Abstract
This study was conducted to evaluate the hepatoprotective effect of Agaricus blazei Murrill extract (ABM) against experimentally induced carbon tetrachloride (CCl4) toxicity in male BALB/c mice. The experiments included a normal group (no induction by CCl4), CCl4-induction group (with hepatotoxicity by CCl4 and without treatment) and experimental groups with low dose (200 mg) or high dose (2,000 mg) of ABM extract (per kilogram mouse weight). All groups other than the normal group were treated with intraperitoneal injections of CCl4 twice a week. Mice were tube-fed with experimental ABM extracts or double-distilled water, accordingly, on the remaining four days each week. The whole experimental protocol lasted 8 weeks; blood and liver samples were collected for biochemical and tissue histochemical analysis. Only administration of a high dose of ABM to treatment groups resulted in a significant abrogation of CCL4-induced increase of serum aspartate aminotransferase (AST) and alanine transaminase (ALT). Post-treatment with ABM also did not significantly reverse the alterations of glutathione peroxidase (GSHPx) and catalase. Both high- and low-dose ABM treatment reduced hepatic necrosis and fibrosis caused by CCl4 in comparison with the CCl4 control group in the histochemical analyses. Our results suggest that the ABM extract affects the levels of ALT and AST in mice.
Mushrooms, primarily basidiomycetous fungi, are a popular and valuable food, low in calories and high in minerals, essential amino acids, vitamins and fiber (1); some produce substances having potential medical effects, and are called medicinal mushrooms. Among the mushroom species of Basidiomycetes, Agaricus blazei Murrill (ABM), a species native to Brazil, where it is generally known as ‘sun mushroom’, has received attention in folk medicine due to its use in the remedies of ailments (2). This esculent mushroom is often consumed as food and tea in different parts of the world, especially on account of its reported medicinal properties (3). It has traditionally been used for the prevention of a range of diseases, including cancer, hepatitis, atherosclerosis, hypercholesterolemia, diabetes and dermatitis (4). Because of its alleged health effects, the mushroom was brought to Japan in the mid 1960s and subjected to biomedical research. In Japan, researchers demonstrated immunostimulation and anticancer effects of ABM extracts experimentally (5), and due to the increased consumption of this mushroom in recent years, considerable effort has been made to investigate these putative effects, with interesting, but still insufficient, clinical studies. Experimental studies increased commercial interest for ABM, especially in Japan, stimulating not only the production, but also the registration of brands with new popular names. This makes it difficult for the public to identify pure ABM strains. ABM was found to be rich in immune-modulating substances, such as β-glucans (6, 7) and proteoglycans (8), and it had anti-infection (9, 10) and antitumor (7, 8) effects in mice. To our knowledge, there are only few published data documenting the antioxidative (11) and hepatoprotective (12) effects of ABM. The purpose of this study was to examine and compare the biological activities of an ABM extract in rats and mice with chronic CCl4-induced liver injury, to evaluate the therapeutic effects of ABM, to probe for potential mechanisms of these effects and to determine whether therapeutic effect occurred in a dose-dependent manner or not.
Materials and Methods
Reagents. Carbon tetrachloride (CCl4), olive oil and other reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Diagnostic kits for assaying alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were purchased from Arkray, Inc. (Kyoto, Japan). Other assay kits were obtained from Cayman Chemical Company (Ann Arbor, MI, USA).
Preparation of ABM extract. Powdered ABM was obtained from S. Canaan Biotechnology Development Co. (Taipei, Taiwan, ROC) and 22.5, 90 or 900 mg were separately suspended in 6 ml distilled water at 60°C for 10 min, then cooled to room temperature.
