated with Agaricus blazei Murrill extract for 90 days.
The final body weight, and absolute and relative organ weights are summarized in Tables 6 and 7. There was no treatment-dependent variation in either sex, except for significant increases in the relative liver weight in males of the 1.25 and 2.5% groups and the relative brain weight in females of the 2.5% group. However, these were not dose-dependent. 3.4. Histopathological examination Histopathological examination showed sporadic microgranuloma formation and extramedullary hematopoiesis in the liver and slight inflammatory cell infiltration in the cardiac muscle in both sexes. Additionally, basophilic tubules and slight hyaline droplet deposition in proximal tubular epithelial cells were infrequently observed in male kidneys. However, the incidences of these lesions were comparable between control and 5% treatment groups. In females, mineralization in the medulla of kidneys was observed without obvious difference in the severity between the control and 5% groups. Thus, no consistent treatment-related histopathological changes were found in either sex.
4. Discussion In the present 90-day subchronic toxicity study of aqueous extract of A. blazei Murrill in F344 rats, no consistent treatment-related changes in clinical signs, body weight and food consumption were observed. The significant increase in MCV in females of the 5% group apparent on hematology could be interpreted as non-treatment-related since individual values were within the physiological range (Mitruka et al., 1983) and the degree of change was slight. Regarding serum biochemistry, significant increases in BUN were observed in males of the 2.5 and 5% groups. It is known that several kinds of mushroom contain nephrotoxins (Karlson-Stiber and Persson, 2003). However, in the present study CRN was rather decreased in males receiving 0.63% or higher doses. In addition, the individual values were within the control range in previous subchronic toxicity studies performed in our facility (Onodera et al., 1997; Takada et al., 1997; Furukawa et al., 1998; Miyauchi et al., 1999). Moreover, no other changes suggesting nephrotoxicity were observed histopathologically. Therefore, the increased BUN in males is unlikely to be of toxicological significance. In females, the significant increase in BUN, observed in the 1.25% group, can also considered
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Table 1 Food consumption and intake of Agaricus blazei Murrill extract Group (%)
0 0.63 1.25 2.5 5
Food consumption (g/rat/day)
Intake of Agaricus blazei Murrill extract (mg/rat/day) (mg/kg b.w./day)
Total intake of Agaricus blazei Murrill extract (g/rat)
Male
Female
Male
Female
Male
Female
Male
Female
13.8 14.1 13.8 14.2 14.2
8.9 9.2 9.0 8.9 9.0
– 89 172 354 711
– 58 113 223 450
– 340 669 1346 2654
– 376 745 1491 2965
– 8.0 15.5 31.9 64.0
– 5.2 10.2 20.1 40.5
Table 2 Hematological data for male F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63
1.25
2.5
5
RBC (·104/lL) Hb (g/dL) Ht (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (·104/lL) WBC (·102/lL)
884 ± 64a 14.8 ± 1.1 47.3 ± 3.4 53.5 ± 0.4 16.8 ± 0.4 31.3 ± 0.6 69.5 ± 7.2 43.8 ± 9.0
860 ± 44 14.4 ± 0.9 46.0 ± 2.5 53.4 ± 0.6 16.8 ± 0.3 31.4 ± 0.7 68.6 ± 6.4 40.6 ± 5.1
887 ± 51 14.8 ± 0.7 47.7 ± 2.9 53.8 ± 0.4 16.7 ± 0.5 31.1 ± 0.5 69.2 ± 4.0 44.1 ± 9.0
860 ± 60 14.3 ± 1.0 45.8 ± 3.1 53.3 ± 0.5 16.6 ± 0.4 31.2 ± 0.7 67.2 ± 6.5 41.4 ± 8.5
871 ± 52 14.5 ± 1.0 46.6 ± 2.9 53.5 ± 0.4 16.7 ± 0.4 31.2 ± 0.9 70.5 ± 3.7 40.6 ± 7.4
Differential cell counts (%) Bandb Seg Eosino Baso Lymph Mono
0.0 ± 0.0 15.4 ± 4.6 1.0 ± 0.7 0.0 ± 0.0 83.0 ± 4.7 0.5 ± 0.5
0.0 ± 0.0 14.0 ± 3.9 0.9 ± 0.8 0.0 ± 0.0 84.3 ± 4.2 0.8 ± 0.8
0.0 ± 0.0 13.4 ± 3.2 0.6 ± 0.5 0.0 ± 0.0 85.6 ± 3.9 0.5 ± 0.7
0.0 ± 0.0 17.2 ± 5.4 1.0 ± 0.9 0.0 ± 0.0 81.2 ± 5.0 0.7 ± 0.5
0.0 ± 0.0 17.6 ± 4.5 0.6 ± 0.4 0.0 ± 0.0 81.3 ± 4.3 0.6 ± 0.7
a
Mean ± S.D. Band, band form leukocytes; seg, segmented leukocytes; eosino, eosinophilic leukocytes; baso, basophilic leukocytes; lymph, lymphocytes; mono, monocytes. b
Table 3 Hematological data for female F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63
1.25
2.5
5
