Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
GENOPROTECTIVE EFFECTS OF SELECTED MUSHROOM SPECIES
Y. Shi1, A. E. James3, I. F.F.Benzie4 and J. A. Buswell1,2
1
Department of Biology and 2 Centre for International Services to Mushroom Biotechnology,
The Chinese University of Hong Kong; 3 Laboratory Animals Services Centre, The Chinese
University of Hong Kong; 4 Department of Nursing and Health Sciences, Hong Kong Polytechnic
University, Hong Kong SAR, China. <jabuswell@cuhk.edu.hk>
ABSTRACT
Although a variety of natural sources have been examined for antioxidant components in order to
develop dietary supplements and preventative treatments for neutralising the genotoxic effects of
reactive oxygen species (ROS), mushrooms have so far received little attention in this context.
Therefore, we have screened eight selected mushroom species for ability to prevent H2O2-induced
oxidative damage to cellular DNA using the single-cell gel electrophoresis (“Comet”) assay. Both
cold (20OC) and hot (100OC) water extracts of mushroom sporophores were tested, and highest
genoprotection was observed with cold water and hot water extracts of Agaricus bisporus and
Ganoderma lucidum fruit bodies, respectively. Material obtained by aqueous extraction (both hot
and cold) of Flammulina velutipes, Auricularia auricula, Hypsizygus marmoreus, Lentinula edodes,
Pleurotus sajor-caju and Volvariella volvacea afforded no genoprotection. The genoprotective
effect of A. bisporus is associated with a heat-labile protein isolated from the mushroom fruit bodies
and identified as tyrosinase.
INTRODUCTION
Reactive oxygen species (ROS) produced during normal metabolic processes, inflammation,
smoking, and after ingestion of certain drugs and pollutants, etc., cause DNA strand breakage and
damage a variety of DNA bases (Halliwell and Gutteridge 1999). Increased levels of oxidative
damage to ROS have been linked to numerous pathological conditions including various types of
cancer (Marnett 2000) and neurodegenerative disorders such as Parkinson’s disease (Ames et al.
1993, Mecocci et al. 1994, Alam et al. 1997).
In Southeast Asia, especially in China and Japan, mushrooms have long been acknowledged for
their medicinal and analeptic qualities in addition to their desirable flavours and nutritional value.
However, very few studies have been conducted on the antioxidant/genoprotective effects of
mushrooms. We have reported earlier that several mushroom species display anti-oxidant power in
the Ferric Reducing Antioxidant Power (FRAP) assay (Benzie and Strain 1996, Benzie et al. 1998),
and mushroom-derived polysaccharoproteins are reported to scavenge active oxygen species (Liu et
al. 1997). Our own research has focused on edible and medicinal mushrooms as sources of
antioxidants that could be used in dietary supplements and preventative treatments for offsetting the
adverse biological effects of ROS. In this investigation, we have used the Comet assay (Singh et al.
1988) to demonstrate the capacity of two mushroom preparations, from A. bisporus and G. lucidum,
to protect against H2O2-induced oxidative damage to cellular DNA in an in vitro cell culture system.
More detailed examination has revealed that the genoprotective effect of A. bisporus is not due to
direct destruction of hydrogen peroxide but instead is linked to the catalytic activity of tyrosinase
present in the mushroom fruit body.
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MATERIALS AND METHODS
Mushroom species and extraction procedures
The following mushroom species were examined in this study: A. bisporus, A. auricula, F.
velutipes, G. lucidum, H. marmoreus, L. edodes, P. sajor-caju and V. volvacea. Pieces of fruit
bodies, (~1 cm2), were suspended in three volumes of distilled water and extracted for 3 hr either
with shaking (150 rpm) at 20OC (cold extraction) or statically at 100OC (hot extraction). After
removing coarse residue material by filtration through cheese cloth, the extracts were centrifuged at
15,300 x g for 30 min at 4OC. Supernatants were freeze-dried and the resultant solid material stored
at –20OC prior to analysis (Shi et al. 2002a).
Purification of genoprotective component from A. bisporus fruit bodies
Fruit bodies of A. bisporus were obtained from a local supermarket. Large-scale extraction
procedures, and the protocol for the purification of the genoprotective component from A. bisporus
fruit bodies were described previously (Shi et al. 2002b).
Cell culture
Raji cells (Burkitt’s lymphoma, ATCC CCL-86) were grown at 37°C under 5% CO2 in RPMI 1640
medium containing 24 mM NaHCO3, 5 mM HEPES, 1.0 mM sodium pyruvate, 50 units ml-1
penicillin G and 50 µg ml-1 streptomycin sulphate and supplemented with 10% foetal bovine serum
(FBS) (Gibco). Cells were subcultured every 2 days.
Assay of genoprotective activity
Genoprotective activity was assayed using the single-cell gel electrophoresis (“Comet”) assay
(Singh et al. 1988). Details of work-up procedures using the Raji cell system are described
elsewhere (Shi et al. 2002b). The Olive Tail moment (integrated value of the percentage of DNA
density of the comet tail multiplied by the relative migration distance which has been corrected for
greyscale calibration) was determined using Comet software version 3.0 (Kinetic Imaging,
Liverpool, UK) as the primary measure of DNA damage (Singh 1996).
H2O2 assay
H2O2 concentrations in buffers and culture media were determined using the PeroXOquant
Quantitative Peroxide Assay Kit (Pierce). Peroxides in the sample oxidize Fe2+ to Fe3+ which then
reacts with xylenol orange. The amount of coloured complex formed was determined by measuring
the absorbance at 595nm in a microplate spectrophotometric reader (Jiang et al. 1992).
Enzyme assays
Laccase, peroxidase and tyrosinase activities were assayed as described previously (Shi et al.
2002b).
Gel electrophoresis
Non-denaturing- and SDS (sodium dodecyl sulphate)- PAGE (polyacrylamide gel electrophoresis)
were performed using the Mini Protean-II system (Bio-Rad) according to the method of Laemmli
(1970). The molecular mass of protein bands was determined using protein Mr standards (Bio-Rad)
358
lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and
phosphorylase b, and staining with Coomassie brilliant blue (Bio-Rad). The polyacrylamide
concentrations of the separating gels for non-denaturing- and SDS-PAGE were 6% and 15%,
respectively.
Protein determination
Protein was determined by the method of Bradford (1976) with bovine serum albumin as standard.
Protein in column effluents was monitored by measuring A280.
Cytotoxicity analysis
Cytotoxic effects of the mushroom extracts were determined by mixing 8µl of sample cell
suspension with 8µl of 0.2% w/v Trypan Blue solution in PBS (pH7.4) containing 3mM NaN3, and
counting the number of viable cells (Hunt 1987).
Statistical analysis and data presentation
DNA damage was expressed as the Mean Olive Tail Moment  standard error. Statistical analysis
was made using Kruskal-Wallis One-Way Analysis of Variance on Ranks (P<0.05), SigmaStat 2.0
(SPSS, Inc., Chicago, IL).
RESULTS
Concentration-response curves relating the H2O2-induced damage to Raji cell DNA showed an
essentially linear response over the range 5-15M (final concentration in test mixture) and
therefore 10M H2O2 was used in subsequent experiments unless indicated otherwise.
Significant protection against H2O2-induced damage was afforded by cold water extracts of A.
bisporus (Ab-cold) and hot water extracts of G. lucidum (Figure 1). Both these crude mushroom
extracts provided virtually complete protection at concentrations of 0.5 mg ml -1. However, no
protection was observed with extracts of the other mushrooms tested (Figure 1) and increased DNA
damage occurred with hot and cold water extracts of A. auricula and H. marmoreus, and hot water
extracts of A. bisporus (Figure 1). Neither of the two DNA protective mushroom extracts produced
any cytotoxic effects after 24 hours treatment at concentrations of 1mg ml -1. However, cold water
extracts of V. volvacea were highly cytotoxic (100% loss of cell viability at 0.025mg ml -1
concentration) and ~10% reduction in viable cells compared with controls was observed with the
cold water extracts of F. velutipes.
359
50
Mean Olive Tail Moment
45
#
#
40
#
35
#
#
30
25
20
15
10
5
*
*
0
C (-)
C (+)
Aa (+) Ab (+)
Fv (+)
Gl (+) Hm (+) Le (+) Psc (+) Vv (+)
Figure 1. Genoprotective effects of different mushroom extracts against H2O2-induced (10M)
damage to Raji cells.
Data are derived from two separate experiments and values are the mean  standard error of the Olive
TailMoment (200 cells). C (-) : negative control (no H2O2 challenge); C (+) : positive control
(H2O2 challenge but without pre-exposure to mushroom extract); Closed bars – pre-exposure to cold
water mushroom extracts (0.5mg ml-1 except Fv where 0.1mg ml-1 was used); Open bars – preexposure to hot water mushroom extracts (0.5mg ml-1).
Aa – A. auricula; Ab – A. bisporus; Fv – F. velutipes; Gl – G. lucidum; Hm – H. marmoreus;
Le – L. edodes; Psc – P. sajor-caju; Vv – V. Volvacea.
*Significant genoprotective effects found at P<0.05 compared with stressed cells without exposure to
mushroom extract; #Significant increased damage to DNA at P<0.05 compared with stressed cells without
exposure to mushroom extract. (Reproduced from: “Mushroom-derived preparations in the prevention of
H2O2-induced oxidative damage to cellular DNA”, Shi et al. 2002. Teratogenesis, Carcinogenesis and
Mutagenesis. Wiley-Liss. Inc. New York).
Subsequent experiments in this study were directed at determining the nature of the genoprotective
effect afforded by Ab-cold extracts, and at identifying the active component(s). Follow up
experiments were performed first to determine if the protective effect was due to the destruction of
H2O2 by Ab-cold extract taken up by the cells during incubation. Cells were incubated with
mushroom extract or catalase (100 U ml-1) for 2 hours, washed and exposed to H2O2 for 30 minutes,
and residual levels of H2O2 in the cell suspension medium were then measured. Residual H2O2
concentrations in mushroom extract-treated samples were virtually identical with those pre-treated
with catalase and with untreated controls. However, whereas the amount of DNA damage in
catalase pre-treated samples remained high (89.3% ± 7.1) compared with untreated controls (100%
± 8.9), the DNA damage in cells treated with mushroom extract was significantly reduced (11.4% ±
360
0.7). Therefore, the protective effect of Ab-cold extracts was not due to cellular uptake or binding
of an extract-associated H2O2-degrading activity.
In addition to its heat-labile nature, preliminary tests to establish the nature of the genoprotective
component of A. bisporus cold-water extracts revealed the bioactive agent to have a molecular
weight in excess of 10 kDa and to be inactivated by treatment with proteinases. We therefore set
about isolating the active component using a series of protein purification procedures involving salt
fractionation anion exchange chromatography using DEAE-Sepharose CL-6B, hydrophobic
interaction chromatography with Phenyl-Sepharose and chromatography on hydroxyapatite. This
resulted in the isolation of FII fraction which, when subjected to polyacrylamide gel
electrophoresis under non-denaturing conditions using 6% gel produced a single homogeneous
band. SDS-PAGE produced a major band of about 42 kDa and a minor band of about 12 kDa. A
typical protocol for the isolation of FII fraction is shown in Table 1.
Table 1. Purification of genoprotective FII-1 fraction from fruit bodies of Agaricus bisporus.
Purification step
Protein
Amount of
Recovery
Specific
Purification
(mg)
protein providing
of activity
activity
factor
one Comet unit of
(%)
(unit/mg)
protection (ng)*
Crude extract
Ultrafiltration
(NH4)2SO4 (40-60%)
DEAE-Sepharose
Phenyl-Sepharose
Hydroxyapatite
2490
2160
646
72
19
1.2
9700
5000
833
167
68
12
100
170
303
169
110
39
103
200
1200
6000
14700
83000
1
1.9
11.6
58.3
143
780
* Defined as the lowest amount of protein providing >90% protection in the standard test system
(Reprinted from Life Sciences, Shi et al. "Role of tyrosinase in the genoprotective effect of the edible
mushroom, Agaricus bisporus", 2002, with permission from Elsevier Science).
Table 2. Relative amount of H2O2 –induced damage to DNA of Raji cells after treatment with
FIIfraction compared with V. volvacea laccase and commercial preparations of tyrosinase and
peroxidase.
Treatment
Relative amount of H2O2 –induced damage (%)
Negative control (no H2O2 challenge)
6.3 ± 2.8
Positive control (no pre-treatment)
100.0 ± 4.9
Laccase
99.3 ± 4.9
Peroxidase
88.9 ± 6.3
Tyrosinase
6.9 ± 1.4 *
FII fraction
11.1 ± 1.4 *
DNA damage is expressed as the Mean Olive Tail Moment  standard error of data obtained from two
replicate experiments (50 cells). Statistical analysis was made using Kruskal-Wallis One-Way Analysis of
Variance on Ranks at p<0.05. Tyrosinase, laccase and peroxidase (2g/ml protein); FII-1 fraction, 30ng/ml
protein. * Difference compared with positive control significant at p<0.05.
361
An indication of the identity of the genoprotective component of Ab-cold extracts was provided by
the observation throughout the purification procedure that genoprotection was associated with a
faint brown colouration appearing in the Raji cell system during the pre-treatment period. This
colouration was similar to, but far less intense than, the dark brown colour that appeared during the
initial extraction phase and which is due to phenoloxidase activity known to be associated with A.
bisporus fruit bodies. All the genoprotective fractions were subsequently found to contain high
levels of tyrosinase, but only very low levels of peroxidase and laccase. The tyrosinase activity of
FII fraction was 64.2 U/mg protein compared to peroxidase and laccase activities of <0.05
U/mg protein. When the genoprotective effects of crude cold-water extracts of A. bisporus fruit
bodies, purified FII fraction, and a commercial preparation of tyrosinase were compared, all
three samples provided ~95-100% protection (Table 2). However, very little genoprotection was
afforded by either horseradish peroxidase or a partially purified laccase from the edible straw
mushroom V. volvacea (Table 2).
We next determined if the genoprotective effect of FII fraction was linked to its catalytic
activity or to some other inherent property of the protein. In order to eliminate possible interference,
the genoprotection assay was carried out with Raji cells suspended in PBS + 10% (v/v) FBS (added
to maintain tissue cell viability) instead of the complex cell culture medium (RPMI 1640) which
contains tyrosine. Under these conditions, no genoprotection was observed when the Raji cells were
pre-treated separately with either FII fraction or tyrosine, whereas 100% protection was
afforded when cells were pre-treated with FII fraction and tyrosine together (Table 3). Further
support for the involvement of the catalytic functions of tyrosinase in genoprotection was provided
by the observed dose-dependent relationship between the tyrosinase activity and the genoprotective
effect of FII fraction. Pre-treatment of Raji cells with protein samples containing increasing
amounts of tyrosinase (0.1 – 0.4 mU/mg protein) resulted in concomitant increases in
genoprotection (Figure 2).
Table 3. Dependence of genoprotective effect on tyrosinase-associated catalytic activities
of Fraction FII-1.
Treatment
Negative control (no H2O2 challenge)
Positive control (no pre-treatment)
FII fraction
Tyrosine (110M)
FII fraction + tyrosine
Relative amount of H2O2 –induced damage (%)
8.1 ± 4.8
100.0 ± 4.8
87.9 ± 4.8
94.4 ± 4.0
5.6 ± 0.1*
DNA damage is expressed as the Mean Olive Tail Moment  standard error of data (indicated by error bars)
obtained from two replicate experiments (50 cells). Statistical analysis was made using Kruskal-Wallis OneWay Analysis of Variance on Ranks at p<0.05. Protein content of FII-1 = 30ng/ml. * Difference compared
to C(+) significant at p<0.05.
362
3
5
3
0
2
5
2
0
MeanOlivTaMoment
1
5
1
0
5
0
0
.
1
0
.
2
0
.
3
0
.
4
3
T
y
r
o
s
i
n
e
h
y
d
r
o
x
y
l
a
t
i
n
g
a
c
t
i
v
i
t
y
(
u
n
i
t
/
m
l
x
1
0
)
Figure 2. Relationship between genoprotective effect and tyrosine hydroxylating.
Activity DNA damage is expressed as the Mean Olive Tail Moment  standard error of data (indicated by
error bars) obtained from two replicate experiments (50 cells). Tyrosine hydroxylating activity was varied by
incorporating different quantities of FII-1 fraction into the assay. (Reprinted from Life Sciences, Shi et al.
"Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus", 2002, with
permission from Elsevier Science).
DISCUSSION
The medicinal properties of mushrooms have long been recognized, especially in Oriental cultures,
and modern techniques have identified numerous bioactive mushroom components which are
variously reported to exhibit anti-cancer, anti-tumour, anti-viral, immunomodulatory,
hypocholesterolaemic and hepatoprotective activities (Chang and Buswell 1996). More recently, we
have shown that fruit bodies of A. bisporus and G. lucidum contained bioactive compounds that
prevented H2O2-induced oxidative damage to cellular DNA (Shi et al. 2002a). The chemical nature
of the active components of the two genoprotective mushrooms appeared to be quite different since
the protective component of A. bisporus extracts is heat labile whereas prolonged extraction with
boiling water was required to obtain the active compound(s) from the fruit bodies of G. lucidum.
Although the medicinal effects of G. lucidum products are well documented (Chang and Buswell
1999), there are few reports attributing medicinal properties to A. bisporus even though it is the
most widely cultivated and consumed edible mushroom (Chang 1999). Quinoid compounds
obtained from this mushroom have been reported to suppress the propagation of mouse ascites
tumour (Graham et al. 1977), and a lectin from this species also reversibly inhibited the
363
proliferation of human colon carcinoma cells (Yu et al. 1993). The genoprotective effect of A.
bisporus is associated with a heat-labile protein present in the fruit body and which has been
identified as tyrosinase. Tyrosinase is one of the major phenoloxidases which cause “browning” in
fruits, vegetables and mushrooms, and is the major phenoloxidase present in A. bisporus (Ratcliffe
et al. 1994). The identity of A. bisporus FII fraction as tyrosinase was confirmed by
electrophoretic analysis, and by enzyme catalysis and inhibition studies. The electrophoretic
properties of the A. bisporus protein are consistent with previous descriptions of A. bisporus
tyrosinase in which the native protein was reported to consist of two heavy sub-units (42 ± 1 kDa)
and two light sub-units (12 ± 1kDa) (Gerritsen et al. 1994, Soler-Rivas et al. 1999).
The active component(s) of A. bisporus MDP protected only against DNA-damage induced by
H2O2 and no protective effect was observed against DNA damage induced by either bleomycin or
ethyl methanesulphonate (Shi et al. 2002a). Hydrogen peroxide reacts with transition metal ions to
form highly reactive OH radicals. These are thought to either attack DNA directly at the sugar
residue leading to fragmentation and base loss (Cohen 1985), or activate DNA-cleaving nucleases
within the cell (Halliwell and Aruoma 1991). The nature of the genoprotective activity of the
FII fraction is dependent upon the two associated catalytic activities of the tyrosinase protein,
namely hydroxylation of tyrosine to L-DOPA (Shi et al. 2002b) and the subsequent oxidation of
this intermediate to L-DOPA oxidation products (Shi et al. 2002b, Shi et al. 2002b). The basis of
the observed genoprotective effects of tyrosinase-generated L-DOPA oxidation products is not yet
known but may be associated with their reported stimulation of cellular antioxidant defence
mechanisms (Han et al. 1996, Mytilineou et al. 1993, Shi et al. 1993, van Muiswinkle et al. 2000).
ACKNOWLEDGEMENTS
This work was supported by a Direct Grant from The Chinese University of Hong Kong, and by the
British Council under the UK/Hong Kong Joint Research Scheme. We thank Park ‘n Shop Limited,
Hong Kong for providing A. bisporus mushrooms.
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