Animals and experimental protocols. Specific pathogen-free male BALB/c mice of similar age (6 weeks) and weight (20-25 g) were obtained from and housed at the Animal Medicine Center, College of Medicine, National Taiwan University animal holding facility, with 12 h light cycle at 20±2°C and humidity of 75±15%, in a filtered laminar air flow-controlled room for acclimatization. The experimental animals were individually earmarked, housed 5 animals per cage, and provided food and water ad libitum. These mice were used for the chronic CCl4-induced liver injury model in all experiments. All animal procedures had been reviewed and approved by the law and regulations for animal experiments of National Science Council of the Republic of China, which are in agreement with the Helsinki declaration, and they were approved by the local Animal Board under the Minister of Agriculture in Taiwan.
In vivo studies for ABM extract as liver injury therapy. Animals were fasted overnight prior to dose administration and randomly assigned to each of the four experimental groups. Mice in group A (n=10) were administered double-distilled water; mice in group B mice (n=10) were administered 40% CCl4/olive oil (1 ml/kg body weight per day, i.p. twice per week) for 8 weeks to induce chronic chemical liver injury as a negative control. Group C was treated with 40% CCl4/olive oil (1 ml/kg body weight per day, i.p. twice per week) and was further subdivided into two different dose-groups of ABM extract treatment (200 mg/kg or 2000 mg/kg, p.o., 4 days per week) for 8 weeks (n=10 for each dose group). Via orbital bleeding at the end of treatment, blood samples were collected (0.2 ml with 10 U/ml heparin), allowed to clot, centrifuged (1500 rpm, 10 min, room temperature) and the serum then stored at −20°C until analysis. At the end of the experiments at week 8, livers were excised immediately after the animals were sacrificed under anesthesia by CO2. Livers were weighed and utilized for the following biochemical and histology assessments described below.
Serum biomarker activity analysis. Serum samples were prepared at the end of the eighth week and analyzed using ALT and AST diagnostic kits, following the manufacturer's protocols. Biochemical analysis of serum ALT and AST activities was performed using standard assay kits (Boehringer Mannheim Corp., Germany). The concentration in samples was measured using a Hitachi 717 (Tokyo, Japan) biochemical instrument (13).
Glutathione peroxidase (GSHPx) assay. Livers of mice were homogenized with cold GSHPx buffer (50 mM Tris-HCl containing 5 mM EDTA and 1 mM dithiothreitol (DTT), pH 7.5). GSHPx activity was measured by a GSHPx assay kit (14). The reaction was initiated mixing glutathione, glutathione reductase, NADPH and the conversion of NADPH to NADP was assessed with a spectrophotometer at 340 nm for 5 min. The specific activity was measured as nanomoles of NADPH oxidized to NADP per minute per milligram protein as described elsewhere (14, 15).
Catalase assays. Liver tissues of mice were homogenized in cold buffer (50 mM potassium phosphate and 1 mM EDTA, pH 7.0). The supernatant was collected after centrifugation and hydrogen peroxide added as exogenous substrate. The activity of catalase was measured using a catalase assay kit (Cayman Chemical Company) as reported previously (16). Changes in the absorbance at 540 nm were monitored according to the manufacturer's protocol.
Histopathological assessment of tissues. Processing of tissue samples for histological assessment followed established procedures. In brief, the tissue samples were rinsed with 0.9% saline solution and fixed in 10% formalin. Then diagonal sections of the liver were obtained and processed (Leica TP1020, Japan) as follows in: (i) 10% neutral buffered formalin for 1 h, twice; (ii) 70% alcohol for 1.5 h; (iii) 80% alcohol for 1.5 h; (iv) 90% alcohol for 1.5 h; (v) absolute alcohol for 1.5 h, twice; (vi) xylene for 1.5 h, twice; (vii) molten wax at 65°C for 2.5 h, two changes. The processed tissues were embedded in paraffin and sectioned at 4 μm thickness, placed on frosted glass slides and dried on a hot plate 70°C for 30 min. The tissues were stained using hematoxylin and eosin (H&E) stain. The sections were dewaxed in two changes of xylene (3 min each), hydrated in two changes of 100% ethanol, followed by 90% ethanol and 70% ethanol, for 3 min each, rinsed with water (3 min) and stained. The stained tissues were dehydrated with 70% ethanol followed by 90% ethanol, placed in two changes of 100% ethanol for 3 min each and cleaned with two changes of xylene (3 min each). Liver pathology was rated on four levels based on Ruwart et al. (17): None (I), no detectable pathological alternation; focal (II), focal and local alteration; multifocal (III), multiple focal alterations; diffuse (IV) represents broad diffuse alteration. Stage assessment of hepatic fibrosis was previously described (18, 19): I, fibrosis only occurring in the portal area; II, portal area fibrosis extends to the hepatic lobule but focal fibrosis does not link these; III, the portal fibrosis extends into the hepatic lobule, a clear pseudo-lobule is formed and bile duct hyperplasia occurs.
Statistical analysis. The experimental results are expressed as the means±SEM and are accompanied by the number of observations. Data were assessed by analysis of variance (ANOVA) and Student's t-test. If the analysis indicated significant differences among the group means, then each group was compared by the Newman-Keuls method.
Results
The changes in toxicity associated with the administration of ABM were assessed at the biochemical, histopathological and antioxidant enzyme activity levels. The serum concentrations of the biochemical markers ALT and AST were obtained to evaluate the liver function. In addition, the histopathological changes in the target organ were semi-quantitatively graded.
Liver function biomarkers. Blood was collected for AST and ALT determination after CCl4 administration to mice, at the end of the eighth week. Both serum AST and ALT levels were markedly increased (Table I) after CCl4 induction as compared to the normal group (double-distilled water treatment) (p<0.05). The mean serum AST level in mice of CCl4 treatment groups was 1137±664 IU/l but it was near 2-fold less (591±234 IU/l) in mice treated with 2000 mg/kg ABM (high dose). AST levels in mice treated with low-dose ABM were not significantly affected by treatment (p>0.05). Similarly, the serum ALT concentrations were measured (Table I). In the normal control group, the ALT serum concentration was substantially lower than that in the CCl4 treatment groups (p<0.05). Increasing dose of ABM caused a decline of serum ALT concentration (low dose: 764±548 IU/l and high dose: 454±137 IU/l). In comparison with the normal control, high or low dose treatment was not able to restore AST and ALT to normal levels.
Assessment of antioxidant enzyme activities. To investigate the role of antioxidant enzyme activities in the liver during CCl4 injury, we analyzed catalase and GPx. CCl4 toxicity did not result in lower GPx and catalase activities. Contrary to expectation, neither low nor high doses of ABM significantly altered catalase and GPx activity (Table I).
Histopathological assessment. Liver tissue sections of mice were stained with H&E (Figure 1). The histopathological assessment in liver was performed for all groups. Mice in the negative control group exhibited normal, well-defined histological structures, without any signs of vascular or inflammatory changes; no cavitations, necrosis or fibrosis were found in normal control sections. The histopathological analysis of the liver revealed signs of toxicity after administration of CCl4. This toxicity was significant in comparison with the negative group and included cavitations, fibrosis in broad areas, mild vascular congestion and moderate inflammatory changes with congested sinusoids, nuclear changes, and centrilobular necrosis (Figure 1B). The broad cavitations and fibrosis in livers were somewhat attenuated in mice treated with low or high doses of ABM during the experimental periods. ABM administration for mice did result in fewer cavitations and less fibrosis in the liver (Figure 1C to 1D). ABM treatment also elevated the survival rate of mice after liver injury induced by CCl4. We also examined the distribution of cavity and fibrosis in different liver regions (from central vein region to hepatic portal veins) for mice, as shown by Table II. ABM reduced apparent liver injury caused by CCl4 for mice by histopathological assessment in a dose-dependent manner.