RBC (·10 /lL) Hb (g/dL) Ht (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (·104/lL) WBC (·102/lL)
a
799 ± 43 14.2 ± 1.1 44.5 ± 2.5 55.7 ± 0.4 17.7 ± 0.5 31.8 ± 0.9 70.5 ± 4.0 27.6 ± 5.3
813 ± 25 14.4 ± 0.6 45.3 ± 1.5 55.7 ± 0.3 17.7 ± 0.3 31.7 ± 0.6 73.6 ± 3.7 28.6 ± 3.6
830 ± 41 14.8 ± 0.8 46.4 ± 2.4 55.9 ± 0.3 17.9 ± 0.4 32.0 ± 0.6 71.3 ± 4.4 29.6 ± 12.3
814 ± 56 14.6 ± 1.2 45.4 ± 3.2 55.7 ± 0.4 17.9 ± 0.5 32.2 ± 0.9 74.2 ± 4.3 29.4 ± 7.0
791 ± 38 14.2 ± 0.9 44.5 ± 2.0 56.2 ± 0.3* 17.9 ± 0.5 31.9 ± 0.9 71.6 ± 4.2 28.7 ± 5.5
Differential cell counts (%) Bandb Seg Eosino Baso Lymph Mono
0.0 ± 0.0 14.6 ± 4.5 1.0 ± 0.6 0.0 ± 0.0 83.1 ± 4.5 1.3 ± 0.7
0.0 ± 0.0 14.9 ± 5.1 1.1 ± 0.7 0.0 ± 0.0 82.7 ± 5.0 1.3 ± 1.3
0.0 ± 0.0 13.8 ± 3.6 0.9 ± 0.6 0.0 ± 0.0 83.8 ± 3.6 1.6 ± 0.7
0.0 ± 0.0 12.0 ± 5.6 1.2 ± 1.3 0.0 ± 0.0 85.0 ± 5.4 1.7 ± 0.8
0.0 ± 0.0 12.4 ± 3.6 1.3 ± 0.8 0.0 ± 0.0 85.0 ± 3.6 1.4 ± 0.9
4
a
Mean ± S.D. Band, band form leukocytes; seg, segmented leukocytes; eosino, eosinophilic leukocytes; baso, basophilic leukocytes; lymph, lymphocytes; mono, monocytes. * Significantly different from the control group at p < 0.05. b
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Table 4 Serum biochemical data for male F344 rats given Agaricus blazei Murrill extract for 90 days Item
TP (g/dL) A/G AlB (g/dL) T. Bil (mg/dL) T. Cho (mg/dL) BUN (mg/dL) CRN (mg/dL) Ca (mg/dL) P (mg/dL) Na (mEq/dL) Cl (mEq/dL) K (mEq/dL) AsT (IU/L) AlT (IU/L) AlP (IU/L) c-GT (IU/L) a
;
Dose level (%) 0
0.63
1.25
2.5
5
6.3 ± 0.1a 2.1 ± 0.1 4.3 ± 0.1 0±0 59.5 ± 4.3 19.7 ± 1.6 0.32 ± 0.02 10.5 ± 0.3 6.4 ± 0.4 144 ± 1 100 ± 1 4.5 ± 0.3 83.4 ± 11.0 51.1 ± 4.3 327 ± 17 <2
6.4 ± 0.1 2.2 ± 0.1 4.3 ± 0.1 0±0 65.0 ± 3.3** 20.7 ± 1.8 0.30 ± 0.02* 10.5 ± 0.1 6.1 ± 0.4 145 ± 1 101 ± 1 4.6 ± 0.4 88.7 ± 13.6 52.3 ± 4.8 318 ± 9 <2
6.4 ± 0.2 2.2 ± 0.1 4.4 ± 0.1* 0±0 66.1 ± 5.4** 21.1 ± 1.7 0.30 ± 0.02* 10.6 ± 0.4 6.3 ± 1.1 145 ± 2 102 ± 2* 4.7 ± 0.5 87.1 ± 13.0 50.7 ± 5.7 340 ± 25 <2
6.3 ± 0.2 2.2 ± 0.1 4.3 ± 0.1 0±0 62.6 ± 3.4 21.2 ± 1.1* 0.30 ± 0.01* 10.6 ± 0.2 6.4 ± 0.5 144 ± 1 102 ± 1** 4.7 ± 0.2 92.7 ± 26.5 55.5 ± 11.2 322 ± 23 <2
6.3 ± 0.2 2.1 ± 0.1 4.3 ± 0.1 0±0 61.4 ± 6.0 21.7 ± 2.0* 0.30 ± 0.02* 10.5 ± 0.2 6.5 ± 0.5 144 ± 1 101 ± 1 4.7 ± 0.2 85.7 ± 11.1 50.0 ± 2.8 335 ± 28 <2
Mean ± S.D. : Significantly different from the control group at p < 0.05, p < 0.01, respectively.
Table 5 Serum biochemical data for female F344 rats given Agaricus blazei Murrill extract for 90 days Item
TP (g/dL) A/G AlB (g/dL) T. Bil (mg/dL) T. Cho (mg/dL) BUN (mg/dL) CRN (mg/dL) Ca (mg/dL) P (mg/dL) Na (mEq/dL) Cl (mEq/dL) K (mEq/dL) AsT (IU/L) AlT (IU/L) AIP (IU/L) c-GT (IU/L) a *
Dose level (%) 0
0.63
1.25
2.5
5
6.3 ± 0.2a 2.3 ± 0.2 4.4 ± 0.2 0.02 ± 0.04 84.3 ± 9.1 17.2 ± 1.4 0.34 ± 0.03 10.3 ± 0.4 5.7 ± 0.7 145 ± 2 105 ± 1 4.2 ± 0.6 84.0 ± 8.1 38.8 ± 3.5 229 ± 21 <2
6.2 ± 0.2 2.4 ± 0.1 4.4 ± 0.1 0.02 ± 0.04 86.0 ± 6.4 17.7 ± 1.1 0.33 ± 0.03 10.1 ± 0.3 5.5 ± 0.5 145 ± 1 105 ± 2 4.1 ± 0.4 88.8 ± 7.6 39.4 ± 3.1 243 ± 21 <2
6.3 ± 0.2 2.5 ± 0.2 4.5 ± 0.2 0.03 ± 0.05 84.2 ± 7.7 18.8 ± 1.2* 0.34 ± 0.03 10.4 ± 0.4 6.1 ± 0.8 145 ± 1 104 ± 2 4.4 ± 0.6 89.0 ± 7.5 39.9 ± 3.1 233 ± 27 <2
6.3 ± 0.2 2.4 ± 0.1 4.5 ± 0.2 0.02 ± 0.04 83.4 ± 7.1 17.4 ± 1.3 0.34 ± 0.03 10.5 ± 0.5 6.1 ± 1.2 145 ± 2 105 ± 2 4.6 ± 1.1 83.9 ± 7.0 39.1 ± 2.4 220 ± 27 <2
6.1 ± 0.2 2.4 ± 0.2 4.3 ± 0.2 0.02 ± 0.04 85.9 ± 5.9 17.6 ± 1.0 0.33 ± 0.03 10.1 ± 0.4 5.4 ± 0.7 145 ± 1 106 ± 2 4.0 ± 0.3 85.7 ± 10.7 42.7 ± 9.2 229 ± 27 <2
Mean ± S.D. Significantly different from the control group at p < 0.05.
incidental given the lack of change in the 5% group. While differences were also observed in several parameters such as AlB, T. Cho and Cl in males, but no dosedependence was observed and the degree of change was slight. We therefore conclude that there were no major treatment-related changes in serum biochemistry. Regarding final body weights and absolute organ weights, no significant differences were observed. The significant increase in the relative liver weight in males of the 1.25 and 2.5% groups and the relative brain weight in females of the 2.5% group were judged to be incidental since such changes were not observed in the 5% group and no dose-dependence was evident.
Histopathologically, several lesions were sporadically observed in the liver, kidney and cardiac muscle in males and/or females. Since the degree and incidences of these lesions were almost the same in both control and 5% groups, and they are well known to occur spontaneously in F344 rats (Boorman et al., 1990), it is reasonable to conclude they were of little toxicological significance. It has been reported that treatment with a crude aqueous extract of A. blazei Murrill exerts a protective effect against liver toxicity as well as initiation of hepatocarcinogenesis by diethylnitrosamine (DEN) (Barbisan et al., 2002). Additionally, feeding of A. blazei Murrill powder exerted a chemopreventive influence against
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Table 6 Organ weight data for male F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63 313 ± 16a
Body weight (g) Absolute (g) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys Testes
1.95 ± 0.04 0.20 ± 0.03 0.98 ± 0.08 0.90 ± 0.05 0.62 ± 0.04 7.13 ± 0.51 0.036 ± 0.004 1.87 ± 0.10 3.07 ± 0.28
Relative (g/100 g b.w.) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys Testes
0.62 ± 0.03 0.06 ± 0.01 0.31 ± 0.02 0.29 ± 0.01 0.20 ± 0.01 2.27 ± 0.09 0.011 ± 0.001 0.60 ± 0.03 0.98 ± 0.08
a *
310 ± 9
1.25
2.5
5
305 ± 12
315 ± 13
314 ± 10
1.95 ± 0.04 0.19 ± 0.04 0.96 ± 0.04 0.88 ± 0.05 0.61 ± 0.02 7.14 ± 0.31 0.037 ± 0.005 1.82 ± 0.11 3.09 ± 0.10
1.95 ± 0.05 0.21 ± 0.04 0.97 ± 0.05 0.86 ± 0.05 0.60 ± 0.03 7.27 ± 0.44 0.037 ± 0.05 1.85 ± 0.11 3.10 ± 0.13
1.94 ± 0.07 0.21 ± 0.05 0.96 ± 0.05 0.90 ± 0.04 0.63 ± 0.04 7.47 ± 0.04 0.037 ± 0.004 1.87 ± 0.10 2.93 ± 0.50
1.95 ± 0.03 0.18 ± 0.03 0.96 ± 0.05 0.89 ± 0.02 0.62 ± 0.03 7.35 ± 0.39 0.037 ± 0.005 1.91 ± 0.08 3.15 ± 0.09
0.63 ± 0.01 0.06 ± 0.01 0.31 ± 0.01 0.28 ± 0.01 0.20 ± 0.01 2.30 ± 0.07 0.012 ± 0.002 0.59 ± 0.02 0.97 ± 0.03
0.63 ± 0.02 0.07 ± 0.01 0.32 ± 0.01 0.28 ± 0.01 0.20 ± 0.01 2.38 ± 0.12* 0.012 ± 0.01 0.61 ± 0.03 1.02 ± 0.04
0.62 ± 0.003 0.07 ± 0.01 0.30 ± 0.01 0.29 ± 0.01 0.20 ± 0.01 2.37 ± 0.09* 0.012 ± 0.02 0.59 ± 0.02 0.93 ± 0.17
0.62 ± 0.02 0.06 ± 0.01 0.31 ± 0.02 0.28 ± 0.01 0.20 ± 0.01 2.34 ± 0.09 0.012 ± 0.01 0.61 ± 0.03 1.00 ± 0.03
2.5
5
Mean ± S.D. Significantly different from the control group at p < 0.05.
Table 7 Organ weight data for female F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63 a
Body weight (g) Absolute (g) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys
1.77 ± 0.05 0.15 ± 0.01 0.69 ± 0.04 0.55 ± 0.02 0.40 ± 0.03 3.69 ± 0.32 0.040 ± 0.004 1.03 ± 0.06
1.80 ± 0.05 0.18 ± 0.03 0.68 ± 0.04 0.55 ± 0.02 0.41 ± 0.02 3.61 ± 0.25 0.037 ± 0.006 1.06 ± 0.05
1.77 ± 0.04 0.16 ± 0.03 0.68 ± 0.08 0.53 ± 0.03 0.41 ± 0.02 3.69 ± 0.32 0.037 ± 0.005 1.06 ± 0.06
1.78 ± 0.04 0.17 ± 0.03 0.69 ± 0.05 0.54 ± 0.03 0.40 ± 0.03 3.62 ± 0.28 0.038 ± 0.006 1.03 ± 0.05
1.78 ± 0.03 0.17 ± 0.03 0.68 ± 0.06 0.54 ± 0.04 0.41 ± 0.04 3.57 ± 0.29 0.042 ± 0.007 1.05 ± 0.07
Relative (g/100 g b.w.) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys
1.06 ± 0.03 0.09 ± 0.01 0.42 ± 0.02 0.33 ± 0.01 0.24 ± 0.01 2.21 ± 0.17 0.024 ± 0.002 0.62 ± 0.02
1.07 ± 0.04 0.11 ± 0.02 0.40 ± 0.03 0.33 ± 0.01 0.24 ± 0.01 2.14 ± 0.08 0.022 ± 0.004 0.63 ± 0.02
1.06 ± 0.05 0.10 ± 0.02 0.41 ± 0.04 0.32 ± 0.02 0.25 ± 0.02 2.21 ± 0.13 0.023 ± 0.02 0.64 ± 0.04
1.10 ± 0.04* 0.10 ± 0.02 0.42 ± 0.02 0.33 ± 0.01 0.25 ± 0.02 2.22 ± 0.14 0.024 ± 0.02 0.63 ± 0.02
1.07 ± 0.05 0.10 ± 0.02 0.41 ± 0.02 0.32 ± 0.02 0.24 ± 0.02 2.13 ± 0.09 0.025 ± 0.05 0.63 ± 0.02
a *
167 ± 7
1.25
169 ± 9
167 ± 9
163 ± 6
167 ± 10
Mean ± S.D. Significantly different from the control group at p < 0.05.
DEN in a medium-term rat liver carcinogenesis assay (Pinheiro et al., 2003). On the other hand, in the same model using crude aqueous extract, no protection was observed (Barbisan et al., 2003a). It has also been reported that the number of pre-neoplastic glutathione S-transferase placental form (GST-P) positive foci was
significantly increased in rats treated with a crude aqueous extract from A. blazei Murrill before initiation (Barbisan et al., 2003b). The ingestion amount of extract in this last report, about 2 g/kg/day, was lower than that in the highest dosage group in the present study. However, we here observed no evidence of liver toxicity in
Y. Kuroiwa et al. / Food and Chemical Toxicology 43 (2005) 1047–1053
the serum chemistry, organ weight and histopathological findings. It is thought that ingredients of A. blazei Murrill extract may differ with various conditions such as the strain, period of mushroom harvest, type of cultivation and extraction method. Since the A. blazei Murrill extracts used have differed in each experiment in the literature, the results cannot be simply compared. In conclusion, in the present 90-day subchronic study, an A. blazei Murrill aqueous extract used as a food additive showed little or no significant toxicity, even with 5% dietary supplementation. Thus, the noobserved-adverse-effect level (NOAEL) was concluded to be 5% in the diet (2654 mg/kg b.w./day for male rats and 2965 mg/kg b.w./day for female rats). Acknowledgements We thank Ms. Machiko Maeda for her expert technical assistance. We are also grateful to Iwade Mushroom Institute for kindly supplying A. blazei Murrill extract. This work was supported by a Grant-in-Aid for Research on Food Sanitation from the Ministry of Health, Labor and Welfare of Japan. References Barbisan, L.F., Miyamoto, M., Scolastici, C., Salvadori, D.M.F., Ribeiro, L.R., Eira, A.F., de Camargo, J.L.V., 2002. Influence of aqueous extract of Agaricus blazei on rat liver toxicity induced by different doses of diethylnitrosamin. Journal of Ethnopharmacology 83, 25–32. Barbisan, L.F., Spinardi-Barbisan, A.L, Moreira, E.L., Salvadori, D.M., Ribeiro, L.R., da Eira, A.F., de Camargo, J.L., 2003a. Agaricus blazei (Himematsutake) does not alter the development of rat diethylnitrosamine-initiated hepatic preneoplastic foci. Cancer Science 94, 188–192. Barbisan, L.F., Scolastici, C., Miyamoto, M., Salvadori, D.M.F., Ribeiro, L.R., da Eira, A.F., de Camargo, J.L.V., 2003b. Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine. Genetics and Molecular Research 2, 295–308. Boorman, G.A., Eustis, S.L., Elwell, M.R., Montgomery, C.A., Jr., Mackenzie, W.F. (Eds.), 1990. Pathology of the Fischer Rat, References and Atlas. Academic Press, San Diego. Furukawa, F., Kasahara, K., Nishikawa, A., Imazawa, T., Hirose, M., 1998. A 13-week subchronic oral toxicity study of chlorophyll in
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F344 rats. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 116, 107–112 (in Japanese). Guterrez, Z.R., Mantovani, M.S., Eira, A.F., Ribeiro, L.R., Jordao, B.Q., 2004. Variation of the antimutagenicity effects of water extracts of Agaricus blazei Murrill in vitro. Toxicology In Vitro 18, 301–309. JETRO (Japan External Trade Organization), 2004. Specifications and Standards for Foods, Food Additives, etc. Under The Food Sanitation Law. Available from:. Karlson-Stiber, C., Persson, H., 2003. Cytotoxic fungi—an overview. Toxicon 42, 339–349. Luiz, R.C., Jordao, B.Q., da Eira, A.F., Ribeiro, L.R., Mantovani, M.S., 2003. Mechanism of anticlastogenicity of Agaricus blazei Murill mushroom organic extracts in wild type CHO (K1) and repair deficient (xrs5) cells by chromosome aberration and sister chromatid exchange assays. Mutation Research 528, 75–79. Menoli, R.C., Mantovani, M.S., Ribeiro, L.R., Speit, G., Jordao, B.Q., 2001. Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells. Mutation Research 496, 5–13. MHLW (Ministry of Health, Labor and Welfare of Japan), 1996a. List of existing food additives, Notification No. 120 of the Ministry of Health and Welfare. MHLW (Ministry of Health, Labor and Welfare of Japan), 1996b. Guidelines for Designation of Food Additives, and for Revision of Standard for Use of Food Additives, Article No. 29 of the Life and Sanitation Bureau. Mitruka, B.M., Rawnsley, H.M., 1983 (T. Ishii, T. Toyama, T. Sakaguchi, Trans.). Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals and Normal Humans, second ed. Seishi Shoin Ltd., Tokyo, p. 64–67 (in Japanese). Miyauchi, M., Furukawa, F., Nishikawa, A., Nakamura, H., Imazawa, T., Hirose, M., 1999. A 13-week subchronic oral toxicity study of orange color in F344 rats. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 117, 123–128 (in Japanese). Onodera, H., Mitsumori, K., Yasuhara, K., Takegawa, K., Takahashi, M., 1997. 13-week subchronic oral toxicity study of phaffia colour in F344 rats. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 115, 99–106 (in Japanese). Osaki, Y., Kato, T., Yamamoto, K., Okubo, J., Miyazaki, T., 1994. Antimutagenic and bacterial substances in the fruit body of a basidiomycete Agaricus blazei. Yakugaku Zasshi 114, 342–350 (in Japanese). Pinheiro, F., Faria, R.R., de Camargo, J.L., Spinardi-Barbisan, A.L., da Eira, A.F., Barbisan, L.F., 2003. Chemoprevention of preneoplastic liver foci development by dietary mushroom Agaricus blazei Murrill in the rat. Food and Chemical Toxicology 41, 1543– 1550. Takada, K., Toyoda, K., Shoda, T., Uneyama, C., Tamura, T., Takahashi, M., 1997. A 13-week subchronic oral toxicity study of carob green colour in F344 rats. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 115, 93–98 (in Japanese).
Lack of subchronic toxicity of an aqueous extract of Agaricus blazei Murrill in F344 rats Y. Kuroiwa, A. Nishikawa *, T. Imazawa, K. Kanki, Y. Kitamura, T. Umemura, M. Hirose Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 24 November 2004; accepted 15 February 2005
Abstract Agaricus blazei Murrill, an edible mushroom, is widely used as a functional food due to its possible medicinal effects. Aqueous extracts are also used as food additive to provide an agreeable bitter taste. As a part of its safety assessment, the present 90-day subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), rats were fed powder diet containing A. blazei Murrill aqueous extract at dose levels of 0 (basal diet), 0.63, 1.25, 2.5 and 5% (maximum) for 90 days. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum blood urea nitrogen was slightly but significantly increased in males of the 2.5 and 5% groups, no related histopathological changes were observed in the kidney, and serum creatinine levels were rather reduced, suggesting the increase of blood urea nitrogen to be of little toxicological significance. Hematology, organ weight measurement and histopathological observation revealed no test compound-related toxicological changes. In conclusion, A. blazei Murrill extract even at 5% in the diet (2654 mg/kg b.w./day for male rats and 2965 mg/kg b.w./day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL was concluded to be 5% in the diet. 2005 Elsevier Ltd. All rights reserved. Keywords: Agaricus blazei; Murrill extract; Subchronic toxicity; F344 rat; Food additive
1. Introduction Agaricus blazei Murrill, an edible mushroom belonging to the Agaricaceae family, is native to southern Bra-
Abbreviations: AlB, albumin; AlP, alkaline phosphatase; AlT, alanine aminotransferase; AsT, aspartate aminotransferase; A/G, albumin/globulin ratio; BUN, blood urea nitrogen; Ca, calcium; Cl, chloride; CRN, creatinine; c-GT, c-glutamyl aminotransferase; Hb, hemoglobin; Ht, hematocrit; K, potassium; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; Na, sodium; NOAEL, no-observedadverse-effect level; P, inorganic phosphate; PLT, platelets; RBC, red blood cell count; T. Bil, total bilirubin; T. Cho, total cholesterol; TP, total protein; WBC, white blood cell count. * Corresponding author. Tel.: +81 3 3700 9819; fax: +81 3 3700 1425. E-mail address: [email protected] (A. Nishikawa). 0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.02.007
zil. It is popularly known as ‘‘Himematsutake’’ in Japan. At present, A. blazei Murrill is widely used as a medicinal and functional food rather than for nutrition proposes because of its potent medicinal properties. Although it was traditionally consumed as tea, various kinds of healthy foods containing A. blazei Murrill and/or its extracts are also produced in some countries such as Brazil, China and Japan and traded in the world. Aqueous extracts from the mycelium, fruit bodies or culture solution of A. blazei Murrill have been approved in Japan as food additive giving a bitter taste (MHLW, 1996a; JETRO, 2004). A. blazei Murrill has tested negative for mutagenicity in the Ames, micronucleus, chromosome aberration, sister chromatid exchange, and comet assays (Osaki et al., 1994; Menoli et al., 2001; Luiz et al., 2003;
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Guterrez et al., 2004), but a comprehensive toxicological evaluation of extracts has hitherto not been conducted. Therefore, a 90-day subchronic toxicity study in F344 rats was here performed as part of a safety assessment for food additive use.
2. Materials and methods 2.1. Experimental animals and housing conditions The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences. Specific pathogen-free F344/DuCrj male and female rats (5 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and acclimated for 1 week prior to commencement of the test. The animals were randomly allocated to five groups, each consisting of 10 males and 10 females, housed in a room with a barrier system, and maintained under the following conditions: temperature of 24 ± 1 C, relative humidity 55 ± 5%, ventilation frequency of 18 times/h and a 12 h light/dark cycle. The animals were housed in plastic cages (5 rats/ cage) on soft chip bedding (Sankyo Labo-service, Tokyo, Japan). Throughout the experiment, chips were renewed every 3 or 4 days and rats were given tap water ad libitum. 2.2. Test compound and administration dose levels A. blazei Murrill extract (ABM-EG3), a brown dry powder, was kindly provided by Iwade Mushroom Institute (Mie, Japan). The mycelium and fruit bodies of A. blazei Murrill (Iwade strain 101) harvested in Paraguay and Brazil were extracted with hot water and solvent, and concentrated in vacuum. The test material ABM-EG3 comprised 30% of the extract, and 70% of soluble dextrin and starch. By an enzymatic analysis, ABM-EG3 proved to include 4.7% of b-glucan as an indicator of mushroom constituents. On the basis of a preliminary 2-week feeding study (data not shown), the maximum dose level was set as 5% in compliance with guidelines for food additives (MHLW, 1996b). As the high, middle and low dose levels, 2.5, 1.25 and 0.63% were set with a common ratio of 2. The requisite amount of test compound for each dose was weighed and mixed with powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo) and the test compound/diet admixture was supplied ad libitum for 90 days. The animals in the control group received the basal diet only. Dry powder of A. blazei Murrill was found to hardly denature over 2 years under refrigeration. Therefore, diets were prepared at a forage supplier Oriental Yeast Co., Ltd., and preserved in the dark at 4 C prior to use.
2.3. Observation and examination methods Clinical signs and general appearance were noted once daily, and body weights and food consumptions were measured once a week. Before the day of necropsy, the animals were deprived of food overnight. They were necropsied after blood samples were collected from the abdominal aorta under ether anesthesia. Hematological parameters, measured with an automated hematology analyzer (Sysmex M-2000, TOA Medical Electronics Co., Ltd., Hyogo, Japan) were: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelets (PLT). The distributions of white blood cells were assessed with a blood cell automatic analyzer (MICROX HEG-120A, Omron Co., Tokyo). Serum biochemical parameters, measured at SRL Inc. (Tokyo, Japan) using sera frozen after centrifugation of blood, were: total protein (TP), albumin/globulin ratio (A/G), albumin (AlB), total bilirubin (T. Bil), total cholesterol (T. Cho), blood urea nitrogen (BUN), creatinine (CRN), calcium (Ca), inorganic phosphate (P), sodium (Na), potassium (K), chloride (Cl), aspartate aminotransferase (AsT), alanine aminotransferase (AlT), alkaline phosphatase (AlP) and c -glutamyl aminotransferase (c-GT). At necropsy, all the organs were examined macroscopically, and the brain, heart, lungs, liver, spleen, adrenals, kidneys and testes were weighed. In addition to the organs mentioned above, the cranium with nasal cavity, pituitary, eyeballs, Harderian glands, spinal cord, salivary glands, thymus, stomach, small and large intestine, caecum, pancreas, urinary bladder, skin, mammary gland, lymph nodes, sternum, trachea, esophagus, thyroid gland, tongue, femoral muscle and bone, trigeminal and ischiatic nerves, epididymis, seminal vesicles, prostate and coagulating glands, uterus, ovary and vagina were fixed in 10% buffered formalin solution. Histopathological examination was carried out only on the 5% and basal diet (control) groups for both sexes. The organs of those groups were routinely processed for embedding in paraffin, sectioned and stained with hematoxylin and eosin. 2.4. Statistical analysis The data for relative organ weights, hematology and serum biochemistry were analyzed statistically as follows. The BartlettÕs test was applied to test homogeneity of variance between groups. If no significant heterogeneity was detected, one-way analysis of variance was applied. If significant heterogeneity of variance was detected, the Kruskal–WallisÕs test was conducted. If parameters were found to significantly differ between
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groups, a Bonferroni/DunnÕs multiple comparison was conducted.
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males, significantly increased BUN was noted in the 1.25% group without change of other serum parameters. 3.3. Organ weights
3. Results 3.1. General signs, body weight and food consumption No deaths occurred and no remarkable changes in general appearance were observed. Changes of body weight during the experiment are shown in Fig. 1. There were no differences between control and any of the dose groups in either sex. Data for food consumption are shown in Table 1. No suppression was evident during the experiment, and average intakes per day were approximately 14 g in male rats and 9 g in female rats. The average daily A. blazei Murrill extract intake in the 0.63, 1.25, 2.5 and 5% groups was 340, 669, 1346 and 2654 mg/kg b.w./day for males and 376, 745, 1491 and 2965 mg/kg b.w./day for females, respectively. The total intakes of A. blazei Murrill extract during the 90 days were respectively 8.0, 15.5, 31.9 and 64.0 g for males and 5.2, 10.2, 20.1 and 40.5 g for females. Thus, the total intake of A. blazei Murrill extract roughly depended on the dose level. 3.2. Hematological and serum biochemical data The results of hematological examinations are summarized in Tables 2 and 3. Except for MCV, which was significantly increased in females of the 5% group, no significant differences were observed for any parameter in either sex. Serum biochemical data are summarized in Tables 4 and 5. Significant increase of AlB was observed in 1.25% males and T. Cho was significantly increased in 0.63 and 1.25% males. BUN was significantly increased in 2.5 and 5% males and Cl in 1.25 and 2.5% males. In contrast, CRN was significantly decreased of 0.63% and higher doses in males. In fe-
350
0% 0.63% 1.25% 2.5% 5%
300
Body weight (g)
250
Males
200 150
Females
100 50 0 0
1
2
3
4
5
6
7
8
9
10
11
12
13
Fig. 1. Body weight curves for male and female F344 rats treated with Agaricus blazei Murrill extract for 90 days.
The final body weight, and absolute and relative organ weights are summarized in Tables 6 and 7. There was no treatment-dependent variation in either sex, except for significant increases in the relative liver weight in males of the 1.25 and 2.5% groups and the relative brain weight in females of the 2.5% group. However, these were not dose-dependent. 3.4. Histopathological examination Histopathological examination showed sporadic microgranuloma formation and extramedullary hematopoiesis in the liver and slight inflammatory cell infiltration in the cardiac muscle in both sexes. Additionally, basophilic tubules and slight hyaline droplet deposition in proximal tubular epithelial cells were infrequently observed in male kidneys. However, the incidences of these lesions were comparable between control and 5% treatment groups. In females, mineralization in the medulla of kidneys was observed without obvious difference in the severity between the control and 5% groups. Thus, no consistent treatment-related histopathological changes were found in either sex.
4. Discussion In the present 90-day subchronic toxicity study of aqueous extract of A. blazei Murrill in F344 rats, no consistent treatment-related changes in clinical signs, body weight and food consumption were observed. The significant increase in MCV in females of the 5% group apparent on hematology could be interpreted as non-treatment-related since individual values were within the physiological range (Mitruka et al., 1983) and the degree of change was slight. Regarding serum biochemistry, significant increases in BUN were observed in males of the 2.5 and 5% groups. It is known that several kinds of mushroom contain nephrotoxins (Karlson-Stiber and Persson, 2003). However, in the present study CRN was rather decreased in males receiving 0.63% or higher doses. In addition, the individual values were within the control range in previous subchronic toxicity studies performed in our facility (Onodera et al., 1997; Takada et al., 1997; Furukawa et al., 1998; Miyauchi et al., 1999). Moreover, no other changes suggesting nephrotoxicity were observed histopathologically. Therefore, the increased BUN in males is unlikely to be of toxicological significance. In females, the significant increase in BUN, observed in the 1.25% group, can also considered
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Table 1 Food consumption and intake of Agaricus blazei Murrill extract Group (%)
0 0.63 1.25 2.5 5
Food consumption (g/rat/day)
Intake of Agaricus blazei Murrill extract (mg/rat/day) (mg/kg b.w./day)
Total intake of Agaricus blazei Murrill extract (g/rat)
Male
Female
Male
Female
Male
Female
Male
Female
13.8 14.1 13.8 14.2 14.2
8.9 9.2 9.0 8.9 9.0
– 89 172 354 711
– 58 113 223 450
– 340 669 1346 2654
– 376 745 1491 2965
– 8.0 15.5 31.9 64.0
– 5.2 10.2 20.1 40.5
Table 2 Hematological data for male F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63
1.25
2.5
5
RBC (·104/lL) Hb (g/dL) Ht (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (·104/lL) WBC (·102/lL)
884 ± 64a 14.8 ± 1.1 47.3 ± 3.4 53.5 ± 0.4 16.8 ± 0.4 31.3 ± 0.6 69.5 ± 7.2 43.8 ± 9.0
860 ± 44 14.4 ± 0.9 46.0 ± 2.5 53.4 ± 0.6 16.8 ± 0.3 31.4 ± 0.7 68.6 ± 6.4 40.6 ± 5.1
887 ± 51 14.8 ± 0.7 47.7 ± 2.9 53.8 ± 0.4 16.7 ± 0.5 31.1 ± 0.5 69.2 ± 4.0 44.1 ± 9.0
860 ± 60 14.3 ± 1.0 45.8 ± 3.1 53.3 ± 0.5 16.6 ± 0.4 31.2 ± 0.7 67.2 ± 6.5 41.4 ± 8.5
871 ± 52 14.5 ± 1.0 46.6 ± 2.9 53.5 ± 0.4 16.7 ± 0.4 31.2 ± 0.9 70.5 ± 3.7 40.6 ± 7.4
Differential cell counts (%) Bandb Seg Eosino Baso Lymph Mono
0.0 ± 0.0 15.4 ± 4.6 1.0 ± 0.7 0.0 ± 0.0 83.0 ± 4.7 0.5 ± 0.5
0.0 ± 0.0 14.0 ± 3.9 0.9 ± 0.8 0.0 ± 0.0 84.3 ± 4.2 0.8 ± 0.8
0.0 ± 0.0 13.4 ± 3.2 0.6 ± 0.5 0.0 ± 0.0 85.6 ± 3.9 0.5 ± 0.7
0.0 ± 0.0 17.2 ± 5.4 1.0 ± 0.9 0.0 ± 0.0 81.2 ± 5.0 0.7 ± 0.5
0.0 ± 0.0 17.6 ± 4.5 0.6 ± 0.4 0.0 ± 0.0 81.3 ± 4.3 0.6 ± 0.7
a
Mean ± S.D. Band, band form leukocytes; seg, segmented leukocytes; eosino, eosinophilic leukocytes; baso, basophilic leukocytes; lymph, lymphocytes; mono, monocytes. b
Table 3 Hematological data for female F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63
1.25
2.5
5
RBC (·10 /lL) Hb (g/dL) Ht (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (·104/lL) WBC (·102/lL)
a
799 ± 43 14.2 ± 1.1 44.5 ± 2.5 55.7 ± 0.4 17.7 ± 0.5 31.8 ± 0.9 70.5 ± 4.0 27.6 ± 5.3
813 ± 25 14.4 ± 0.6 45.3 ± 1.5 55.7 ± 0.3 17.7 ± 0.3 31.7 ± 0.6 73.6 ± 3.7 28.6 ± 3.6
830 ± 41 14.8 ± 0.8 46.4 ± 2.4 55.9 ± 0.3 17.9 ± 0.4 32.0 ± 0.6 71.3 ± 4.4 29.6 ± 12.3
814 ± 56 14.6 ± 1.2 45.4 ± 3.2 55.7 ± 0.4 17.9 ± 0.5 32.2 ± 0.9 74.2 ± 4.3 29.4 ± 7.0
791 ± 38 14.2 ± 0.9 44.5 ± 2.0 56.2 ± 0.3* 17.9 ± 0.5 31.9 ± 0.9 71.6 ± 4.2 28.7 ± 5.5
Differential cell counts (%) Bandb Seg Eosino Baso Lymph Mono
0.0 ± 0.0 14.6 ± 4.5 1.0 ± 0.6 0.0 ± 0.0 83.1 ± 4.5 1.3 ± 0.7
0.0 ± 0.0 14.9 ± 5.1 1.1 ± 0.7 0.0 ± 0.0 82.7 ± 5.0 1.3 ± 1.3
0.0 ± 0.0 13.8 ± 3.6 0.9 ± 0.6 0.0 ± 0.0 83.8 ± 3.6 1.6 ± 0.7
0.0 ± 0.0 12.0 ± 5.6 1.2 ± 1.3 0.0 ± 0.0 85.0 ± 5.4 1.7 ± 0.8
0.0 ± 0.0 12.4 ± 3.6 1.3 ± 0.8 0.0 ± 0.0 85.0 ± 3.6 1.4 ± 0.9
4
a
Mean ± S.D. Band, band form leukocytes; seg, segmented leukocytes; eosino, eosinophilic leukocytes; baso, basophilic leukocytes; lymph, lymphocytes; mono, monocytes. * Significantly different from the control group at p < 0.05. b
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Table 4 Serum biochemical data for male F344 rats given Agaricus blazei Murrill extract for 90 days Item
TP (g/dL) A/G AlB (g/dL) T. Bil (mg/dL) T. Cho (mg/dL) BUN (mg/dL) CRN (mg/dL) Ca (mg/dL) P (mg/dL) Na (mEq/dL) Cl (mEq/dL) K (mEq/dL) AsT (IU/L) AlT (IU/L) AlP (IU/L) c-GT (IU/L) a
;
Dose level (%) 0
0.63
1.25
2.5
5
6.3 ± 0.1a 2.1 ± 0.1 4.3 ± 0.1 0±0 59.5 ± 4.3 19.7 ± 1.6 0.32 ± 0.02 10.5 ± 0.3 6.4 ± 0.4 144 ± 1 100 ± 1 4.5 ± 0.3 83.4 ± 11.0 51.1 ± 4.3 327 ± 17 <2
6.4 ± 0.1 2.2 ± 0.1 4.3 ± 0.1 0±0 65.0 ± 3.3** 20.7 ± 1.8 0.30 ± 0.02* 10.5 ± 0.1 6.1 ± 0.4 145 ± 1 101 ± 1 4.6 ± 0.4 88.7 ± 13.6 52.3 ± 4.8 318 ± 9 <2
6.4 ± 0.2 2.2 ± 0.1 4.4 ± 0.1* 0±0 66.1 ± 5.4** 21.1 ± 1.7 0.30 ± 0.02* 10.6 ± 0.4 6.3 ± 1.1 145 ± 2 102 ± 2* 4.7 ± 0.5 87.1 ± 13.0 50.7 ± 5.7 340 ± 25 <2
6.3 ± 0.2 2.2 ± 0.1 4.3 ± 0.1 0±0 62.6 ± 3.4 21.2 ± 1.1* 0.30 ± 0.01* 10.6 ± 0.2 6.4 ± 0.5 144 ± 1 102 ± 1** 4.7 ± 0.2 92.7 ± 26.5 55.5 ± 11.2 322 ± 23 <2
6.3 ± 0.2 2.1 ± 0.1 4.3 ± 0.1 0±0 61.4 ± 6.0 21.7 ± 2.0* 0.30 ± 0.02* 10.5 ± 0.2 6.5 ± 0.5 144 ± 1 101 ± 1 4.7 ± 0.2 85.7 ± 11.1 50.0 ± 2.8 335 ± 28 <2
Mean ± S.D. : Significantly different from the control group at p < 0.05, p < 0.01, respectively.
