- Email: [email protected]
Effect of baking and freeze-drying on the direct and indirect mutagenicity of extracts from the edible mushroom Agaricus bisporus
Food and Chemical Toxicology 36 (1998) 315±320
Eect of Baking and Freeze-drying on the Direct and Indirect Mutagenicity of Extracts from the Edible Mushroom Agaricus bisporus K. WALTON, R. WALKER and C. IOANNIDES* School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH,UK (Accepted 29 October 1997) AbstractÐThe objectives of the study were to evaluate the eect of baking and freeze-drying on the direct and indirect mutagenicity in the Ames test of ethanolic extracts from the edible mushroom Agaricus bisporus. Direct mutagenicity was not in¯uenced by baking for 10 min at 2258C, but more prolonged baking, for example 4 hr at 1008C reduced mutagenicity. Hepatic cytosol from Aroclor 1254-induced rats and mushroom tyrosinase potentiated the mutagenic response elicited by the mushroom extracts. Baking did not in¯uence either of these processes. Finally, freeze-drying in¯uenced neither the direct nor the indirect mutagenicity of the mushroom extracts. It is concluded that mutagenic and premutagenic compounds present in mushroom are generally not heat labile. # 1998 Elsevier Science Ltd. All rights reserved Keywords: mushroom; Agaricus bisporus; Ames test; mutagenicity.
INTRODUCTION
Agaricus bisporus is the most extensively consumed type of mushroom worldwide (Gry and Pilegaard, 1991). Concern about the safety of this mushroom was expressed following the studies of Toth and Erickson (1986), who demonstrated that lifetime administration of uncooked A. bisporus to mice induced tumours at a number of sites. However, when air-dried mushroom were fed to rats for 500 days, no carcinogenic eect was evident (Matsumoto et al., 1991). It is not yet clear whether this marked dierence in carcinogenic response re¯ects a species dierence in susceptibility or whether it is due to the dierent protocols employed in the two studies. Extracts from this mushroom can induce a mutagenic response in the Ames mutagenicity test, in the absence of an activation system, particularly when TA104 is used as the Salmonella typhimurium tester strain (Papaparaskeva et al., 1991; Sterner et al., 1982; von Wright et al., 1982). This mutagenic response cannot be ascribed to traces of the histidine present in the ethanolic extracts of the mushroom since the mutagenic potential was also evident in studies using histidine-independent bacterial strains (GruÈter et al., 1991). Moreover, mutagenic response was suppressed by the nucleophile glutathione and the hydroxyl radical scavenger dimethyl sulf*Author for correspondence.
oxide (Papaparaskeva et al., 1991; PapaparaskevaPetrides et al., 1993). The presence of the standard activation system, Aroclor 1254-induced rat hepatic S-9, failed to enhance signi®cantly the mutagenic response. However, the mutagenic eect was elevated in the presence of hepatic cytosol derived from Aroclor 1254-induced rats, and supplemented with NADPH (Papaparaskeva-Petrides et al., 1993). This eect was prevented by the addition of dicoumarol and menadione, an inhibitor and substrate respectively, of the cytosolic DT-diaphorase. It was concluded that phenols and quinones present in the mushroom could be converted to semiquinones that could directly interact with DNA or act as generators of reactive oxygen species following interaction with molecular oxygen. The mutagenicity of the mushroom extracts was also enhanced in the presence of mushroom tyrosinase (Papaparaskeva-Petrides et al., 1993). This polyphenol oxidase catalyses the conversion of phenols to quinones through the formation of dihydroxycompounds. A monophenolase activity facilitates the o-hydroxylation of the monophenol to the o-diphenol, and a diphenolase activity converts the o-diphenol to the o-quinone (Sanchez-Ferrer et al., 1995). Mushrooms are commonly baked before consumption, for example in products such as pizzas. The majority of the consumption is in the form of baked mushroom found in many products such as pizzas. This prompted us to investigate the
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(97)00161-0
316
K. Walton et al.
eect of baking on the mutagenicity of extracts from A. bisporus using the S. typhimurium strain TA104 which shows the clearest response to the direct mutagenicity of mushroom extracts. Earlier studies (Toth et al., 1992), showed that short-term baking of mushrooms did not in¯uence their direct mutagenicity in S. typhimurium strains TA1535 and 1537. Moreover, freeze-dried mushrooms are extensively used in the production of soups and sauces; consequently, the eect of freeze-drying on the mutagenicity of mushroom extracts was also investigated.
MATERIALS AND METHODS
Aroclor 1254 (Robens Institute, Guildford, UK), mushroom tyrosinase and all cofactors (Sigma Chemical Co., Poole, Dorset, UK) were all purchased. A. bisporus mushrooms were obtained locally. Mushrooms were rinsed, blotted dry and then homogenized in 96% ethanol (v/v) at a concentration of 1 g/ml. The extract was ®ltered through muslin and Whatman ®lter paper (No. 10). The ®ltrate was evaporated down to about 10% of its
original volume, and was stored at ÿ208C until use. Aliquots of mushroom were baked evenly for either 10 min at 2258C or 4 hr at 1008C. The mushrooms were then reweighed and reconstituted to the original weight using water. Finally, an aliquot of mushroom was snap-frozen in liquid nitrogen and freeze-dried for 12 hr. Mushrooms were reweighed and the original weight reconstituted by the addition of water. Extracts of the baked and freezedried mushrooms were prepared as described for raw mushroom (see above). Male Wistar albino rats (about 200 g) were obtained from Harlan Olac (Bicester, Oxon., UK). Treatment with Aroclor 1254 comprised a single ip administration (500 mg/kg), the animals being killed on the 5th day following administration. Hepatic cytosolic fractions were prepared as previously described (Ioannides and Parke, 1975). The mutagenicity of the mushroom extracts was determined using the Ames mutagenicity test and employing S. typhimurium strain TA104 (Maron and Ames, 1983). Activation systems contained 10% (v/v) hepatic cytosol from Aroclor 1254-induced rats or mushroom tyrosinase (8000 units).
Fig. 1. Eect of baking and freeze-drying on the direct mutagenicity of extracts from the mushroom A. bisporus. The study was carried out using S. typhimurium strain TA104. Results are presented as mean 2SD for triplicates. The spontaneous reversion rate of 419213 has already been subtracted. 4Nitroquinoline N-oxide (0.5 mg) induced a reversion rate of 1118299. (*) Raw mushroom; (Q) mushroom baked at 1008C for 4 hr; (R) mushroom baked at 2258C for 10 min, and (W) freeze-dried mushroom.
Mutagenicity of mushroom RESULTS
As expected, raw mushroom extracts were mutagenic in S. typhimurium strain TA104. A concentration-dependent eect was evident, with doubling of the spontaneous reversion rate achieved at the highest concentration studied, that is, 100 ml/plate
317
(Fig. 1). Mutagenic potential was unaected by baking at 2258C for 10 min. However, a more prolonged baking at a lower temperature, that is at 1008C for 4 hr, suppressed the mutagenic response. Freeze-drying of the mushroom did not alter signi®cantly the mutagenic response (Fig. 1).
Fig. 2. Eect of baking and freeze-drying on the cytosol-mediated mutagenicity of extracts from the mushroom A. bisporus. The study was carried out using S. typhimurium strain TA104 and an activation system (10% v/v) derived from Aroclor 1254-induced rats. Results are presented as mean2SD for triplicates. The spontaneous reversion rate of 388232 has already been subtracted. 4-Nitroquinoline Noxide (0.5 mg) induced a reversion rate of 1401276. (W) Without cytosol; (Q) with cytosol. (A) Raw mushroom; (B) mushroom baked at 1008C for 4 hr; (C) mushroom baked at 2258C for 10 min and (D) freeze-dried mushroom.
318
K. Walton et al.
Inclusion of an activation system comprising hepatic cytosol from Aroclor 1254-induced rats, forti®ed with an NADPH-generating system, clearly enhanced the mutagenic response of raw mushroom extracts (Fig. 2). When extracts from mushroom baked at 2258C for 10 min were used, hepatic cyto-
sol still increased the mutagenic response, the eect being of a similar magnitude to that observed with the raw mushroom. Baking at 1008C for 4 hr reduced the direct mutagenic response but mutagenicity was still increased following the addition of hepatic cytosol. Finally, freeze-drying did not in¯u-
Fig. 3. Eect of baking and freeze-drying on the tyrosinase-mediated mutagenicity of extracts from the mushroom A. bisporus. The study was carried out using S. typhimurium strain TA104 and an activation system comprising mushroom tyrosinase (8000 units). Mushroom extracts, tyrosinase and bacteria were preincubated for 30 min at 378C in a shaking water-bath. Results are presented as mean2SD for triplicates. The spontaneous reversion rate of 3902 30 has already been subtracted. 4-Nitroquinoline Noxide (0.5 mg) induced a reversion rate of 1237285. (W) Without tyrosinase; (Q) with tyrosinase. (A) Raw mushroom; (B) mushroom baked at 1008C for 4 hr; (C) mushroom baked at 2258C for 10 min and (D) freeze-dried mushroom.
Mutagenicity of mushroom
ence signi®cantly the cytosol-mediated mutagenic response (Fig. 2). The mutagenicity of raw mushroom extract was approximately doubled in the presence of 8000 units of mushroom tyrosinase as an activation system (Fig. 3). Baking, whether for a short time at high temperature or longer at lower temperature, as well as freeze-drying did not modulate the potentiation of the mutagenic response by tyrosinase (Fig. 3). DISCUSSION
The constituent(s) responsible for the carcinogenic and mutagenic activity of the edible mushroom A. bisporus has not been identi®ed. Initially, it was considered that the hydrazine, agaritine, b-N(g-L(+)glutamyl)-4-(hydroxymethyl) phenylhydrazine, present in substantial amounts in this mushroom, could be responsible, at least partly, for the genotoxic and carcinogenic eects. Indeed, agaritine can be converted to genotoxic intermediates that can bind covalently to proteins and induce mutations in the Ames test (Price et al., 1996; Walton et al., 1997a,b). Loss of the glutamyl group is catalysed by g-glutamyl transpeptidase in the kidney, the resulting free hydrazine being oxidized to a reactive intermediate, presumably the diazonium ion, which is a potent mutagen (Walton et al., 1997a,b). However, agaritine failed to display carcinogenicity in mice, whereas mushrooms were carcinogenic, and moreover, it was not responsible for the mutagenic activity of mushroom extracts (Papaparaskeva et al., 1991; Toth et al., 1981). It is likely that phenols and quinones present in this mushroom play a signi®cant role in the mutagenicity of mushroom extracts (Papaparaskeva-Petrides et al., 1993). As mushrooms are largely consumed in cooked form, it is pertinent to evaluate the eect of cooking on the direct and indirect mutagenicity of mushroom extracts. Mushrooms are consumed commonly in baked form. Two conditions for baking were employed: a short baking of 10 min at 2258C, as employed by Toth et al. (1992), and a prolonged baking for 4 hr, but at the lower temperature of 1008C. Extracts from the baked mushroom were clearly mutagenic in S. typhimurium strain TA104. Short-term baking did not signi®cantly in¯uence the mutagenic response compared with raw mushroom. Similar observations have been made when the tester bacterial strains were TA1535 and TA1537 (Toth et al., 1992). However, baking for 4 hr at 1008C decreased the mutagenic response, indicating that the direct mutagens present in mushroom cannot survive this cooking process. Inclusion of an activation system containing hepatic cytosol from Aroclor 1254-induced rats enhanced the mutagenic response elicited by the raw mushroom extracts in agreement with our previous observations (Papaparaskeva-Petrides et al.,
319
1993). A similar increase in mutagenic response was also seen with the extracts from the baked mushroom. The increase in mutagenic response caused by the presence of hepatic cytosol is believed to be the consequence of the formation of semiquinones which can either interact directly with DNA or may generate genotoxic reactive oxygen species following interaction with molecular oxygen. Indeed, the cytosol-mediated increases in mutagenic potency is inhibited by reduced glutathione and the antioxidant enzymes catalase and superoxide dismutase (Papaparaskeva-Petrides et al., 1993). Baking appears not to in¯uence the availability of phenols and quinones. As we have previously reported (PapaparaskevaPetrides et al., 1993), mushroom tyrosinase, a polyphenol oxidase, elevated the mutagenic response of raw mushroom extracts. A similar increase was observed with extracts from baked mushroom. It may be inferred that the cooking procedures investigated in this study do not destroy the tyrosinase substrates that are converted by this enzyme to mutagenic intermediates in the Ames test. Finally, freeze-drying had no signi®cant eect on the direct mutagenicity of mushroom extracts or the potentiation of the mutagenic response induced by the presence of hepatic cytosol from Aroclor 1254induced rats or mushroom tyrosinase. It may be concluded that freeze-dried mushroom utilized in the preparation of soups and sauces may retain the mutagenic activity of the fresh mushroom. In conclusion, it has been demonstrated that the constituents of the mushroom A. bisporus responsible for its direct and indirect mutagenicity in the Ames test are not heat labile. Direct mutagens were only destroyed partly by prolonged baking for 4 hr at 1008C. Baking had no major eect on the mutagenicity of mushroom extracts elicited by rat hepatic cytosol or mushroom tyrosinase. Finally, neither direct nor indirect mutagenicity of mushrooms were in¯uenced by freeze-drying. It is noteworthy to point out that in recent studies, published in abstract form, mice fed on diets containing mushroom, baked for 10 min at 220±2308C, exhibited a higher tumour incidence in many organs compared with controls, but the eect was not statistically signi®cant (Toth et al., 1997). The contribution of cooked or uncooked mushroom to human cancer is likely to be minimal since the mutagenic response is weak and the mutagens are water soluble and consequently their absorption through the gastrointestinal tract may be poor. Finally, the mutagenic compounds will be subject to deactivation by nucleophilic constituents of the diet and/or during their transport to the target cell. AcknowledgementsÐThe authors thank the Food Contaminants Division, Ministry of Agriculture, Fisheries and Food, for supporting this work (Project Ref: FS2058).
320
K. Walton et al. REFERENCES
Gry J. and Pilegaard K. (1991) Hydrazines in the cultivated mushroom (Agaricus bisporus). VaÊr FoÈda 43 (Suppl. 1), 1±38. GruÈter A., Friederich U. and WuÈrgler F. E. (1991) The mutagenicity of edible mushrooms in histidine-independent bacterial test system. Food and Chemical Toxicology 29, 159±165. Ioannides C. and Parke D. V. (1975) Mechanism of induction of hepatic drug metabolising enzymes by a series of barbiturates. Journal of Pharmacy and Pharmacology 27, 739±749. Maron D. M. and Ames B. N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173±215. Matsumoto K., Ita M., Yagyu S., Ogino H. and Hirono H. (1991) Carcinogenicity examination of Agaricus bisporus, edible mushroom in rats. Cancer Letters 58, 87± 90. Papaparaskeva C., Ioannides C. and Walker R. (1991) Agaritine does not mediate the mutagenicity of the edible mushroom Agaricus bisporus. Mutagenesis 6, 213± 317. Papaparaskeva-Petrides C., Ioannides C. and Walker R. (1993) Contribution of phenolic and quinonoid structures in the mutagenicity of the edible mushroom Agaricus bisporus. Food and Chemical Toxicology 31, 561±567. Price R. J., Walters D. G., Ho C., Mistry H., Renwick A. B., Wield P. T., Beamand J. A. and Lake B. G. (1996) Metabolism of [Ring-U-14C]agaritine by precision-cut rat, mouse and human liver and lung slices. Food and Chemical Toxicology 34, 603±609. Sanchez-Ferrer A., Rodriguez-Lopez J. N., GarciaCanovas F. and Garcia-Carmona F. (1995) Tyrosinase:
a comprehensive review of its mechanism. Biochimica et Biophysica Acta 1247, 1±11. Sterner O., Bergman R., Kesler E., Magnusson G., Nilsson L., Wickberg B. and Zimerson E. (1982) Mutagens in larger fungi. Forty-eight species screened for mutagenic activity in the Salmonella/microsome assay. Mutation Research 101, 269±281. Toth B. and Erickson J. (1986) Cancer induction in mice by feeding the uncooked cultivated mushroom of commerce Agaricus bisporus. Cancer Research 46, 4007± 4011. Toth B., Erickson J., Gannett T. and Lawson T. (1997) Baked Agaricus bisporus (AB) mushroom carcinogenesis in mice: another experimental approach (Abstract). FASEB Journal 11, 3341. Toth B., Gannet P., Rogan E. and Williamson J. (1992) Bacterial mutagenicity of extracts of the baked and raw Agaricus bisporus mushroom. In Vivo 6, 487±490. Toth B., Raha C. R., Wallcave L. and Nagel D. (1981) Attempted tumor induction with agaritine in mice. Anticancer Research 1, 255±258. von Wright A., Knuutinen J., Lindroth S., Pellinen M., Widen K.-G. and Seppa E. L. (1982) The mutagenicity of some edible mushrooms in the Ames test. Food and Chemical Toxicology 20, 265±267. Walton K., Coombs M. M., Catterall F. S., Walker R. and Ioannides C. (1997a) Bioactivation of the mushroom hydrazine, agaritine, to intermediates that bind covalently to proteins and induce mutation in the Ames test. Carcinogenesis 18, 1603±1608. Walton K., Coombs M. M., Walker R. and Ioannides C. (1997b) Bioactivation of mushroom hyrazines to mutagenic products by mammalian and fungal enzymes. Mutation Research 381, 131±139.
Eect of Baking and Freeze-drying on the Direct and Indirect Mutagenicity of Extracts from the Edible Mushroom Agaricus bisporus K. WALTON, R. WALKER and C. IOANNIDES* School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH,UK (Accepted 29 October 1997) AbstractÐThe objectives of the study were to evaluate the eect of baking and freeze-drying on the direct and indirect mutagenicity in the Ames test of ethanolic extracts from the edible mushroom Agaricus bisporus. Direct mutagenicity was not in¯uenced by baking for 10 min at 2258C, but more prolonged baking, for example 4 hr at 1008C reduced mutagenicity. Hepatic cytosol from Aroclor 1254-induced rats and mushroom tyrosinase potentiated the mutagenic response elicited by the mushroom extracts. Baking did not in¯uence either of these processes. Finally, freeze-drying in¯uenced neither the direct nor the indirect mutagenicity of the mushroom extracts. It is concluded that mutagenic and premutagenic compounds present in mushroom are generally not heat labile. # 1998 Elsevier Science Ltd. All rights reserved Keywords: mushroom; Agaricus bisporus; Ames test; mutagenicity.
INTRODUCTION
Agaricus bisporus is the most extensively consumed type of mushroom worldwide (Gry and Pilegaard, 1991). Concern about the safety of this mushroom was expressed following the studies of Toth and Erickson (1986), who demonstrated that lifetime administration of uncooked A. bisporus to mice induced tumours at a number of sites. However, when air-dried mushroom were fed to rats for 500 days, no carcinogenic eect was evident (Matsumoto et al., 1991). It is not yet clear whether this marked dierence in carcinogenic response re¯ects a species dierence in susceptibility or whether it is due to the dierent protocols employed in the two studies. Extracts from this mushroom can induce a mutagenic response in the Ames mutagenicity test, in the absence of an activation system, particularly when TA104 is used as the Salmonella typhimurium tester strain (Papaparaskeva et al., 1991; Sterner et al., 1982; von Wright et al., 1982). This mutagenic response cannot be ascribed to traces of the histidine present in the ethanolic extracts of the mushroom since the mutagenic potential was also evident in studies using histidine-independent bacterial strains (GruÈter et al., 1991). Moreover, mutagenic response was suppressed by the nucleophile glutathione and the hydroxyl radical scavenger dimethyl sulf*Author for correspondence.
oxide (Papaparaskeva et al., 1991; PapaparaskevaPetrides et al., 1993). The presence of the standard activation system, Aroclor 1254-induced rat hepatic S-9, failed to enhance signi®cantly the mutagenic response. However, the mutagenic eect was elevated in the presence of hepatic cytosol derived from Aroclor 1254-induced rats, and supplemented with NADPH (Papaparaskeva-Petrides et al., 1993). This eect was prevented by the addition of dicoumarol and menadione, an inhibitor and substrate respectively, of the cytosolic DT-diaphorase. It was concluded that phenols and quinones present in the mushroom could be converted to semiquinones that could directly interact with DNA or act as generators of reactive oxygen species following interaction with molecular oxygen. The mutagenicity of the mushroom extracts was also enhanced in the presence of mushroom tyrosinase (Papaparaskeva-Petrides et al., 1993). This polyphenol oxidase catalyses the conversion of phenols to quinones through the formation of dihydroxycompounds. A monophenolase activity facilitates the o-hydroxylation of the monophenol to the o-diphenol, and a diphenolase activity converts the o-diphenol to the o-quinone (Sanchez-Ferrer et al., 1995). Mushrooms are commonly baked before consumption, for example in products such as pizzas. The majority of the consumption is in the form of baked mushroom found in many products such as pizzas. This prompted us to investigate the
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(97)00161-0
316
K. Walton et al.
eect of baking on the mutagenicity of extracts from A. bisporus using the S. typhimurium strain TA104 which shows the clearest response to the direct mutagenicity of mushroom extracts. Earlier studies (Toth et al., 1992), showed that short-term baking of mushrooms did not in¯uence their direct mutagenicity in S. typhimurium strains TA1535 and 1537. Moreover, freeze-dried mushrooms are extensively used in the production of soups and sauces; consequently, the eect of freeze-drying on the mutagenicity of mushroom extracts was also investigated.
MATERIALS AND METHODS
Aroclor 1254 (Robens Institute, Guildford, UK), mushroom tyrosinase and all cofactors (Sigma Chemical Co., Poole, Dorset, UK) were all purchased. A. bisporus mushrooms were obtained locally. Mushrooms were rinsed, blotted dry and then homogenized in 96% ethanol (v/v) at a concentration of 1 g/ml. The extract was ®ltered through muslin and Whatman ®lter paper (No. 10). The ®ltrate was evaporated down to about 10% of its
original volume, and was stored at ÿ208C until use. Aliquots of mushroom were baked evenly for either 10 min at 2258C or 4 hr at 1008C. The mushrooms were then reweighed and reconstituted to the original weight using water. Finally, an aliquot of mushroom was snap-frozen in liquid nitrogen and freeze-dried for 12 hr. Mushrooms were reweighed and the original weight reconstituted by the addition of water. Extracts of the baked and freezedried mushrooms were prepared as described for raw mushroom (see above). Male Wistar albino rats (about 200 g) were obtained from Harlan Olac (Bicester, Oxon., UK). Treatment with Aroclor 1254 comprised a single ip administration (500 mg/kg), the animals being killed on the 5th day following administration. Hepatic cytosolic fractions were prepared as previously described (Ioannides and Parke, 1975). The mutagenicity of the mushroom extracts was determined using the Ames mutagenicity test and employing S. typhimurium strain TA104 (Maron and Ames, 1983). Activation systems contained 10% (v/v) hepatic cytosol from Aroclor 1254-induced rats or mushroom tyrosinase (8000 units).
Fig. 1. Eect of baking and freeze-drying on the direct mutagenicity of extracts from the mushroom A. bisporus. The study was carried out using S. typhimurium strain TA104. Results are presented as mean 2SD for triplicates. The spontaneous reversion rate of 419213 has already been subtracted. 4Nitroquinoline N-oxide (0.5 mg) induced a reversion rate of 1118299. (*) Raw mushroom; (Q) mushroom baked at 1008C for 4 hr; (R) mushroom baked at 2258C for 10 min, and (W) freeze-dried mushroom.
Mutagenicity of mushroom RESULTS
As expected, raw mushroom extracts were mutagenic in S. typhimurium strain TA104. A concentration-dependent eect was evident, with doubling of the spontaneous reversion rate achieved at the highest concentration studied, that is, 100 ml/plate
317
(Fig. 1). Mutagenic potential was unaected by baking at 2258C for 10 min. However, a more prolonged baking at a lower temperature, that is at 1008C for 4 hr, suppressed the mutagenic response. Freeze-drying of the mushroom did not alter signi®cantly the mutagenic response (Fig. 1).
Fig. 2. Eect of baking and freeze-drying on the cytosol-mediated mutagenicity of extracts from the mushroom A. bisporus. The study was carried out using S. typhimurium strain TA104 and an activation system (10% v/v) derived from Aroclor 1254-induced rats. Results are presented as mean2SD for triplicates. The spontaneous reversion rate of 388232 has already been subtracted. 4-Nitroquinoline Noxide (0.5 mg) induced a reversion rate of 1401276. (W) Without cytosol; (Q) with cytosol. (A) Raw mushroom; (B) mushroom baked at 1008C for 4 hr; (C) mushroom baked at 2258C for 10 min and (D) freeze-dried mushroom.
318
K. Walton et al.
Inclusion of an activation system comprising hepatic cytosol from Aroclor 1254-induced rats, forti®ed with an NADPH-generating system, clearly enhanced the mutagenic response of raw mushroom extracts (Fig. 2). When extracts from mushroom baked at 2258C for 10 min were used, hepatic cyto-
sol still increased the mutagenic response, the eect being of a similar magnitude to that observed with the raw mushroom. Baking at 1008C for 4 hr reduced the direct mutagenic response but mutagenicity was still increased following the addition of hepatic cytosol. Finally, freeze-drying did not in¯u-
Fig. 3. Eect of baking and freeze-drying on the tyrosinase-mediated mutagenicity of extracts from the mushroom A. bisporus. The study was carried out using S. typhimurium strain TA104 and an activation system comprising mushroom tyrosinase (8000 units). Mushroom extracts, tyrosinase and bacteria were preincubated for 30 min at 378C in a shaking water-bath. Results are presented as mean2SD for triplicates. The spontaneous reversion rate of 3902 30 has already been subtracted. 4-Nitroquinoline Noxide (0.5 mg) induced a reversion rate of 1237285. (W) Without tyrosinase; (Q) with tyrosinase. (A) Raw mushroom; (B) mushroom baked at 1008C for 4 hr; (C) mushroom baked at 2258C for 10 min and (D) freeze-dried mushroom.
Mutagenicity of mushroom
ence signi®cantly the cytosol-mediated mutagenic response (Fig. 2). The mutagenicity of raw mushroom extract was approximately doubled in the presence of 8000 units of mushroom tyrosinase as an activation system (Fig. 3). Baking, whether for a short time at high temperature or longer at lower temperature, as well as freeze-drying did not modulate the potentiation of the mutagenic response by tyrosinase (Fig. 3). DISCUSSION
The constituent(s) responsible for the carcinogenic and mutagenic activity of the edible mushroom A. bisporus has not been identi®ed. Initially, it was considered that the hydrazine, agaritine, b-N(g-L(+)glutamyl)-4-(hydroxymethyl) phenylhydrazine, present in substantial amounts in this mushroom, could be responsible, at least partly, for the genotoxic and carcinogenic eects. Indeed, agaritine can be converted to genotoxic intermediates that can bind covalently to proteins and induce mutations in the Ames test (Price et al., 1996; Walton et al., 1997a,b). Loss of the glutamyl group is catalysed by g-glutamyl transpeptidase in the kidney, the resulting free hydrazine being oxidized to a reactive intermediate, presumably the diazonium ion, which is a potent mutagen (Walton et al., 1997a,b). However, agaritine failed to display carcinogenicity in mice, whereas mushrooms were carcinogenic, and moreover, it was not responsible for the mutagenic activity of mushroom extracts (Papaparaskeva et al., 1991; Toth et al., 1981). It is likely that phenols and quinones present in this mushroom play a signi®cant role in the mutagenicity of mushroom extracts (Papaparaskeva-Petrides et al., 1993). As mushrooms are largely consumed in cooked form, it is pertinent to evaluate the eect of cooking on the direct and indirect mutagenicity of mushroom extracts. Mushrooms are consumed commonly in baked form. Two conditions for baking were employed: a short baking of 10 min at 2258C, as employed by Toth et al. (1992), and a prolonged baking for 4 hr, but at the lower temperature of 1008C. Extracts from the baked mushroom were clearly mutagenic in S. typhimurium strain TA104. Short-term baking did not signi®cantly in¯uence the mutagenic response compared with raw mushroom. Similar observations have been made when the tester bacterial strains were TA1535 and TA1537 (Toth et al., 1992). However, baking for 4 hr at 1008C decreased the mutagenic response, indicating that the direct mutagens present in mushroom cannot survive this cooking process. Inclusion of an activation system containing hepatic cytosol from Aroclor 1254-induced rats enhanced the mutagenic response elicited by the raw mushroom extracts in agreement with our previous observations (Papaparaskeva-Petrides et al.,
319
1993). A similar increase in mutagenic response was also seen with the extracts from the baked mushroom. The increase in mutagenic response caused by the presence of hepatic cytosol is believed to be the consequence of the formation of semiquinones which can either interact directly with DNA or may generate genotoxic reactive oxygen species following interaction with molecular oxygen. Indeed, the cytosol-mediated increases in mutagenic potency is inhibited by reduced glutathione and the antioxidant enzymes catalase and superoxide dismutase (Papaparaskeva-Petrides et al., 1993). Baking appears not to in¯uence the availability of phenols and quinones. As we have previously reported (PapaparaskevaPetrides et al., 1993), mushroom tyrosinase, a polyphenol oxidase, elevated the mutagenic response of raw mushroom extracts. A similar increase was observed with extracts from baked mushroom. It may be inferred that the cooking procedures investigated in this study do not destroy the tyrosinase substrates that are converted by this enzyme to mutagenic intermediates in the Ames test. Finally, freeze-drying had no signi®cant eect on the direct mutagenicity of mushroom extracts or the potentiation of the mutagenic response induced by the presence of hepatic cytosol from Aroclor 1254induced rats or mushroom tyrosinase. It may be concluded that freeze-dried mushroom utilized in the preparation of soups and sauces may retain the mutagenic activity of the fresh mushroom. In conclusion, it has been demonstrated that the constituents of the mushroom A. bisporus responsible for its direct and indirect mutagenicity in the Ames test are not heat labile. Direct mutagens were only destroyed partly by prolonged baking for 4 hr at 1008C. Baking had no major eect on the mutagenicity of mushroom extracts elicited by rat hepatic cytosol or mushroom tyrosinase. Finally, neither direct nor indirect mutagenicity of mushrooms were in¯uenced by freeze-drying. It is noteworthy to point out that in recent studies, published in abstract form, mice fed on diets containing mushroom, baked for 10 min at 220±2308C, exhibited a higher tumour incidence in many organs compared with controls, but the eect was not statistically signi®cant (Toth et al., 1997). The contribution of cooked or uncooked mushroom to human cancer is likely to be minimal since the mutagenic response is weak and the mutagens are water soluble and consequently their absorption through the gastrointestinal tract may be poor. Finally, the mutagenic compounds will be subject to deactivation by nucleophilic constituents of the diet and/or during their transport to the target cell. AcknowledgementsÐThe authors thank the Food Contaminants Division, Ministry of Agriculture, Fisheries and Food, for supporting this work (Project Ref: FS2058).
320
K. Walton et al. REFERENCES
Gry J. and Pilegaard K. (1991) Hydrazines in the cultivated mushroom (Agaricus bisporus). VaÊr FoÈda 43 (Suppl. 1), 1±38. GruÈter A., Friederich U. and WuÈrgler F. E. (1991) The mutagenicity of edible mushrooms in histidine-independent bacterial test system. Food and Chemical Toxicology 29, 159±165. Ioannides C. and Parke D. V. (1975) Mechanism of induction of hepatic drug metabolising enzymes by a series of barbiturates. Journal of Pharmacy and Pharmacology 27, 739±749. Maron D. M. and Ames B. N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173±215. Matsumoto K., Ita M., Yagyu S., Ogino H. and Hirono H. (1991) Carcinogenicity examination of Agaricus bisporus, edible mushroom in rats. Cancer Letters 58, 87± 90. Papaparaskeva C., Ioannides C. and Walker R. (1991) Agaritine does not mediate the mutagenicity of the edible mushroom Agaricus bisporus. Mutagenesis 6, 213± 317. Papaparaskeva-Petrides C., Ioannides C. and Walker R. (1993) Contribution of phenolic and quinonoid structures in the mutagenicity of the edible mushroom Agaricus bisporus. Food and Chemical Toxicology 31, 561±567. Price R. J., Walters D. G., Ho C., Mistry H., Renwick A. B., Wield P. T., Beamand J. A. and Lake B. G. (1996) Metabolism of [Ring-U-14C]agaritine by precision-cut rat, mouse and human liver and lung slices. Food and Chemical Toxicology 34, 603±609. Sanchez-Ferrer A., Rodriguez-Lopez J. N., GarciaCanovas F. and Garcia-Carmona F. (1995) Tyrosinase:
a comprehensive review of its mechanism. Biochimica et Biophysica Acta 1247, 1±11. Sterner O., Bergman R., Kesler E., Magnusson G., Nilsson L., Wickberg B. and Zimerson E. (1982) Mutagens in larger fungi. Forty-eight species screened for mutagenic activity in the Salmonella/microsome assay. Mutation Research 101, 269±281. Toth B. and Erickson J. (1986) Cancer induction in mice by feeding the uncooked cultivated mushroom of commerce Agaricus bisporus. Cancer Research 46, 4007± 4011. Toth B., Erickson J., Gannett T. and Lawson T. (1997) Baked Agaricus bisporus (AB) mushroom carcinogenesis in mice: another experimental approach (Abstract). FASEB Journal 11, 3341. Toth B., Gannet P., Rogan E. and Williamson J. (1992) Bacterial mutagenicity of extracts of the baked and raw Agaricus bisporus mushroom. In Vivo 6, 487±490. Toth B., Raha C. R., Wallcave L. and Nagel D. (1981) Attempted tumor induction with agaritine in mice. Anticancer Research 1, 255±258. von Wright A., Knuutinen J., Lindroth S., Pellinen M., Widen K.-G. and Seppa E. L. (1982) The mutagenicity of some edible mushrooms in the Ames test. Food and Chemical Toxicology 20, 265±267. Walton K., Coombs M. M., Catterall F. S., Walker R. and Ioannides C. (1997a) Bioactivation of the mushroom hydrazine, agaritine, to intermediates that bind covalently to proteins and induce mutation in the Ames test. Carcinogenesis 18, 1603±1608. Walton K., Coombs M. M., Walker R. and Ioannides C. (1997b) Bioactivation of mushroom hyrazines to mutagenic products by mammalian and fungal enzymes. Mutation Research 381, 131±139.