Introduction

Ganoderma lucidum (Fr.) Karst. (Polyporaceae) is a medicinal mushroom known to the Japanese as ‘Reishi’ or ‘Mannentake,’ or to the Chinese as ‘Lingzhi.’ Its fruiting bodies have been used for their medicinal properties in traditional Chinese medicine for more than 2,000 years. The use of this mushroom for promotion of vitality and as an anti-aging agent was described in detail in the classic compendium of traditional Chinese medicine Shen Nung Ben Cao Jin (dated 206 BC–8 AD), and G. lucidum has been used more recently in China and other oriental countries for the treatment of debility and weakness, hypertension, cardiovascular disease, bronchitis, arthritis, neurasthenia, insomnia, hepatopathy, chronic hepatitis, nephritis, gastric ulcer, asthma, diabetes, altitude sickness, acquired immunodeficiency syndrome (AIDS), and cancer [1–3]. Of particular interest among the reported biological/pharmacological properties of G. lucidum are its antitumor activities, including cell-cycle arrest, induction of apoptosis, inhibition of motility, anti-angiogenesis, and anti-mutagenesis [3–9]. The fruiting bodies and mycelium of the mushroom have very similar composition, but as the mycelium contains several additional nutrients and components it is considered the “essence” of the mushroom organism responsible for its beneficial functions. The use of Ganoderma mycelia as a “designer food” means that culture techniques for this organism have been well established.

A water-soluble extract from the culture medium of G. lucidum mycelia (designated as MAK) after fermentation on a solid medium containing bagasse contains various substances such as polysaccharides, proteins, water-soluble lignin, and triterpenes. Previously, we have reported that MAK exerts preventive effects against the development of chemical carcinogen-induced aberrant crypt foci (ACF), colon adenomas, colon adenocarcinomas, and pulmonary adenocarcinomas in rats [10–13], and against X-ray irradiation-induced small-intestinal injury in mice [14]. Chemotherapy is highly cytotoxic, causing a number of severe adverse effects such as nausea and vomiting. Recently, we reported that MAK was able to protect against small-intestinal injury after chemotherapy [15]. Many previous studies using animals have investigated the effects of various agents against implanted tumors, such as sarcoma 180 [16–18]. So far, however, no studies employing tumors induced by chemical agents have been reported.

The present study was conducted to assess the effects of MAK on ACF and small-intestinal injury induced in rats by azoxymethane after administration of the anticancer agent fluorouracil (5-FU).

Materials and methods

Animals

Fifty-one male F344/DuCrj rats, 6 weeks of age at study commencement, were purchased from Charles River Japan (Hino, Japan). They were housed 5 to a polycarbonate cage and kept under constant conditions of temperature (24° ± 2 °C) and relative humidity (55 ± 10 %) with a 12 h light:12 h dark cycle. The animals were maintained in accordance with the “Guidelines for the Care and Use of Laboratory Animals” and fed a commercial diet (MF; Oriental Yeast, Tokyo, Japan), either alone or supplemented with 2.5 % (w/w) MAK. Tap water was also provided ad libitum.

MAK

MAK was prepared by Noda Shokkin-Kogyo (Chiba, Japan). In brief, G. lucidum mycelia were cultured in a solid medium composed mainly of sugar cane bagasse for 3 months, and then the entire medium containing the mycelia was extracted with hot water. The extract was filtered and spray-dried to obtain MAK.

Azoxymethane

Azoxymethane (AOM) was purchased from Nard Institute (Hyogo, Japan). Starting at 7 weeks of age, the rats were given a subcutaneous injection of AOM (15 mg/kg body wt) weekly for 3 weeks to induce ACF.

5-FU

5-FU was obtained from Kyowa Hakko (Tokyo, Japan) (5-FU Injection 250 Kyowa). Starting 1 day after the final AOM treatment, 25 or 80 mg/kg 5-FU was injected intraperitoneally three times at 5-day intervals.

Autopsy

Rats were killed 3.5 days after the last injection of 5-FU (Fig. 1). Immediately after death, segments of the jejunum from the ileal junction were removed and fixed in Carnoy’s solution. The segments were cut into several pieces, bundled together, embedded in paraffin, sectioned at a thickness of 3 μm, and stained with hematoxylin and eosin. To quantify regenerating crypts, the number of crypts per circumference was determined in cross-sections. In each rat, the number of regenerative crypts in ten gut cross-sections was scored. Immunohistochemistry was performed using an anti-proliferating cell nuclear antigen (PCNA) antibody (Dako, Kyoto, Japan) by the avidin–biotin complex method. Tissue sections were deparaffinized with xylene, rehydrated through a graded ethanol series, and incubated with 0.3 % hydrogen peroxide for 30 min to block endogenous peroxidase activity. They were then incubated with 10 % normal horse serum at room temperature for 30 min to block background staining, and then reacted with the anti-PCNA antibody.