Discussion
The histopathological changes in the liver together with markers of hepatic function biochemical, were evaluated after of induction CCl4 injury and ABM treatment. In the current study, a significant rise in serum AST and ALT levels was observed after administration of CCl4 in comparison with the negative control group. This indicates that the experiment was successful at inducing liver injury. This is a consistent finding with previous studies where increases of serum AST and ALT concentrations after administration of CCl4 in rats were demonstrated (18). In addition, the histopathological analysis also showed vascular and inflammatory changes, such as centrilobular necrosis, congested sinusoids and nuclear changes, which were similar to a previous study in rats (18). In contrast, after a high dose of ABM was administered, the serum AST and ALT levels were only slightly elevated and mild liver damage with slight vascular congestion and signs of hepatocyte regeneration were found. It should be noted that low-dose treatment with ABM cannot restore the serum AST and ALT levels to normal but can normalize the histopathological analysis. Comparison of the data of transaminases and histopathological assessment is very difficult and should be cautiously understated on account of the significant disagreement of administration results. If results are judged only by transaminases, it could be concluded that a low dose of ABM is not able to abrogate chemical liver injury in mice. However, this conclusion is not in agreement with the histopathological assessment. Analysis of antioxidant enzymes showed that ABM extracts had no significant effect on the activities of GSHPx and catalase under CCl4 treatment. These results indicate that the recovery/reparative effects of ABM extracts shown by the histopathological results do not take place through an antioxidant mechanism. Removal of free radicals is not likely to contribute to the effects of ABM.
Of particular importance, we suggested that a rat model is superior to a mouse model because cavitations and fibrosis in broad areas are more severe in liver sections from the corresponding rat model of CCl4-induced liver injury (18). Sections from CCl4-only treated mice did not display such apparent cavitations and fibrosis in broad areas compared with those of the rat model. We can conclude that the rat model can substitute for a mouse model to evaluate treatment effect of ABM (18).
We have shown that ABM is able to abrogate chemical liver injury in mice and rats. There were no consistent treatment-related changes in clinical signs, body weight and food consumption at a dose of 2,654 mg/kg b.w./day ABM for male and 2,965 mg/kg b.w./day ABM for female rats in a study to evaluate 90-day subchronic toxicity of an aqueous extract of ABM in F344 rats (20). Although toxicity and carcinogenicity of ABM has not been established for humans, a main problem for the administration of ABM remains the problem of the presence of aromatic hydrazines (i.e. agaritine and its derivatives) whose carcinogenicity and chronic systemic effects are well known in animals (21) and are probably due to metabolized toxic intermediates capable of damaging cellular macromolecules and stimulating proteolysis, giving rise to hydrazine-mediated DNA strand breaks (22). Both these studies demonstrated that the administration of hydrazine analogs s.c. to Swiss mice induced fibrosarcoma in 24% of males, and soft tissue tumors in both sexes (23); while in another article, agaritine administered in drinking water at 0.062% and 0.031% did not give rise to cancer, although a substantial number of animals (Swiss mice) developed convulsive seizures (24). Hydrazine analogs (from A. bisporus and Gyromitra esculenta) administered in drinking water to Swiss mice and Syrian hamsters, in contrast, did give rise to liver neoplasms (benign hepatomas, liver cell carcinomas, angiomas, angiosarcomas) and adenomas and adenocarcinomas of lungs (25).
Medicinal agents derived from plants have been used for health maintenance and disease management since the dawn of time. A main safety concern is represented by the toxicity and carcinogenicity of ABM and its derivatives that should be completely evaluated. In addition, it would probably be useful for these mushrooms, like other herbal remedies, to completely define the problem of heavy metal content. Due to the large consumption of ABM in popular remedies, more data are needed on the mechanisms of action of its components and safety before contemplating the use of ABM for prevention and treatment of cancer and immunodepressive disorders.
A high dose of ABM extract was found to reduce the activities of serum ALT and AST, which were elevated by CCl4 in mice, as found previously for rats. This conclusion is in agreement with the histopathological assessment. This suggests that ABM extract plays a role in ALT/AST release and may improve the plasma transaminase activities and histopathology in liver injury of mice.
Acknowledgements
This study was supported by a research grant from China Medical University, Taichung. Taiwan and supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004).
Footnotes
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↵* These Authors contributed equally to this study.
- Received May 4, 2011.
- Revision received June 20, 2011.
- Accepted June 27, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved