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Mushroom Biology and Mushroom Products. Snchez 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 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>
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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 Parkinsons 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 (Burkitts lymphoma, ATCC CCL-86) were grown at 37C 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 g 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)

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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 8l of sample cell suspension with 8l 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 5-15M (final concentration in test mixture) and therefore 10M 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.

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Figure 1. Genoprotective effects of different mushroom extracts against H2O2-induced (10M) 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%

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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 FII1 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 FII1 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 FII1 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 * FII1 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 (2g/ml protein); FII-1 fraction, 30ng/ml protein. * Difference compared with positive control significant at p<0.05.

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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 FII1 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 FII1 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 FII1 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 FII1 fraction or tyrosine, whereas 100% protection was afforded when cells were pre-treated with FII1 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 FII1 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) FII1 fraction Tyrosine (110M) FII1 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.

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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

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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 FII1 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 FII1 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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