Fig. 1
figure 1

Experimental schedule. d days, MAK water-soluble extract from culture medium of Ganoderma lucidum mycelia, AOM azoxymethane, 5-FU fluorouracil

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Each colon was removed, flushed with saline, slit open longitudinally from the cecum to the anus, placed on a paper towel, and fixed in 10 % buffered formalin for 24 h. Following the protocol reported by Magnuson et al. [19], the fixed colons were stained with 0.5 % methylene blue for 30 min, and then placed on a glass slide with the luminal side up. The stained colons were viewed under a microscope at a magnification of 20× to 30× and assessed for the presence of ACF. The numbers of ACF per colon, the numbers of AC (aberrant crypt) per colon, and the numbers of AC per focus were determined. Segments from the proximal to the descending colon were cut, and sections of paraffin-embedded tissue were stained with hematoxylin and eosin, Alcian blue–periodic acid–Schiff, and also for PCNA.

Cell counting

PCNA-positive cells in the small-intestinal crypts were counted in single vertically sectioned crypts from the bed of the crypt to the top, and crypt length was also measured using an image analyzer.

The PCNA positivity index and germinal region in the large intestine were measured by the method reported previously [10]. The PCNA-positive cells nearest to and furthest from the crypt bed were defined as the base and the top, respectively, and the distance between them (germinal region) was measured. The numbers of cells observed in single crypts, and from the crypt bed at the top, were counted and converted into percentage control values for half crypts. The numbers of positively stained nuclei were counted and divided by the total number of nuclei to give the PCNA index (%).

Statistical analysis

Statistical significance was determined by Dunnett’s method for multiple comparisons.

Results

Administration of AOM led to a significant decrease in body weight as compared with nontreated animals. After the first and second administrations of 80 mg 5-FU, body weight was significantly decreased as compared with animals receiving AOM, and body weight loss was especially evident in the AOM+5FU80+MAK group (Fig. 2).

Fig. 2
figure 2

Body weight

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The histological appearance of ACF is shown in Fig. 3. Many mitoses were evident in the crypts, and the mucin components revealed by Alcian blue–PAS staining differed from those in normal crypts. PCNA-positive cells were found in abnormal crypts. Table 1 summarizes the data for ACF, total numbers of AC, and numbers of AC per ACF. Numbers of ACF, total numbers of AC, and mean numbers of AC per ACF in the AOM + 5-FU and AOM + 5-FU + MAK groups were significantly decreased in comparison to rats that had received AOM alone, and the numbers of ACF and total numbers of AC in the AOM + 5-FU25 + MAK group were significantly decreased in comparison to the AOM + 5-FU25 group. In the 5-FU80 groups, the numbers of ACF and total numbers of AC were significantly decreased as compared to rats that had received AOM alone. However, the differences between the AOM + 5-FU80 and AOM + 5-FU + MAK groups were not significant.

Fig. 3
figure 3

Left: aberrant crypt foci (ACF) (arrows) in colon. Hematoxylin and eosin (H&E), ×100. Middle: Alcian blue–periodic acid–Schiff (PAS), ×100. Right: proliferating cell nuclear antigen (PCNA), ×200

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Table 1 Number of aberrant crypt foci (ACF), aberrant crypts (AC), and AC per ACF
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The PCNA positivity index and the height of the germinal region in the AOM + 5-FU groups were significantly increased as compared with the MAK groups (Fig. 4; Table 2).

Fig. 4
figure 4

Length of germinal region. Proliferating cell nuclear antigen (PCNA) staining. Left: AOM + 5-FU25 in colon, ×200. Right: AOM + 5-FU25 + MAK, ×200

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Table 2 Proliferating cell nuclear antigen (PCNA) labeling index and height of germinal region in colon crypt
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Tissue specimens from the small intestine were prepared and the numbers of regenerative crypts in them were counted (Table 3). Regenerated crypts in the AOM + 5-FU25 + MAK group (168 ± 130) tended to be increased in comparison with the AOM + 5-FU25 group (145 ± 71, p > 0.05). Histologically evident crypt damage in the AOM + 5-FU80 group was more severe than that in the AOM + 5-FU25 group, and the regenerative crypts in the AOM + 5-FU80 + MAK group were longer than those in the AOM + 5-FU group, which still retained damaged villi (Fig. 5). Crypt length and the number of PCNA-positive cells per crypt in the MAK groups (Fig. 6) were significantly increased as compared with the 5-FU groups (Table 4), and were significantly increased in the AOM + 5-FU80 + MAK group relative to the AOM + 5-FU25 + MAK group.