Table 5 Serum biochemical data for female F344 rats given Agaricus blazei Murrill extract for 90 days Item
TP (g/dL) A/G AlB (g/dL) T. Bil (mg/dL) T. Cho (mg/dL) BUN (mg/dL) CRN (mg/dL) Ca (mg/dL) P (mg/dL) Na (mEq/dL) Cl (mEq/dL) K (mEq/dL) AsT (IU/L) AlT (IU/L) AIP (IU/L) c-GT (IU/L) a *
Dose level (%) 0
0.63
1.25
2.5
5
6.3 ± 0.2a 2.3 ± 0.2 4.4 ± 0.2 0.02 ± 0.04 84.3 ± 9.1 17.2 ± 1.4 0.34 ± 0.03 10.3 ± 0.4 5.7 ± 0.7 145 ± 2 105 ± 1 4.2 ± 0.6 84.0 ± 8.1 38.8 ± 3.5 229 ± 21 <2
6.2 ± 0.2 2.4 ± 0.1 4.4 ± 0.1 0.02 ± 0.04 86.0 ± 6.4 17.7 ± 1.1 0.33 ± 0.03 10.1 ± 0.3 5.5 ± 0.5 145 ± 1 105 ± 2 4.1 ± 0.4 88.8 ± 7.6 39.4 ± 3.1 243 ± 21 <2
6.3 ± 0.2 2.5 ± 0.2 4.5 ± 0.2 0.03 ± 0.05 84.2 ± 7.7 18.8 ± 1.2* 0.34 ± 0.03 10.4 ± 0.4 6.1 ± 0.8 145 ± 1 104 ± 2 4.4 ± 0.6 89.0 ± 7.5 39.9 ± 3.1 233 ± 27 <2
6.3 ± 0.2 2.4 ± 0.1 4.5 ± 0.2 0.02 ± 0.04 83.4 ± 7.1 17.4 ± 1.3 0.34 ± 0.03 10.5 ± 0.5 6.1 ± 1.2 145 ± 2 105 ± 2 4.6 ± 1.1 83.9 ± 7.0 39.1 ± 2.4 220 ± 27 <2
6.1 ± 0.2 2.4 ± 0.2 4.3 ± 0.2 0.02 ± 0.04 85.9 ± 5.9 17.6 ± 1.0 0.33 ± 0.03 10.1 ± 0.4 5.4 ± 0.7 145 ± 1 106 ± 2 4.0 ± 0.3 85.7 ± 10.7 42.7 ± 9.2 229 ± 27 <2
Mean ± S.D. Significantly different from the control group at p < 0.05.
incidental given the lack of change in the 5% group. While differences were also observed in several parameters such as AlB, T. Cho and Cl in males, but no dosedependence was observed and the degree of change was slight. We therefore conclude that there were no major treatment-related changes in serum biochemistry. Regarding final body weights and absolute organ weights, no significant differences were observed. The significant increase in the relative liver weight in males of the 1.25 and 2.5% groups and the relative brain weight in females of the 2.5% group were judged to be incidental since such changes were not observed in the 5% group and no dose-dependence was evident.
Histopathologically, several lesions were sporadically observed in the liver, kidney and cardiac muscle in males and/or females. Since the degree and incidences of these lesions were almost the same in both control and 5% groups, and they are well known to occur spontaneously in F344 rats (Boorman et al., 1990), it is reasonable to conclude they were of little toxicological significance. It has been reported that treatment with a crude aqueous extract of A. blazei Murrill exerts a protective effect against liver toxicity as well as initiation of hepatocarcinogenesis by diethylnitrosamine (DEN) (Barbisan et al., 2002). Additionally, feeding of A. blazei Murrill powder exerted a chemopreventive influence against
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Table 6 Organ weight data for male F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63 313 ± 16a
Body weight (g) Absolute (g) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys Testes
1.95 ± 0.04 0.20 ± 0.03 0.98 ± 0.08 0.90 ± 0.05 0.62 ± 0.04 7.13 ± 0.51 0.036 ± 0.004 1.87 ± 0.10 3.07 ± 0.28
Relative (g/100 g b.w.) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys Testes
0.62 ± 0.03 0.06 ± 0.01 0.31 ± 0.02 0.29 ± 0.01 0.20 ± 0.01 2.27 ± 0.09 0.011 ± 0.001 0.60 ± 0.03 0.98 ± 0.08
a *
310 ± 9
1.25
2.5
5
305 ± 12
315 ± 13
314 ± 10
1.95 ± 0.04 0.19 ± 0.04 0.96 ± 0.04 0.88 ± 0.05 0.61 ± 0.02 7.14 ± 0.31 0.037 ± 0.005 1.82 ± 0.11 3.09 ± 0.10
1.95 ± 0.05 0.21 ± 0.04 0.97 ± 0.05 0.86 ± 0.05 0.60 ± 0.03 7.27 ± 0.44 0.037 ± 0.05 1.85 ± 0.11 3.10 ± 0.13
1.94 ± 0.07 0.21 ± 0.05 0.96 ± 0.05 0.90 ± 0.04 0.63 ± 0.04 7.47 ± 0.04 0.037 ± 0.004 1.87 ± 0.10 2.93 ± 0.50
1.95 ± 0.03 0.18 ± 0.03 0.96 ± 0.05 0.89 ± 0.02 0.62 ± 0.03 7.35 ± 0.39 0.037 ± 0.005 1.91 ± 0.08 3.15 ± 0.09
0.63 ± 0.01 0.06 ± 0.01 0.31 ± 0.01 0.28 ± 0.01 0.20 ± 0.01 2.30 ± 0.07 0.012 ± 0.002 0.59 ± 0.02 0.97 ± 0.03
0.63 ± 0.02 0.07 ± 0.01 0.32 ± 0.01 0.28 ± 0.01 0.20 ± 0.01 2.38 ± 0.12* 0.012 ± 0.01 0.61 ± 0.03 1.02 ± 0.04
0.62 ± 0.003 0.07 ± 0.01 0.30 ± 0.01 0.29 ± 0.01 0.20 ± 0.01 2.37 ± 0.09* 0.012 ± 0.02 0.59 ± 0.02 0.93 ± 0.17
0.62 ± 0.02 0.06 ± 0.01 0.31 ± 0.02 0.28 ± 0.01 0.20 ± 0.01 2.34 ± 0.09 0.012 ± 0.01 0.61 ± 0.03 1.00 ± 0.03
2.5
5
Mean ± S.D. Significantly different from the control group at p < 0.05.
Table 7 Organ weight data for female F344 rats given Agaricus blazei Murrill extract for 90 days Item
Dose level (%) 0
0.63 a
Body weight (g) Absolute (g) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys
1.77 ± 0.05 0.15 ± 0.01 0.69 ± 0.04 0.55 ± 0.02 0.40 ± 0.03 3.69 ± 0.32 0.040 ± 0.004 1.03 ± 0.06
1.80 ± 0.05 0.18 ± 0.03 0.68 ± 0.04 0.55 ± 0.02 0.41 ± 0.02 3.61 ± 0.25 0.037 ± 0.006 1.06 ± 0.05
1.77 ± 0.04 0.16 ± 0.03 0.68 ± 0.08 0.53 ± 0.03 0.41 ± 0.02 3.69 ± 0.32 0.037 ± 0.005 1.06 ± 0.06
1.78 ± 0.04 0.17 ± 0.03 0.69 ± 0.05 0.54 ± 0.03 0.40 ± 0.03 3.62 ± 0.28 0.038 ± 0.006 1.03 ± 0.05
1.78 ± 0.03 0.17 ± 0.03 0.68 ± 0.06 0.54 ± 0.04 0.41 ± 0.04 3.57 ± 0.29 0.042 ± 0.007 1.05 ± 0.07
Relative (g/100 g b.w.) Brain Thymus Lungs Heart Spleen Liver Adrenals Kidneys
1.06 ± 0.03 0.09 ± 0.01 0.42 ± 0.02 0.33 ± 0.01 0.24 ± 0.01 2.21 ± 0.17 0.024 ± 0.002 0.62 ± 0.02
1.07 ± 0.04 0.11 ± 0.02 0.40 ± 0.03 0.33 ± 0.01 0.24 ± 0.01 2.14 ± 0.08 0.022 ± 0.004 0.63 ± 0.02
1.06 ± 0.05 0.10 ± 0.02 0.41 ± 0.04 0.32 ± 0.02 0.25 ± 0.02 2.21 ± 0.13 0.023 ± 0.02 0.64 ± 0.04
1.10 ± 0.04* 0.10 ± 0.02 0.42 ± 0.02 0.33 ± 0.01 0.25 ± 0.02 2.22 ± 0.14 0.024 ± 0.02 0.63 ± 0.02
1.07 ± 0.05 0.10 ± 0.02 0.41 ± 0.02 0.32 ± 0.02 0.24 ± 0.02 2.13 ± 0.09 0.025 ± 0.05 0.63 ± 0.02
a *
167 ± 7
1.25
169 ± 9
167 ± 9
163 ± 6
167 ± 10
Mean ± S.D. Significantly different from the control group at p < 0.05.
DEN in a medium-term rat liver carcinogenesis assay (Pinheiro et al., 2003). On the other hand, in the same model using crude aqueous extract, no protection was observed (Barbisan et al., 2003a). It has also been reported that the number of pre-neoplastic glutathione S-transferase placental form (GST-P) positive foci was
significantly increased in rats treated with a crude aqueous extract from A. blazei Murrill before initiation (Barbisan et al., 2003b). The ingestion amount of extract in this last report, about 2 g/kg/day, was lower than that in the highest dosage group in the present study. However, we here observed no evidence of liver toxicity in
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the serum chemistry, organ weight and histopathological findings. It is thought that ingredients of A. blazei Murrill extract may differ with various conditions such as the strain, period of mushroom harvest, type of cultivation and extraction method. Since the A. blazei Murrill extracts used have differed in each experiment in the literature, the results cannot be simply compared. In conclusion, in the present 90-day subchronic study, an A. blazei Murrill aqueous extract used as a food additive showed little or no significant toxicity, even with 5% dietary supplementation. Thus, the noobserved-adverse-effect level (NOAEL) was concluded to be 5% in the diet (2654 mg/kg b.w./day for male rats and 2965 mg/kg b.w./day for female rats). Acknowledgements We thank Ms. Machiko Maeda for her expert technical assistance. We are also grateful to Iwade Mushroom Institute for kindly supplying A. blazei Murrill extract. This work was supported by a Grant-in-Aid for Research on Food Sanitation from the Ministry of Health, Labor and Welfare of Japan. References Barbisan, L.F., Miyamoto, M., Scolastici, C., Salvadori, D.M.F., Ribeiro, L.R., Eira, A.F., de Camargo, J.L.V., 2002. Influence of aqueous extract of Agaricus blazei on rat liver toxicity induced by different doses of diethylnitrosamin. Journal of Ethnopharmacology 83, 25–32. Barbisan, L.F., Spinardi-Barbisan, A.L, Moreira, E.L., Salvadori, D.M., Ribeiro, L.R., da Eira, A.F., de Camargo, J.L., 2003a. Agaricus blazei (Himematsutake) does not alter the development of rat diethylnitrosamine-initiated hepatic preneoplastic foci. Cancer Science 94, 188–192. Barbisan, L.F., Scolastici, C., Miyamoto, M., Salvadori, D.M.F., Ribeiro, L.R., da Eira, A.F., de Camargo, J.L.V., 2003b. Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine. Genetics and Molecular Research 2, 295–308. Boorman, G.A., Eustis, S.L., Elwell, M.R., Montgomery, C.A., Jr., Mackenzie, W.F. (Eds.), 1990. Pathology of the Fischer Rat, References and Atlas. Academic Press, San Diego. Furukawa, F., Kasahara, K., Nishikawa, A., Imazawa, T., Hirose, M., 1998. A 13-week subchronic oral toxicity study of chlorophyll in
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F344 rats. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 116, 107–112 (in Japanese). Guterrez, Z.R., Mantovani, M.S., Eira, A.F., Ribeiro, L.R., Jordao, B.Q., 2004. Variation of the antimutagenicity effects of water extracts of Agaricus blazei Murrill in vitro. Toxicology In Vitro 18, 301–309. JETRO (Japan External Trade Organization), 2004. Specifications and Standards for Foods, Food Additives, etc. Under The Food Sanitation Law. Available from: