Antimutagenic Effect of Aqueous Extract from Agaricus brasiliensis on Culture of Human Lymphocytes

Abstract

The mushroom Agaricus brasiliensis (sun mushroom), native from the southeast of Brazil, is well known by its medicinal properties that include effects on diabetes, cholesterol levels, and osteoporosis. The antimutagenic effects of A. brasiliensis has been investigated recently and revealed some controversial results depending on the temperature by which the A. brasiliensis tea is obtained. In the present study, we evaluated the effect of the A. brasiliensis extract prepared in two different temperatures, 4°C and 25°C, on the doxorubicin-induced DNA strand breaks and chromosomal aberrations (CAs) in human lymphocytes. The results demonstrated that A. brasiliensis was able to reduce the DXR-induced DNA damage in both temperatures; however, the CA test was more sensitive to demonstrate a better reduction when the cells were treated with an extract obtained at 25°C. A. brasiliensis extract obtained in different temperatures exhibited antigenotoxic and anticlastogenic effects in human lymphocytes.

R esearchers worldwide have conducted several studies searching for substances that can cure or even reduce the risk of cancer. Agaricus brasiliensis is a mushroom known as sun mushroom because of its nutritional and medicinal properties. It was discovered in the 1960s, by the Japanese immigrant T. Furumoto, in the highlands of the Atlantic Forest of southern Brazilian state of Sao Paulo, in the city of Piedade.¹ This mushroom presents some similarities with Agaricus blazei Murrill and Agaricus sylvaticus, found in Japan and England, respectively.²

It has been reported that treatment with tea of the A. brasiliensis mushroom results in beneficial effects for treating physical and emotional stress, stimulating the immune system, improving health in diabetes, lowering cholesterol, and reducing the symptoms of gastric ulcer.^3,4 The antitumoral activities of this mushroom have been related to the action of some antitumor polysaccharides, especially the glycoprotein complex-binding (1→6)-β-d-glucan-protein extracted from A. brasiliensis. ^5,6 Since the early steps of carcinogenesis include the disruption of genomic instability and that the genotoxic stress is involved in this process, the searching for compounds that reduce the damage to DNA molecule is of great importance.

There are different biomarkers to evaluate the antimutagenic properties of a such compound, including the chromosomal aberration (CA) assay, which indicates the occurrence of DNA damage by the present of chromosome breaks and rearrangements,⁴ and the comet assay that detects single and double breaks in DNA molecules, which are induced by wide range of genotoxic compounds.⁷ The comet assay is less labor-intensive and costly than other methods such as CA analysis or the micronucleus assay.⁸ The chemotherapeutic agent doxorubicin (DXR) has been extensively used as an inducer of genotoxic stress in antimutagnenic approaches⁹ because of its genotoxic properties resulting from the generation of reactive oxygen species and inhibition of the DNA topoisomerase II complex.¹⁰

The objective of the present study was to evaluate in vitro the effects of A. brasiliensis extracts obtained in different temperatures on the modulation of the DXR-induced genotoxic effects. To address this question, temporary cultures of peripheral human lymphocytes were treated with A. brasiliensis extract obtained at 4°C or 25°C, alone or in combination with DXR. DNA damage was detected by assessing extension of DNA strand breaks detected by comet assay, and chromosomal damage by the frequency of CAs. We observed that the extract from A. brasiliensis exhibited protective effects when obtained at 4°C or 25°C; however, at 25°C, these effects were more significant.

Human peripheral blood was collected using heparinized vials from three healthy individuals. Lymphocytes were isolated with plasma and cultured in an RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 20% fetal calf serum (Cultilab, Campinas, SP, Brazil), penicillin (5 μg/mL), streptomycin (10 μg/mL), and 2% phytohemagglutinin (Life Technologies, Grand Island, NY, USA). Cells were cultured at 37°C in culture flasks containing 5 mL of complete medium. The National Research Ethics Committee approved the protocol of the experiments, and written consent was obtained from each blood donor before joining the study.

The aqueous extracts from A. brasiliensis (Fungibras, Botucatu, SP, Brazil) were prepared from the powder (7.5 g in 100 mL of sterilized water) at temperatures of 4°C and 25°C for 2 h. Afterward, the extracts were filtered by Millipore^® filter (0.22 μm).

After 24 h of cell culture, lymphocytes were treated with 100 μL of A. brasiliensis extract obtained at 4°C or 25°C alone or in combination with DXR (0.15 μg/mL) for more 24 or 26 h according to the following groups: T1: negative control; T2: positive control DXR (0.15 μg/mL of culture medium); T3: solution of A. brasiliensis at 4°C; T4: solution of A. brasiliensis at 25°C; T5: solution of A. brasiliensis at 4°C + DXR; and T6: solution of A. brasiliensis at 25°C + DXR. After that, cell cultures were harvested for comet assay when incubation completed at 48 h or CA test when incubation completed at 50 h.

An aliquot of 300 μL from each culture was taken after 48 h of incubation to test for cell viability by trypan blue exclusion and for the alkaline version of the comet assay as described by Singh et al. ⁷ Briefly, 300 μL of the cell suspension was centrifuged for 5 min (2000 g) in a refrigerated microcentrifuge (Eppendorff). The resulting pellet was homogenized with 80 μL of a low-melting-point agarose (0.5%), spread onto microscope slides precoated with a normal-melting-point agarose (1.5%), and covered with a coverslip. After 5 min at 4°C, the coverslip was removed, and the slides were immersed in a cold lysis solution (2.4 M NaCl; 100 mM EDTA; 10 mM Tris, 10% DMSO, and 1% Triton-X, pH 10) for 24 h. After lysis, the slides were placed in an electrophoresis chamber and covered with an electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH >13), for a remaining 20 min to allow for unwinding of DNA. The electrophoresis proceeded for 20 min (25 V and 300 mA). Afterward, the slides were submerged for 15 min in a neutralization buffer (0.4 M Tris–HCl, pH 7.5), dried at room temperature, and fixed in 100% ethanol for 5 min. Slides were stained immediately before analyzing using ethidium bromide (20 μg/mL). Slides were prepared in duplicate, and 100 cells were screened per sample (50 cells from each slide) in a fluorescent microscope (ZEISS, Jena, Germany) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm using a 40×objective.¹¹ The nucleoids were classified visually according to the migration of the fragments in class 0 (no damage); class 1 (little damage with a short tail length smaller than the diameter of the nucleus); class 2 (medium damage with a tail length one or two times the diameter of the nucleus); 3 (significant damage with a tail length between two and a half to three times the diameter of the nucleus); class 4 (significant damage with a long tail of damage greater than three times the diameter of the nucleus).

To analyze CAs, a metaphase preparation was performed after 50 h of cell culture. About 90 min before harvesting, 12.5 μL colchicine (0.016%, Sigma) was added to each culture. The conventional cell harvest procedure was followed according to Moorhead et al. ¹² The cells were treated with hypotonic KCl solution (0.075 M) for 10 min, fixed with methanol:acetic acid (3:1), air-dried, and stained with Giemsa:Sörensen buffer (1:30) for 5 min. All slides were coded. The same scorer analyzed the slides in a blind test, and a total of 100 cells per treatment per individual were scored for the presence of CAs classified according to Savage,¹³ and 2000 cells per treatment per individual were scored for MI.

The percentage of damage reduction in response to A. brasiliensis treatments is calculated as recommended by:¹⁴ % of reduction=(DXR−ABL)/(DXR−NC)×100%, where DXR is damage in response to positive control (DXR alone), ABL is damage in response to A. brasiliensis extract combined with DXR, and NC is damage in response to negative control.

The data were submitted to two types of parametric analyses, where all the variables were submitted to the analysis of variance, after a test of comparison of means (Duncan's test, at the level of 5% of probability) when the treatment resulted in a significant effect. The statistical analyses were executed by the program WinStat System of Statistic Analysis for Windows—Beta version.¹⁴

In the present study, we investigated the antigenotoxic and anticlastogenic activities of A. brasiliensis extracts obtained from different temperatures of infusions at 4°C and 25°C. The results obtained demonstrated that in vitro exposure of peripheral lymphocytes to A. brasiliensis resulted in neither a genotoxic nor a clastogenic effect, but showed antigenotoxic activity.

Results obtained from CA test showed that treatment with A. brasiliensis at 4°C or 25°C did not increase the frequency of cells with aberrations when compared to the negative control (P>.05; Table 1). Treatment with DXR significantly increased the frequency of cells with aberration; however, A. brasiliensis at 4°C or 25°C resulted in a significant reduction of these abnormal cells (54.05% and 77.55%, respectively) after DXR treatment, and at 25°C, the frequency of normal cells was restored to values statistically similar to the negative control (Table 1). The mitotic index obtained after all treatments was statistically similar to the negative control (data not shown).

Table 1.

Chromosomal Aberrations in 300 Metaphases, Frequency of Cells with Aberrations, and Percentage of Reduction Observed After In Vitro Treatment with Different Aqueous Extracts of Agaricus brasiliensis and Chemotherapeutic Doxorubicin

	Chromosomal aberrations (%)
		Breaks
Treatment	Gaps	C	IC	Others	Normal cells (%)	Cells with aberrations (%)	% of reduction	\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} $$\bar {x}$$ \end{document} ±s
Negative control	1.3^a	0.7^b	—	—	98.0^c	2.0	-	2.0±1.15
DXR	17.0^c	13.0^c	5.7^c	4.6^c	59.7^a	40.3	-	40.3±15.37
ABL 4°C	1.3^a	—	—	—	98.7^c	1.3	101.83	1.3±0.58
ABL 25°C	2.7^a	1.0^b	—	—	96.3^c	3.7	95.56	3.7±1.53
ABL 4°C+DXR	9.0^b	4.3^b	5.0^c	1.3^b	80.4^b	19.6	54.05	19.6±2.65
ABL 25°C+DXR	7.3^bc	2.3^b	0.7^b	0.3^ab	89.4^bc	10.6	77.55	10.6±1.00

x indicates the mean and s indicates the standard deviation.

abc

Values in the same column followed by different superscript letters displayed statistically significant differences (α=0.05, Duncan's test).

DXR, chemotherapeutic doxorubicin; ABL, A. brasiliensis; C, chromatid break; IC, isochromatid break.

The distribution of different types of CAs was very uniform among the different groups and gaps represented the most frequent aberration detected, followed by chromatid and isochromatid breaks and other types of aberrations such as complex rearrangements.

The extension of DNA damage was also evaluated using comet assay. Cell cultures treated with A. brasiliensis at 4°C or 25°C did not present an increased number of comet cells when compared to the negative control (Table 2). The combination of A. brasiliensis with DXR resulted in a percentage of reduction of 137.8% at 25°C and 35.1% at 4°C. These results showed that A. brasiliensis exhibited a differential effect against DXR as a function of extraction temperature.

Table 2.

Percentage of Comet Cells, DNA Damage Index, and Percentage of Damage Reduction Observed After Treatment with Different Aqueous Extracts of Agaricus brasiliensis and Chemotherapeutic Doxorubicin in 300 Cells by Comet Assay

	Distribution of comet classes (%)
Treatment	0	1	2	3 and 4	Normal cells (%)	Cells with comet (%)	% of reduction	\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} $$\bar {x}$$ \end{document} ±s
Negative control	75.7^a	14.0^ab	8.3^ab	2.0^c	75.7^a	24.3	—	24.3±14.01
DXR	63.3^a	16.7^ab	14.0^a	6.0^a	63.3^a	36.7	—	36.7±29.74
ABL 4°C	74.7^a	15.7^ab	8.3^ab	1.3^c	74.7^a	25.3	91.94	25.3±20.65
ABL 25°C	82.3^a	9.3^b	5.7^b	2.7^c	82.3^a	17.7	153.23	17.7±3.06
ABL 4°C+DXR	67.7^a	19.3^a	9.7^ab	3.3^b	67.7^a	32.3	35.48	32.3±13.50
ABL 25°C+DXR	80.3^a	13.3^ab	4.7^b	1.7^b	80.3^a	19.7	137.10	19.7±16.17

abc

Values in the same column followed by different superscript letters displayed statistically significant differences (α=0.05, Duncan's test).

The antigenotoxic effect against DXR evidenced in the results was also observed in cultured Chinese hamster V79 cells through the comet and micronucleus assays by Oliveira et al.,¹⁵ studying the aqueous extract of A. blazei, at temperatures 4°C, 25°C, and 60°C with three treatment protocols (simultaneous, pretreatment, and post-treatment) against the mutagen metylmethane sulfonate (MMS). The results indicated that all treatments resulted in significant reduction of micronuclei, but this reduction was more effective at 4°C and 25°C when compared to 60°C. Our results did not show significant difference in the percentage of DNA damage reduction between temperatures 4°C and 25°C. Delmanto et al. ¹⁶ evaluated aqueous extracts of a mixture of lineage A. blazei, obtained at three temperatures (4°C, 21°C, and 60°C) that showed antimutagenic activity against cyclophosphamide in vivo. Many researchers reveal the relationship between the temperature of the preparation of the extract and the protective effect of the mushroom, because very high temperatures for preparation of the extracts can influence the efficiency of the fungus, and they are able to affect the active principles responsible for its protective effect.¹⁶

The percentage of damage reduction obtained by the CA assay, which is an indicator of the possible antigenotoxic effect of A. brasiliensis, showed significant difference in relation to the positive control (DXR), and the number of normal cells was higher for extract obtained at 25°C than 4°C, with 89.9% and 80.9%, respectively. These results were also confirmed by the comet assay. These results are consistent with those found by Guterrez et al. ¹⁷ when studying the effects of aqueous extract of different strains of A. brasiliensis obtained at temperatures of 4, 25, and 60°C on Chinese hamster V79 cells pre-, post-, and simultaneously treated. A significant reduction of chromosome damage induced by MMS was found and assessed by micronucleus test; however, the reduction exhibited by comet assay was not statistically significant. On the other hand, Ishii et al.,¹⁸ using a Swiss mouse model, demonstrated that the mushroom A. blazei exhibited antigenotoxic activity. According to these authors, supplementation increased the number of monocytes and the phagocytic activity promoting immunomodulation, which can account for the destruction of cells with DNA alterations.

The antitumoral DXR is considered efficient in the induction of CA in vitro test systems of short duration, and has been extensively employed in studies of chemoprotection.^19,20 The most frequent type of CA was chromatid breaks and gaps. The antimutagenic activity of A. brasiliensis is still subject to several studies, being attributed to various chemical compounds. In addition to the aforementioned complex glycoprotein, other compounds, such as linoleic acid, are suggested as responsible for the antimutagenic activities of this mushroom.^4,21 During the process of discovering new compounds with chemopreventive properties, especially those obtained from natural sources, it is important to take into account the method by which those compounds are extracted. Machado et al. ^22,23 demonstrated that extracts from Tirmania pinoyi, obtained in a sequence of boiling water, chloroform, and ethanol, exhibited different effects: the chloroform extract presented mutagenic effects by the Ames Salmonella/microsome assay, whereas the ethanolic extract inhibited the mutagenic effects of some carcinogens.

In conclusion, in the present study, an extract of A. brasiliensis obtained at different temperatures was effective in reducing the DXR-induced DNA damage in peripheral blood lymphocytes treated in vitro, and the reduction of CAs was more significant when cell cultures were treated with extract obtained at 25°C. The antigenotoxic effects of A. brasiliensis observed here were demonstrated by both CA test and comet assay.

Footnotes

Author Disclosure Statement

The authors declare no conflict of interest.

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