Table 3 Number of surviving crypts
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Fig. 5
figure 5

Small intestine. Hematoxylin and eosin (H&E). Left: AOM + 5-FU80, ×100. Right: AOM + 5-FU80 + MAK, ×100

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Fig. 6
figure 6

Position of PCNA-positive cells in a small-intestinal crypt. Left: AOM + 5-FU25. Right: 5–2 AOM + 5-FU25 + MAK, ×100

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Table 4 Crypt length and position of PCNA in small intestine
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Discussion

In this study, the presence of ACF, which is a precancerous state, as induced by AOM was shown to be decreased by 5-FU and MAK. Many previous studies of cancer prevention have used transplanted tumors, such as sarcoma 180 [16–18], whereas here we developed a new assay model for evaluation of cancer prevention using tumors induced in a short time by AOM.

We found that 5-FU decreased the formation of ACF induced by AOM, and that combination with MAK further decreased the numbers of ACF. At a high dose of 5-FU (80 mg), AOM-induced ACFs were decreased (to 61.1) by 5-FU alone, and to 9.8 and 8.1, respectively, when MAK was used in combination. The decrease of ACFs following treatment with 5-FU was marked, but the effect of the combination treatment was not clear. Most of the intestinal mucosa containing ACFs could have been ablated as a result of the cytotoxic effect of 5-FU alone. By contrast, with a low dose of 5-FU (25 mg), the decrease of ACFs resulting from treatment with 5-FU alone was moderate (to 46.9), and an additional decrease of ACFs (to 26.4) was clearly observed when 5-FU was combined with MAK. Previously we reported that dietary application of MAK clearly inhibited the induction of ACF and adenoma in F344 rats [10]. Recently, using follow-up colonoscopy, Oka et al. [20] reported that the number and total size of adenomas in humans to whom MAK was administered were significantly decreased from the baseline. It is suggested that MAK suppresses the development of colorectal adenomas in the large bowel, and that MAK might decrease the effects of tumor prevention by 5-FU. Moreover, in the present study, 5-FU decreased ACF induced by AOM, and the decrease induced by 5-FU + MAK was greater than that with 5-FU alone. This finding suggests that MAK might decrease ACF without interfering with the effects of 5-FU. In fact, the two agents appeared to produce an additive antitumor effect, suggesting that MAK used in combination with chemotherapeutic drugs might improve the effectiveness of cancer treatment. Wang et al. [21] have reported that a G. lucidum extract ameliorated cisplatin (CDDP)-induced nausea and vomiting, and improved food intake, in a concentration-dependent manner in a rat pica model based on measurement of kaolin intake. In a clinical study reported by Zhuang et al. [22], a Chinese medicinal herb complex containing G. lucidum extract decreased leucopenia and neutropenia induced by chemotherapy. Nonaka et al. [23] recently reported that an antlered form of G. lucidum (Rokkaku-reishi) relieved cyclophosphamide (CPA)-induced weight loss and suggested that G. lucidum might be useful for reducing the adverse effects of anticancer drugs. Furthermore, Nonaka et al. [18] have reported that G. lucidum inhibited the growth of transplanted tumors and prolonged survival when administered orally to mice, as well as exerting antitumor activity when administered after tumor inoculation.

In the present study, MAK prevented small-intestinal injury by increasing the number of regenerative crypts induced by 5-FU. We reported previously that MAK ameliorated the small-intestinal injury caused by 5-FU, CDDP, CPA, and gefitinib [15]. We suggest that MAK may reduce the gastrointestinal adverse effects of anticancer drugs without attenuating their beneficial antitumor activity. Further studies are required to elucidate any differences in effects between the mycelia and fruiting bodies of G. lucidum.

In this experiment, the PCNA positivity index and the height of the germinal region in the AOM + 5-FU + MAK group were significantly decreased in comparison with the AOM + 5-FU group. Previously, we reported that MAK acted as a preventive agent against colon carcinogenesis by suppressing cell proliferation [10] and that MAK was able to immediately promote cell growth for repair of acute small-intestinal injury caused by X-irradiation [14] or administration of chemotherapeutic drugs [15]. Oral administration of dextran sulfate sodium also caused colon mucosal injury, which was rapidly ameliorated by application of MAK [24]. It is considered that MAK might have two contrasting actions: in the case of abnormal cell growth, renewal may be decreased, thus inhibiting tumor growth, whereas in the case of damage, cell proliferation may be increased to effect emergency repair. Similar results were obtained in the present study. Further studies are required to clarify how the switching of these different actions occurs.

In conclusion, our results suggest that 5-FU decreases the formation of ACF induced by AOM, and that combination with MAK further decreases the number of ACF and the degree of acute small-intestinal damage induced by 5-FU. Further longer-term studies are required to assess the value of MAK in the treatment of other adverse effects of anticancer drugs, such as organ injury and alopecia.