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Contribution of phenolic and quinonoid structures in the mutagenicity of the edible mushroom Agaricus bisporus
Fd Chem. Toxic. Vol. 31, No. 8, pp. 561-567, 1993 Printed in Great Britain. All rights reserved
027S-6915/93 $6.00-~ 0.00 Copyright ,t' 1993Pergamon Press Ltd
C O N T R I B U T I O N OF P H E N O L I C A N D Q U I N O N O I D S T R U C T U R E S IN THE M U T A G E N I C I T Y OF THE EDIBLE MUSHROOM AGARICUS BISPORUS C. PAPAPARASKEVA-PETRIDES,C. IOANNIDES* and R. WALKER Division of Toxicology, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK (Accepted 8 April I993) Abstract The objectives of this work were to establish the contribution of agaritine in the mutagenicity of ethanolic extracts from Agaricus bisporus and to examine the possible involvement of phenolic and quinonoid compounds in the mutagenic response to mushrooms. The mutagenic profile of agaritine in the Ames test, in the absence of an activation system, was different from that of the mushroom ethanolic extracts. Incorporation of rat hepatic cytosolic fractions as the activation system increased the mutagenicity of the mushroom ethanolic extracts in Salmonella typhimurium strain TA104 but did not influence the mutagenicity of agaritine. It was concluded that agaritine is not the principal mutagenic component in the mushroom. The cytosol-induced mutagenicity of the mushroom extracts required NADPH, and was inhibited by dicoumarol and menadione. Moreover, the mutagenic response in the presence of cytosolic fractions was inhibited by superoxide dismutase, catalase, glutathione and dimethyl sulfoxide, thus implicating reactive oxygen species. Finally, tyrosinase, the enzyme converting mushroom phenols to quinones, increased the mutagenicity of the mushroom extracts. Collectively, the above results indicate that phenolic and quinonoid compounds, presumably through the generation of reactive oxygen species, may play a significant role in the mutagenicity of mushroom extracts.
INTRODUCTION
Many chemicals with carcinogenic potential have been detected in the diet; they are inherent to food (e.g. hydrazines) and contaminants (e.g. mycotoxins), or generated during the cooking process (e.g. aminoimidazoazaarenes). It is rarely, however, that an item of daily food displays itself carcinogenic potential in animal long-term lifetime bioassays. Toth and Erickson (1986) reported that excessive consumption of the edible mushroom Agaricus bisporus, by far the most extensively cultivated and used mushroom in the Western hemisphere, induced tumours at multiple sites in Swiss mice. Recent studies using the same protocol revealed that a wild edible mushroom of the false morel family Gyromitra esculenta also induced tumours at many sites in Swiss mice (Toth et al., 1992). In contrast, when Agaricus bisporus was tested in rats using a different protocol no carcinogenic response was evident; this finding raises the possibility that mushroom-induced tumours may be specific to mice (Matsumoto et al., 1991). Extracts from A. bisporus have been shown to elicit a positive but weak mutagenic response in mutagenicity systems, such as the Ames test, using a variety of Salmonella typhimurium strains (Friedrich et al., *To whom correspondence and reprint requests should be addressed. Abbreviations: DMSO=dimethyl sulfoxide; GHB=7-Lglutaminyl-4-hydroxybenzene. 561
1986; Rogan et al., 1982), but a more pronounced mutagenic response was evident when the T A I 0 4 bacterial strain was used (Papaparaskeva et al., 1991). In contrast, A. bisporus extracts failed to provoke a positive mutagenic response in other in vitro mutagenicity tests and in vivo tests such as the micronucleus test (Morales et al., 1990; Pool-Zobel et al., 1990). Pool-Zobel et al. (1990) considered the possibility that the mutagenicity of the A. bisporus extracts represented nothing more than the presence of a histidine artefact which interfered with the test. However, subsequent investigations using a histidineindependent bacterial strain confirmed the mutagenic potential of this mushroom (Gr/iter et al., 1991). The naturally occurring hydrazide agaritine (L-glutamic acid-5-[2-4(hydroxymethyl)]phenyl hydrazide) has been implicated as the component responsible for the mutagenicity/carcinogenicity of A. bisporus. In contrast to this hypothesis, however, the mutagenicity of various mushroom samples did not correlate with the agaritine content and, moreover, exogenous supply of 7-glutamyl transpeptidase, the enzyme catalysing the activation of agaritine, did not influence the mutagenic potential of A. bisporus extracts, thus casting doubt on the role of hydrazide agaritine in mushroom mutagenicity (Papaparaskeva et al., 1991). Furthermore, agaritine, when administered to mice for life in the drinking water or following sc administration, did not provoke a positive carcinogenic response (Toth et al., 1981; Toth and Sornson, 1984). Clearly, agaritine cannot
562
( ' . PAPAPARASKEVA=PETRII)ES el gl]. Table I C'omparison of the mutagenicities of agaritine and Agaru'us hts'porus extracts in the Ames tesl Histidine revertants plate Compound Sponlaneoas reversion rate Agaritine {2861~g) M u s h r o o m extract 1100 ~1 corresponding to 286 l*g agaritine)
TAI535
TAI537
TAI538
TA97
TA98
TAI00
TAI02
TA. 104
20+3 22+ I 39 ~ 4
II ~ I 30~2 22 I
7÷ I S+ I 14 ~ I
113+7 241 + 3 4 226 ~ 2
12+1 t6+2 29 + I
81+9 96+5 194+ 17
259+17 329+29 519+35
337 ~ 16 562 5 6 4 1193+206
The mutagenicity of m u s h r o o m extracts was compared with that of agaritine (2861~g). The volume of ethanolic m u s h r o o m extract containing 2861~g agaritme (i.e. 100t, i) was calculated o n the assumption that I kg fresh m u s h r o o m contains 0.05g agaritine (Stijve ct al., 19861. The numbers of histidine revertants are presented as means + SD of three plates (triplicate assays).
] able 2. Effect of the activation h_v hepatic Qtosolic fractions on the mutagenicilies of agaritine and ethanolic extracts of Agaricu.s hi.sporu.s Histidine revertants/plate Compound
No activation
With cytosolic activation
344 ~- 19 713 + 20 1268 + 69
325 + 11 733 + 38 156g + 42
Spontaneous reversion rate Agaritine (2861~g) M u s h r o o m extract (100 l~l corresponding to 2~6 l~g agaritine)
The activation system contained 10% (v/v) hepatic cytosolic fractions from Aroclor 1254-treated rats. M ushroom extracts corresponding to an agaritine level of 286 ~g, authentic agaritine (286/.tg), and S. typhimurium T A 104 were used. Results are presented as means ~ SD of three plates (triplicate assays).
A. his7~orus mushrooms were obtained locally and were always used within 24 hr following purchase. They were rinsed with water and then homogenized in 96% (v/v) ethanol at a concentration of l g/ml using an Osterizer blender. The homogenate was filtered through muslin and Whatman filter paper (Qualitative No. 1). Ethanol was finally evaporated in a rotary evaporator, and the final volume was aboul 20% of the original tiltrate. Male Wistar albino rats (about 200 g) were obtained from the Experimental Biology Unit (University of Surrey, Guildford, Surrey, UK). Induction of the hepatic mixed-function oxidases was achieved by a single ip injection of Aroclor 1254 (500 mg/kg body weight) dissolved in corn oil (200 mg/ml); the animals were killed on the fifth day following dosing. Hepatic cytosolic fractions were prepared as previously described (Ioannides and Parke, 1975). The mutagenicity of mushroom extracts was determined using the Ames mutagenicity test (Maron and Ames, 1983). All activation systems used contained 10% (v/v) hepatic cytosolic fractions from Aroclor
account for the mutagenic and carcinogenic effects of
A. bisporus. In the present study we provide further experimental evidence that agaritine is not the major contributor to the mutagenicity of A. bisporus and that quinonoid and phenolic chemicals may be largely responsible for the mulagenic response. MATERIALS AND METHODS
Oxidized and reduced glutathione, menadione, catalase, superoxide dismutase, mushroom tyrosinase and all cofactors (Sigma Chemical Co., Poole, Dorset, UK), Aroclor 1254 (Robens Institute, Guildford, UK) as well as dicoumarol (Aldrich Chemical Co., Ltd, Gillingham, Dorset, UK) were all purchased. Pure agaritine and the various Salmonella typhimurium strains were generous gifts from Professor B. Toth (The Eppley Institute of Research in Cancer, University of Nebraska Medical Center, USA) and from Professor B. N. Ames (Berkeley, CA, USA), respectively.
Table 3. Effect of the presence of hepatic cytosolic fractions in the mutagenic activity of ethanolic extracts from Agaricus bisporus H st d ne revertants'plate Compound Spontaneous reversion rate Mushroom extracts
TA97
TA98
--cyt
+cyl
98+24
90- 8
184+8
190±4
T A 100
cyt
+cyt
c vt
17-4
18-+1
95~1
44_+9
44+12
201=24
+c.vt
T A 102 cyt
T A 104 q- cyt
108- I
259+17
289+29
202+9
419±35
457+2
cyt
TA1537 +cyl
cyt
+cyt
337+16
394:+:41
I1+1
15-+2
1[93-+96
1506±91
16+1
18_+2
The activation system contained I1)% (v,v) hepatic cytosolic fraction (cyt) from Aroclor 1254-treated rats• Results are expressed as means +_ SD o f three p[ates (triplicate assays).
Mutagenicity of edible mushroom
563
Table 4. Role of NADPH and NADH in the cytosolic activation of ethanolic extracts of Agaricm
hisporus Extract Type of assay No activation Cytosolic activation system +NADPHgeneratingsystem Cytosolic activation system -- N A D P H generating system
947 -~ 152
Histidine revertants'plate Extract Extract Extract q+ + 1.5mM-NADH 3.0mM-NADH 4.5mM-NADH 953 + 101
909 ± 92
919 + 121
1223+41
787±53
829±67
859+62
764 + 75
755 + 25
766 ± 67
7(17 _+ 53
Salmonella typhhnurium TA 104 and mushroom extracts corresponding to 0.6 g flesh mushroom were used. The activation system contained hepatic cytosolic fractions (10%. vv) fi-om Aroclor 1254-treated rats. The spontaneous reversion rate of 417_+_27 has already been subtracted. Results are expressed as mean + SD of three plates (triplicate assays).
1254-treated rats. V a r i a t i o n s o f the p r o c e d u r e are d e s c r i b e d in the f o o t n o t e s to the a p p r o p r i a t e tables. RESULTS
T h e m u t a g e n i c i t y o f the e t h a n o l i c extracts o f A.
bisporus was e v a l u a t e d in the A m e s test in the a b s e n c e o f an a c t i v a t i o n system, using eight S. typhimurium strains, a n d was c o m p a r e d with the m u t a g e n i c i t y o f the c o r r e s p o n d i n g a m o u n t o f agaritine a s s u m i n g that m u s h r o o m has an agaritine c o n t e n t o f 50 m g / k g (Stijve et al., 1986). A g a r i t i n e elicited a clear positive m u t a g e n i c r e s p o n s e in strain T A 9 7 a n d its predecessor TA1537 (Table 1). A n increase in the reversion rate was also seen in T A I 0 4 , but did n o t attain d o u b l i n g o f the s p o n t a n e o u s reversion rate. T h e m u s h r o o m e t h a n o l i c extracts, in c o n t r a s t , p r o v o k e d
F--] M u s h r o o m extract
4000
Extract + DMSO
Extract
dicoumarol
3500 -0J
3000 -250(I --
-7
I
"T-
2000 1500 1000
//
i::i::i::iiil~i ii
///
a positive m u t a g e n i c r e s p o n s e in all bacterial strains, especially in T A I 0 4 (Table 1). In a s e c o n d study, agaritine achieved a positive m u t a g e n i c r e s p o n s e in T A I 0 4 as indicated by a d o u b l i n g o f the s p o n t a n e o u s reversion rate, but the m u t a g e n i c i t y was still m u c h lower t h a n that o f m u s h r o o m extracts c o n t a i n i n g the equivalent a m o u n t o f agaritine (Table 2). I n c o r p o r a t i o n o f rat hepatic cytosolic fractions p o t e n t i a t e d the m u t a g e n i c i t y o f the m u s h r o o m extracts but did not influence the m u t a g e n i c i t y o f agaritine (Table 2). T h e ability o f hepatic cytosolic fractions to increase m u s h r o o m m u t a g c n i c i t y was f u r t h e r evaluated in a n u m b e r o f bacterial strains (Table 3). T h e cytosolic fractions p o t e n t i a t e d the m u t a g e n i c r e s p o n s e only in bacterial strain T A 104. T h e c y t o s o l - m e d i a t e d m u s h r o o m m u t a g e n i c i t y required N A D P H a n d could n o t be s u p p o r t e d by N A D H (Table 4). M o r e o v e r , N A D H inhibited the c y t o s o l - i n d u c e d m u t a g e n i c i t y in the presence o f N A D P H (Table 4). T h e c y t o s o l - i n d u c e d m u t a g e n i c ity was i m p a i r e d by d i c o u m a r o l , which h o w e v e r had no effect on the direct m u t a g e n i c i t y o f the m u s h r o o m e t h a n o l i c extracts (Fig. 1). It is w o r t h n o t i n g that d i m e t h y l sulfoxide ( D M S O ) , the vehicle for dicoum a r o l , also r e d u c e d m u t a g e n i c response. Similar to d i c o u m a r o l , the c y t o s o l - m e d i a t e d m u t a g e n i c i t y o f the m u s h r o o m ethanolic extracts was c o m p l e t e l y inhibited by m e n a d i o n e , which h o w e v e r did n o t p e r t u r b the direct m u t a g e n i c i t y o f the m u s h r o o m extracts in the a b s e n c e o f an a c t i v a t i o n system (Table 5). T h e e n z y m e s catalase a n d s u p e r o x i d e d i s m u t a s e inhibited m u s h r o o m mutagenicity, b o t h in the presence a n d
500 0 A
B
Fig. 1. Inhibition of the cytosol-mediated activation of Agaricus bisporus extracts by dicoumarol. The activation system contained hepatic cytosolic fractions (2.5%, v/v) derived from Aroclor 1254-treated rats. Ethanolic mushroom extracts (100pl), dicoumarol dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 30 ,UM and S. typhimurium strain TAI04 were used. The spontaneous reversion rate of 506 + 38 has already been subtracted. Results are presented as means _+ SD of triplicate assays. A: in the absence of cytosok B: in the presence of cytosol. FCI 3 1 / ~
Table 5. Modulation of the mutagenicity of ethanolic extracts from Agaricus hisporus by menadione Histidine reverants/plate Compound No activation With activation Mushroom extracts 1462 i 97 1938 + 23 Mushroom extracts and menadione 1363 + 45 1172 ± 149 The activation system contained 2.5% (v/v) hepatic cytosolic fractions from Aroclor 1254-treated rats. Ethanolic mushroom extracts (100 p I), 9 mM-menadione dissolved in dimethyl sulfoxide and S. typhimurium TAI04 were used. The spontaneous reversion rate of 418 + 28 has already been subtracted. The numbers of histidine revertants are presented as means + SD of three plates (triplicate assays).
564
el
C. PAPAPARASKEVA-PETRIDES I
al.
[•
No acti,,ation
L t~llO R
1400
--0--
('alalasc
--+--
SO1)
- - . - -
('ala hlsc,'SOD
1400
Mushroum
cxtracl
Mushrmm~
extract
+ GStl
Mushroom
cxHact
+ GSS(I
120()
120O
~,~.
lOl)O
T.
T
I0()0
i "o
@tO
,-'~
4(}{}
-"
#::;:i.[iiii
.~
~
c\'tosnlic
"==& 1 5 ( I 0
>
I{)0
150
2(1(I
4(111
250 :#::#.Uii~ !!i:p!%%:i:i~:
I 2(I(I
activation
('atahlsc
--+--
St)It
~'--
('atalasc"SOD T
Fig. 3. Inhibition of AgarictB bi.worus mutagenicity by glutathione. The mutagenicity was determined with S. typhimurium TA104 and mushroom ethanolic extracts (100,ul), without {At or with (B) an activation system containing hepatic cytosolic fractions (10%, v/v) derived from Aroclor 1254-treated rats, in the presence of oxidized or reduced glutathione (5 mM). The spontaneous reversion rate of 401 _+ 26 has already been subtracted. Results are presented as means + SD of triplicate assays.
b 5011
i 5(1
I00
150
B
A
--o--
tO01}
m ~-
I
.2_
t)
20110
~;~'i:i:i:i~i:?i:
!
E n z y m e a c t i v i t y {units/plate)
2. W i t h
ii!ili!i~i~iii~i~i
600
20() L 0
i
bit)l}
i
]
2(10
250
Enzyme activity (units/plate)
Fig. 2. Inhibition of Agaricus bi~porus mutagenicity by catalase and superoxide dismutase. The test was carried out with S. typhimurium T A I 0 4 and mushroom ethanolic extracts (100 ,u 1) with or without an activation system containing hepatic cytosolic fractions (10%, v/v) derived from Aroclor 1254-treated rats, in the presence of catalase and/or superoxide dismutase (SOD). The spontaneous reversion rate of 390 _+ 17 has already been subtracted. Each point represents the mean _+ SD of a triplicate assay. a b s e n c e o f c y t o s o l i c fractions (Fig. 2). S i m u l t a n e o u s i n c o r p o r a t i o n of both e n z y m e s w a s the m o s t effective w a y o f inhibiting m u t a g e n c i t y . T h e c y t o s o l - i n d u c e d m u t a g e n i c i t y of m u s h r o o m extrracts w a s m a r k e d l y inhibited by both o x i d i z e d and reduced g l u t a t h i o n e Table 6. Effect of pH on the tyrosinasemediated activation of ethanolic extracts of Agaricus bisporu.~
(Fig. 3). In the a b s e n c e o f c y t o s o l i c fractions, o n l y reduced g l u t a t h i o n e decreased the m u t a g e n i c res p o n s e . T h e inhibitory effect of D M S O on the mutagenicity of the m u s h r o o m extracts, both in the a b s e n c e and presence of rat hepatic c y t o s o l i c fractions w a s investigated in m o r e detail. As s h o w n in Fig. 4, m u t a g e n i c r e s p o n s e w a s inhibited whether c y t o s o l i c fractions were present or not. 201)0
=
1500
>
I(1011
-:tq
.-j •-
511(1
Histidine revertants/plate pH
tyrosinase
+ tyrosinase
7.4 8.0
1037 ± 51 1024 ± 44
1323_+40 1768 _+ 164
Ethanolic mushroom extracts (1001~l) were preincubated with S. typhimurium (TAI04) in (it the absence of tyrosinase or (ii) the presence of tyrosinase (8000 units) for 25 min at 37 C in a shaking water-bath. The spontaneous reversion rate of 272 + 4 has already been subtracted. The numbers of histidine revertants are presented as means+ SD of three plates (
__l 1()0
i
i
2Im
30[)
400
500
7oncentration of DMSO (BI/plate) Fig. 4. Inhibition of Agaricus bisporus mutagenicity by dimethyl sulfoxide (DMSO). The assay was performed with S. typhimurium TA104 and mushroom ethanolic extracts (100 ttl) and with ( + ) or without ([Z]) an activation system containing hepatic cytosolic fractions (10%, v/v) from Aroclot 1254-treated rats. The spontaneous reversion rate of 394 4- 36 has already been subtracted. Each point represents the mean + SD of a triplicate assay.
Mutagenicity of edible mushroom 2000 151)0 > 1It(1(I
f
(a)
T/T/T
.~_ 51111 7~ 0 ~/ 11
I
I
I
I
I
!
21)
40
60
NO
ll)O
120
Concentration of extract (pd/plate) o
"~
101)0
U~> 800 ~u
(b)
9(R) /
700 600
4oo
I I J I I i I I 1) l 2 3 4 6 7 S 9 I() Tyrosinase concn (units/plate x 1000)
Fig. 5. Mutagenicity of Agaricus bisporus ethanolic extracts by tyrosinase. (at Mushroom extracts were preincubated with tyrosinase (8000 units) for 25 min at 37C in a shaking water-bath: ( + ) with tyrosinase and (Q) without tyrosinase. The spontaneous reversion rate of 353 +_9 has already been subtracted. (b) Mushroom extracts (75/~1) were preincubated with S. o'phimurium TAI04 and tyrosinase for 25 min at 37"C in a shaking water-bath. Each point represents the mean _+SD of a triplicate assay. Incorporation of purified mushroom tyrosinase as an activation system increased the mutagenicity of mushroom extracts (Fig. 5at. The increase in mutagenie response was dependent on tyrosinase concentration up to a level of 2000 units/plate (Fig. 5b). Increasing the pH from 7.4 to 8.0 increased the mutagenicity of the mushroom extracts in the presence of tyrosinase (Table 6). Finally, in the presence of cytosol, tyrosine (1 mM) failed to influence the mushroom mutagenicity in TAI04 (results not shown). DISCUSSION Agaritine is the mushroom constituent that was implicated in the mutagenictty and carcinogenicity of A. bisporus despite the fact that it was not carcinogenic in mice (Toth et al., 1981; Toth and Sornson, 1984). In the present study, we provided direct evidence complementing our previous observations (Papaparaskeva et al., 1991) that agaritine is not the major mutagenic component of this mushroom. The mutagenicity of the mushroom ethanolic extracts was compared with that of the equivalent amount of agaritine, calculated on the basis of a content of 50mg agaritine/kg fresh mushroom (Stijve et al.,
565
1986), in eight different S. typhimurium strains. The mutagenic profile induced by agaritine was markedly different from that induced by the mushroom ethanolic extracts, in that bacterial strains reverted by the extracts were not reverted by agaritine itself. Moreover, incorporation of hepatic cytosolic fractions as an activation system increased the direct mutagenic potential of mushroom extracts but not that of agaritine. These observations provide unequivocal evidence that the contribution of agaritine to the mutagenicity of the ethanolic extracts of A. bisporus is minimal and that this compound does not constitute the major mutagenic mushroom component. Our initial observation (Papaparaskeva et al., 1991) that by far the most susceptible bacterial strain to the mutagenicity of mushroom was TA104, a bacterial strain particularly sensitive to reactive oxygen-generating species such as hydroperoxides and quinones (Levin et al., 1982), led us to consider the possibility that such structures, rather than hydrazincs, may be responsible for the mutagenicity/ carcinogenicity of mushroom. As A. bisporus contains such phenolic and quinonoid compounds (Boekelheide et al., 1980) associated with the pink-tobrown pigmentation change in the gill tissue, the role of these components in mushroom mutagenicity was considered. Such compounds may be converted enzymically through one- or two-electron transfers to yield the reactive semiquinone radical or hydroquinone, respectively. Quinones are capable of inducing DNA damage with A : T sequences (Lee et al., 1991), the base pairs present at the critical sites of TA 104. The presence of rat hepatic cytosolic fractions potentiated the mutagenicity of the mushroom extracts TAI04; the fact that these fractions did not increase the mutagenic potential in all bacterial strains mirrors the multiplicity of mutagenic compounds present in the mushroom. The potentiation of the mutagenicity by cytosolic fractions required NADPH, but could not be supported by NADH. In the presence of NADPH, NADH decreased the cytosol-mediated mutagenicity of the mushroom extracts, presumably by diverting electron flow. The cytosolic potentiation of the mutagenicity of the mushroom extract was effectively inhibited by menadione and dicoumarol, an excellent substrate and a potent inhibitor, respectively, of the cytosolic enzyme DTdiaphorase. Although DT-diaphorase is largely linked with the deactivation of quinones (Prochaska and Talalay, 1986), it has been associated with activation in certain cases (Lee et al., 1991; Siegel et al., 1990; Talcott et al., 1983). In order to confirm the capacity of mushroom to convert phenols to quinones in the mushroom itself, mushroom tyrosinase was incorporated into the Ames assay. This enzyme is responsible for the metabolism of phenols, such as 7-L-glutaminyl'4-hydroxybenzene (GHB) to 7-L-glutaminyl-3,4-benzoquinone through the formation of 7-L-glutaminyl-
566
C. PAPAPARASKEVA-PETRIDESet al.
3,4-dihydroxybenzene (agaridoxin). The benzoquinone undergoes a base-catalysed non-enzymic conversion to the 2-hydroxy-4-iminoquinone also known as 490 quinone (Boekelheide et al., 1980). Furthermore, raising the pH to 8, the optimum for the first metabolic step (i.e. conversion of G H B to agaridoxin), stimulated the tyros(he-mediated mutagenic response. These data show that m u s h r o o m tyrosinase can mediate the formation of mutagens in the Ames test. The level of tyrosinase in the ethanolic extracts is likely to be low as a result of denaturation during the extraction, thus necessitating the addition of an exogenous supply. However, the possibility that some of the m u s h r o o m phenol quinones were activated to genotoxic species by endogenous tyrosinase during the extraction procedure c a n n o t be excluded. A m a m m a l i a n tyrosinase is unlikely to be involved in the cytosolic potentiation of the mutagenicity of m u s h r o o m extracts since tyrosine failed to inhibit the mutagenic response. Semiquinones may interact with molecular oxygen to generate reactive oxygen species which are themselves genotoxic (Totter, 1980). In the present study, superoxide dismutase caused a decrease in the directacting and particularly in the cytosol-mediated mutagenicity of the m u s h r o o m extracts, thus implicating superoxide anions in m u s h r o o m mutagenicity. Superoxide anions in m u s h r o o m may also dismutate to yield hydrogen peroxide, which itself possesses mutagenic capacity (Levin et al., 1982). Such a mechanism is supported by our observation that catalase and reduced glutathione inhibited the mutagenicity of m u s h r o o m extracts, especially in the presence of cytosolic fractions. The observation that oxidized glutathione was effective in decreasing the cytosolmediated, but not the direct-acting mutagenicity of m u s h r o o m , may be attributed to the reduction of glutathione by glutathione reductase in the cytosol. The most reactive oxygen species is the hydroxyl radical (Hsie et al., 1986), which may be formed t¥om hydrogen peroxide and superoxide anion in metalcatalysed reactions; in the present study, its scavenger D M S O inhibited the mutagenicity of the m u s h r o o m ethanolic extracts, establishing its role in the mutagenic mechanism. In s u m m a r y , the present study provides direct evidence that agaritine is not the m a j o r m u t a g e n in the ethanolic extracts of A. bisporus and that phenolic and q u i n o n o i d c o m p o u n d s , t h r o u g h generation of genotoxic oxygen species, may play a significant role in the induction of the mutagenic response. Acknowledgements The authors thank the Food Science Group, Ministry of Agriculture, Fisheries and Food, for supporting this work (grant no. 501).
REFERENCES
Boekelheide K., Graham D. G., Mize P. D. and Jeffs P. W. (1980) The metabolic pathway catalysed by tyrosinase of Agaricus bisporus. Journal of Biological Chemistr.v 255, 4766 4771.
Friederich U., Fischer B., Luthy J., Hazm D., Schlaner C. and Wfirgler E. (19863 The mutagenic activity of agaritine a constituent of the cultivated mushroom Agaricus hisporus -and its derivatives detected with the Sahnonella/mammalian microsome assay (Ames test). Zeitschr([~ [~ir Lchensmittel Untersuchun,~ und For~chung 183, 85 89. GrOter A., Friedcrich U. and Wiirglcr F. [i. (19913 The mutagenioty of edible mushrooms in a histidine-independent bacterial test system. Food and Chemical To.~:icolo~.r 29, 159 165. Hsie A. W., Recto L., Katz D. S., Lee C. Q., Wagner M. and Schenley R. L. (I9863 Evidence for rettctive oxygen species inducing mutations in mammalian cells. Proceedings (!lithe National .4cadenly (}/S('iel, tce.'~ (~['[h[' ~:.,~..I. 83, 9616 9620. loannides C. and Parke D. V. (19753 Mechanism of induction of hepatic drug metabolising enzymes by a series of barbiturates. Journal 0[ Pharntac)' and Pharmacolog.r 27, 739 749. Lee C.-S., Hartley J. A., Berardini M. D., Butler J. Siegel D., Ross D., and Gibson N. W. (199I) Alteralton in DNA crosslinking and sequence selectivity of a series of aziridinyl-benzoquinones after enzymatic reduction by DT-diaphorase. Biochcnlistry 31, 3019 3025. Levin D. E., Hollstein M., Christman M. F., Schwiers f'L A. and Ames B. N. (1982) A new Sahmmella tester strain (TA 102) with A:T base pairs at the site of mutation detects oxidative mutagens. Proceedings ~[ the National ,4cadcmy ~)/ Sciences 0[' the U.S.A. 79, 7445 7449. Maron D. M. and Ames B. N. (1983) Revised methods for Salmonella mutagenicity test. Mutation Research 113, 173 215. Matsumoto K., lta M., Yagyu S., Ogmo 11. and Hirono I. (1991) Carcinogen(city examination of Aguricu.s hi.~porus, edible mushroom in rats. Cancer Letters 58, 87 90. Morales P., Bermudez E., Sanz B. and Hermindez P. E. (1990) A study of the mutagenicity of some commercially canned Spanish mushrooms. Food and Chemical Tovico/ogy 28, 607 611. Papaparaskeva C., loannides C. and Walker R. (199I) Agaritine does not mediate the mutagenicity of the edible mushroom Agaricus hi.sporus'. Mutagenesis 6, 213 217. Pool-Zobel B. L., Schmezer P., Sinrachatanant Y., Kliagasioglu F., Reinhart K., Martin R., Klein P. and Tricker A. R. (19903 Mutagenic and genotoxic activities of extracts derived from the cooked and raw edible mushroom Agaricus bisporu.~. Journal Of Cancer Research and Clinical Ontology 116, 475 479. Prochaska H. J. and Talalay P. (19863 Puritication and character(sat(on of two isofunctional forms of NAD(P)Hquinone reductase from mouse liver. Journal ~!/'Biological Chenlistrv 261, 137l 1378. Rogan E. G., Walker B. A., Gingell R., Nagel D. L. and Toth B. (1982) Microbial mutagenicity of selected hydrazines. Mutation Research 102, 413 424. Siegerl D., Gibson N. W,, Preusch P-C. and Ross D. (1990) Metabolism of mitomycin C by DT-diaphorase. Role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Research 50, 7483 7489. Stijve T., Fumeaux R. and Phillippossian G. (19863 Agaratine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus) and in some of its wild-growing relatives. Deutsche Lehensmittel Rundeschau 8, 243 248. Talcott R. E., Rosenblum M. and Levin V. A. (19833 Possible role of DT-diaphorase in the bioactivation of antitumor quinones. Biochemical and Biophysical Research Comnnmications I I l, 346 351.
Mutagenicity of edible mushroom Toth B. and Erickson K. (1986) Cancer induction in mice by feeding of the uncooked cultivated mushroom of commerce Agaricus bisporus. Cancer Research 46, 4007~4011. Toth B., Patti K., Pyysalo H., Stessman C. and Gannett P. (1992) Cancer induction in mice by feeding the raw false morel mushroom Gyromitra esculenta. Cancer Research 52, 2279 2284. Toth B., Raha C. R., Wallcave L. and Nagel D. (1981)
567
Attempted tumor induction with agaritine in mice. Anticancer Research 1, 255 258. Toth B. and Sornson H. (1984) Lack of carcinogenicity of agaritine by subcutaneous administration in mice. Mycopathologia 85, 75 79 Totter J. R. (1980) Spontaneous cancer and its possible relationship to oxygen metabolism. Proceedings ~/ the National Academy ~[ Seiences of the U.S.A. 77, 1763 1767.
027S-6915/93 $6.00-~ 0.00 Copyright ,t' 1993Pergamon Press Ltd
C O N T R I B U T I O N OF P H E N O L I C A N D Q U I N O N O I D S T R U C T U R E S IN THE M U T A G E N I C I T Y OF THE EDIBLE MUSHROOM AGARICUS BISPORUS C. PAPAPARASKEVA-PETRIDES,C. IOANNIDES* and R. WALKER Division of Toxicology, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK (Accepted 8 April I993) Abstract The objectives of this work were to establish the contribution of agaritine in the mutagenicity of ethanolic extracts from Agaricus bisporus and to examine the possible involvement of phenolic and quinonoid compounds in the mutagenic response to mushrooms. The mutagenic profile of agaritine in the Ames test, in the absence of an activation system, was different from that of the mushroom ethanolic extracts. Incorporation of rat hepatic cytosolic fractions as the activation system increased the mutagenicity of the mushroom ethanolic extracts in Salmonella typhimurium strain TA104 but did not influence the mutagenicity of agaritine. It was concluded that agaritine is not the principal mutagenic component in the mushroom. The cytosol-induced mutagenicity of the mushroom extracts required NADPH, and was inhibited by dicoumarol and menadione. Moreover, the mutagenic response in the presence of cytosolic fractions was inhibited by superoxide dismutase, catalase, glutathione and dimethyl sulfoxide, thus implicating reactive oxygen species. Finally, tyrosinase, the enzyme converting mushroom phenols to quinones, increased the mutagenicity of the mushroom extracts. Collectively, the above results indicate that phenolic and quinonoid compounds, presumably through the generation of reactive oxygen species, may play a significant role in the mutagenicity of mushroom extracts.
INTRODUCTION
Many chemicals with carcinogenic potential have been detected in the diet; they are inherent to food (e.g. hydrazines) and contaminants (e.g. mycotoxins), or generated during the cooking process (e.g. aminoimidazoazaarenes). It is rarely, however, that an item of daily food displays itself carcinogenic potential in animal long-term lifetime bioassays. Toth and Erickson (1986) reported that excessive consumption of the edible mushroom Agaricus bisporus, by far the most extensively cultivated and used mushroom in the Western hemisphere, induced tumours at multiple sites in Swiss mice. Recent studies using the same protocol revealed that a wild edible mushroom of the false morel family Gyromitra esculenta also induced tumours at many sites in Swiss mice (Toth et al., 1992). In contrast, when Agaricus bisporus was tested in rats using a different protocol no carcinogenic response was evident; this finding raises the possibility that mushroom-induced tumours may be specific to mice (Matsumoto et al., 1991). Extracts from A. bisporus have been shown to elicit a positive but weak mutagenic response in mutagenicity systems, such as the Ames test, using a variety of Salmonella typhimurium strains (Friedrich et al., *To whom correspondence and reprint requests should be addressed. Abbreviations: DMSO=dimethyl sulfoxide; GHB=7-Lglutaminyl-4-hydroxybenzene. 561
1986; Rogan et al., 1982), but a more pronounced mutagenic response was evident when the T A I 0 4 bacterial strain was used (Papaparaskeva et al., 1991). In contrast, A. bisporus extracts failed to provoke a positive mutagenic response in other in vitro mutagenicity tests and in vivo tests such as the micronucleus test (Morales et al., 1990; Pool-Zobel et al., 1990). Pool-Zobel et al. (1990) considered the possibility that the mutagenicity of the A. bisporus extracts represented nothing more than the presence of a histidine artefact which interfered with the test. However, subsequent investigations using a histidineindependent bacterial strain confirmed the mutagenic potential of this mushroom (Gr/iter et al., 1991). The naturally occurring hydrazide agaritine (L-glutamic acid-5-[2-4(hydroxymethyl)]phenyl hydrazide) has been implicated as the component responsible for the mutagenicity/carcinogenicity of A. bisporus. In contrast to this hypothesis, however, the mutagenicity of various mushroom samples did not correlate with the agaritine content and, moreover, exogenous supply of 7-glutamyl transpeptidase, the enzyme catalysing the activation of agaritine, did not influence the mutagenic potential of A. bisporus extracts, thus casting doubt on the role of hydrazide agaritine in mushroom mutagenicity (Papaparaskeva et al., 1991). Furthermore, agaritine, when administered to mice for life in the drinking water or following sc administration, did not provoke a positive carcinogenic response (Toth et al., 1981; Toth and Sornson, 1984). Clearly, agaritine cannot
562
( ' . PAPAPARASKEVA=PETRII)ES el gl]. Table I C'omparison of the mutagenicities of agaritine and Agaru'us hts'porus extracts in the Ames tesl Histidine revertants plate Compound Sponlaneoas reversion rate Agaritine {2861~g) M u s h r o o m extract 1100 ~1 corresponding to 286 l*g agaritine)
TAI535
TAI537
TAI538
TA97
TA98
TAI00
TAI02
TA. 104
20+3 22+ I 39 ~ 4
II ~ I 30~2 22 I
7÷ I S+ I 14 ~ I
113+7 241 + 3 4 226 ~ 2
12+1 t6+2 29 + I
81+9 96+5 194+ 17
259+17 329+29 519+35
337 ~ 16 562 5 6 4 1193+206
The mutagenicity of m u s h r o o m extracts was compared with that of agaritine (2861~g). The volume of ethanolic m u s h r o o m extract containing 2861~g agaritme (i.e. 100t, i) was calculated o n the assumption that I kg fresh m u s h r o o m contains 0.05g agaritine (Stijve ct al., 19861. The numbers of histidine revertants are presented as means + SD of three plates (triplicate assays).
] able 2. Effect of the activation h_v hepatic Qtosolic fractions on the mutagenicilies of agaritine and ethanolic extracts of Agaricu.s hi.sporu.s Histidine revertants/plate Compound
No activation
With cytosolic activation
344 ~- 19 713 + 20 1268 + 69
325 + 11 733 + 38 156g + 42
Spontaneous reversion rate Agaritine (2861~g) M u s h r o o m extract (100 l~l corresponding to 2~6 l~g agaritine)
The activation system contained 10% (v/v) hepatic cytosolic fractions from Aroclor 1254-treated rats. M ushroom extracts corresponding to an agaritine level of 286 ~g, authentic agaritine (286/.tg), and S. typhimurium T A 104 were used. Results are presented as means ~ SD of three plates (triplicate assays).
A. his7~orus mushrooms were obtained locally and were always used within 24 hr following purchase. They were rinsed with water and then homogenized in 96% (v/v) ethanol at a concentration of l g/ml using an Osterizer blender. The homogenate was filtered through muslin and Whatman filter paper (Qualitative No. 1). Ethanol was finally evaporated in a rotary evaporator, and the final volume was aboul 20% of the original tiltrate. Male Wistar albino rats (about 200 g) were obtained from the Experimental Biology Unit (University of Surrey, Guildford, Surrey, UK). Induction of the hepatic mixed-function oxidases was achieved by a single ip injection of Aroclor 1254 (500 mg/kg body weight) dissolved in corn oil (200 mg/ml); the animals were killed on the fifth day following dosing. Hepatic cytosolic fractions were prepared as previously described (Ioannides and Parke, 1975). The mutagenicity of mushroom extracts was determined using the Ames mutagenicity test (Maron and Ames, 1983). All activation systems used contained 10% (v/v) hepatic cytosolic fractions from Aroclor
account for the mutagenic and carcinogenic effects of
A. bisporus. In the present study we provide further experimental evidence that agaritine is not the major contributor to the mutagenicity of A. bisporus and that quinonoid and phenolic chemicals may be largely responsible for the mulagenic response. MATERIALS AND METHODS
Oxidized and reduced glutathione, menadione, catalase, superoxide dismutase, mushroom tyrosinase and all cofactors (Sigma Chemical Co., Poole, Dorset, UK), Aroclor 1254 (Robens Institute, Guildford, UK) as well as dicoumarol (Aldrich Chemical Co., Ltd, Gillingham, Dorset, UK) were all purchased. Pure agaritine and the various Salmonella typhimurium strains were generous gifts from Professor B. Toth (The Eppley Institute of Research in Cancer, University of Nebraska Medical Center, USA) and from Professor B. N. Ames (Berkeley, CA, USA), respectively.
Table 3. Effect of the presence of hepatic cytosolic fractions in the mutagenic activity of ethanolic extracts from Agaricus bisporus H st d ne revertants'plate Compound Spontaneous reversion rate Mushroom extracts
TA97
TA98
--cyt
+cyl
98+24
90- 8
184+8
190±4
T A 100
cyt
+cyt
c vt
17-4
18-+1
95~1
44_+9
44+12
201=24
+c.vt
T A 102 cyt
T A 104 q- cyt
108- I
259+17
289+29
202+9
419±35
457+2
cyt
TA1537 +cyl
cyt
+cyt
337+16
394:+:41
I1+1
15-+2
1[93-+96
1506±91
16+1
18_+2
The activation system contained I1)% (v,v) hepatic cytosolic fraction (cyt) from Aroclor 1254-treated rats• Results are expressed as means +_ SD o f three p[ates (triplicate assays).
Mutagenicity of edible mushroom
563
Table 4. Role of NADPH and NADH in the cytosolic activation of ethanolic extracts of Agaricm
hisporus Extract Type of assay No activation Cytosolic activation system +NADPHgeneratingsystem Cytosolic activation system -- N A D P H generating system
947 -~ 152
Histidine revertants'plate Extract Extract Extract q+ + 1.5mM-NADH 3.0mM-NADH 4.5mM-NADH 953 + 101
909 ± 92
919 + 121
1223+41
787±53
829±67
859+62
764 + 75
755 + 25
766 ± 67
7(17 _+ 53
Salmonella typhhnurium TA 104 and mushroom extracts corresponding to 0.6 g flesh mushroom were used. The activation system contained hepatic cytosolic fractions (10%. vv) fi-om Aroclor 1254-treated rats. The spontaneous reversion rate of 417_+_27 has already been subtracted. Results are expressed as mean + SD of three plates (triplicate assays).
1254-treated rats. V a r i a t i o n s o f the p r o c e d u r e are d e s c r i b e d in the f o o t n o t e s to the a p p r o p r i a t e tables. RESULTS
T h e m u t a g e n i c i t y o f the e t h a n o l i c extracts o f A.
bisporus was e v a l u a t e d in the A m e s test in the a b s e n c e o f an a c t i v a t i o n system, using eight S. typhimurium strains, a n d was c o m p a r e d with the m u t a g e n i c i t y o f the c o r r e s p o n d i n g a m o u n t o f agaritine a s s u m i n g that m u s h r o o m has an agaritine c o n t e n t o f 50 m g / k g (Stijve et al., 1986). A g a r i t i n e elicited a clear positive m u t a g e n i c r e s p o n s e in strain T A 9 7 a n d its predecessor TA1537 (Table 1). A n increase in the reversion rate was also seen in T A I 0 4 , but did n o t attain d o u b l i n g o f the s p o n t a n e o u s reversion rate. T h e m u s h r o o m e t h a n o l i c extracts, in c o n t r a s t , p r o v o k e d
F--] M u s h r o o m extract
4000
Extract + DMSO
Extract
dicoumarol
3500 -0J
3000 -250(I --
-7
I
"T-
2000 1500 1000
//
i::i::i::iiil~i ii
///
a positive m u t a g e n i c r e s p o n s e in all bacterial strains, especially in T A I 0 4 (Table 1). In a s e c o n d study, agaritine achieved a positive m u t a g e n i c r e s p o n s e in T A I 0 4 as indicated by a d o u b l i n g o f the s p o n t a n e o u s reversion rate, but the m u t a g e n i c i t y was still m u c h lower t h a n that o f m u s h r o o m extracts c o n t a i n i n g the equivalent a m o u n t o f agaritine (Table 2). I n c o r p o r a t i o n o f rat hepatic cytosolic fractions p o t e n t i a t e d the m u t a g e n i c i t y o f the m u s h r o o m extracts but did not influence the m u t a g e n i c i t y o f agaritine (Table 2). T h e ability o f hepatic cytosolic fractions to increase m u s h r o o m m u t a g c n i c i t y was f u r t h e r evaluated in a n u m b e r o f bacterial strains (Table 3). T h e cytosolic fractions p o t e n t i a t e d the m u t a g e n i c r e s p o n s e only in bacterial strain T A 104. T h e c y t o s o l - m e d i a t e d m u s h r o o m m u t a g e n i c i t y required N A D P H a n d could n o t be s u p p o r t e d by N A D H (Table 4). M o r e o v e r , N A D H inhibited the c y t o s o l - i n d u c e d m u t a g e n i c i t y in the presence o f N A D P H (Table 4). T h e c y t o s o l - i n d u c e d m u t a g e n i c ity was i m p a i r e d by d i c o u m a r o l , which h o w e v e r had no effect on the direct m u t a g e n i c i t y o f the m u s h r o o m e t h a n o l i c extracts (Fig. 1). It is w o r t h n o t i n g that d i m e t h y l sulfoxide ( D M S O ) , the vehicle for dicoum a r o l , also r e d u c e d m u t a g e n i c response. Similar to d i c o u m a r o l , the c y t o s o l - m e d i a t e d m u t a g e n i c i t y o f the m u s h r o o m ethanolic extracts was c o m p l e t e l y inhibited by m e n a d i o n e , which h o w e v e r did n o t p e r t u r b the direct m u t a g e n i c i t y o f the m u s h r o o m extracts in the a b s e n c e o f an a c t i v a t i o n system (Table 5). T h e e n z y m e s catalase a n d s u p e r o x i d e d i s m u t a s e inhibited m u s h r o o m mutagenicity, b o t h in the presence a n d
500 0 A
B
Fig. 1. Inhibition of the cytosol-mediated activation of Agaricus bisporus extracts by dicoumarol. The activation system contained hepatic cytosolic fractions (2.5%, v/v) derived from Aroclor 1254-treated rats. Ethanolic mushroom extracts (100pl), dicoumarol dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 30 ,UM and S. typhimurium strain TAI04 were used. The spontaneous reversion rate of 506 + 38 has already been subtracted. Results are presented as means _+ SD of triplicate assays. A: in the absence of cytosok B: in the presence of cytosol. FCI 3 1 / ~
Table 5. Modulation of the mutagenicity of ethanolic extracts from Agaricus hisporus by menadione Histidine reverants/plate Compound No activation With activation Mushroom extracts 1462 i 97 1938 + 23 Mushroom extracts and menadione 1363 + 45 1172 ± 149 The activation system contained 2.5% (v/v) hepatic cytosolic fractions from Aroclor 1254-treated rats. Ethanolic mushroom extracts (100 p I), 9 mM-menadione dissolved in dimethyl sulfoxide and S. typhimurium TAI04 were used. The spontaneous reversion rate of 418 + 28 has already been subtracted. The numbers of histidine revertants are presented as means + SD of three plates (triplicate assays).
564
el
C. PAPAPARASKEVA-PETRIDES I
al.
[•
No acti,,ation
L t~llO R
1400
--0--
('alalasc
--+--
SO1)
- - . - -
('ala hlsc,'SOD
1400
Mushroum
cxtracl
Mushrmm~
extract
+ GStl
Mushroom
cxHact
+ GSS(I
120()
120O
~,~.
lOl)O
T.
T
I0()0
i "o
@tO
,-'~
4(}{}
-"
#::;:i.[iiii
.~
~
c\'tosnlic
"==& 1 5 ( I 0
>
I{)0
150
2(1(I
4(111
250 :#::#.Uii~ !!i:p!%%:i:i~:
I 2(I(I
activation
('atahlsc
--+--
St)It
~'--
('atalasc"SOD T
Fig. 3. Inhibition of AgarictB bi.worus mutagenicity by glutathione. The mutagenicity was determined with S. typhimurium TA104 and mushroom ethanolic extracts (100,ul), without {At or with (B) an activation system containing hepatic cytosolic fractions (10%, v/v) derived from Aroclor 1254-treated rats, in the presence of oxidized or reduced glutathione (5 mM). The spontaneous reversion rate of 401 _+ 26 has already been subtracted. Results are presented as means + SD of triplicate assays.
b 5011
i 5(1
I00
150
B
A
--o--
tO01}
m ~-
I
.2_
t)
20110
~;~'i:i:i:i~i:?i:
!
E n z y m e a c t i v i t y {units/plate)
2. W i t h
ii!ili!i~i~iii~i~i
600
20() L 0
i
bit)l}
i
]
2(10
250
Enzyme activity (units/plate)
Fig. 2. Inhibition of Agaricus bi~porus mutagenicity by catalase and superoxide dismutase. The test was carried out with S. typhimurium T A I 0 4 and mushroom ethanolic extracts (100 ,u 1) with or without an activation system containing hepatic cytosolic fractions (10%, v/v) derived from Aroclor 1254-treated rats, in the presence of catalase and/or superoxide dismutase (SOD). The spontaneous reversion rate of 390 _+ 17 has already been subtracted. Each point represents the mean _+ SD of a triplicate assay. a b s e n c e o f c y t o s o l i c fractions (Fig. 2). S i m u l t a n e o u s i n c o r p o r a t i o n of both e n z y m e s w a s the m o s t effective w a y o f inhibiting m u t a g e n c i t y . T h e c y t o s o l - i n d u c e d m u t a g e n i c i t y of m u s h r o o m extrracts w a s m a r k e d l y inhibited by both o x i d i z e d and reduced g l u t a t h i o n e Table 6. Effect of pH on the tyrosinasemediated activation of ethanolic extracts of Agaricus bisporu.~
(Fig. 3). In the a b s e n c e o f c y t o s o l i c fractions, o n l y reduced g l u t a t h i o n e decreased the m u t a g e n i c res p o n s e . T h e inhibitory effect of D M S O on the mutagenicity of the m u s h r o o m extracts, both in the a b s e n c e and presence of rat hepatic c y t o s o l i c fractions w a s investigated in m o r e detail. As s h o w n in Fig. 4, m u t a g e n i c r e s p o n s e w a s inhibited whether c y t o s o l i c fractions were present or not. 201)0
=
1500
>
I(1011
-:tq
.-j •-
511(1
Histidine revertants/plate pH
tyrosinase
+ tyrosinase
7.4 8.0
1037 ± 51 1024 ± 44
1323_+40 1768 _+ 164
Ethanolic mushroom extracts (1001~l) were preincubated with S. typhimurium (TAI04) in (it the absence of tyrosinase or (ii) the presence of tyrosinase (8000 units) for 25 min at 37 C in a shaking water-bath. The spontaneous reversion rate of 272 + 4 has already been subtracted. The numbers of histidine revertants are presented as means+ SD of three plates (
__l 1()0
i
i
2Im
30[)
400
500
7oncentration of DMSO (BI/plate) Fig. 4. Inhibition of Agaricus bisporus mutagenicity by dimethyl sulfoxide (DMSO). The assay was performed with S. typhimurium TA104 and mushroom ethanolic extracts (100 ttl) and with ( + ) or without ([Z]) an activation system containing hepatic cytosolic fractions (10%, v/v) from Aroclot 1254-treated rats. The spontaneous reversion rate of 394 4- 36 has already been subtracted. Each point represents the mean + SD of a triplicate assay.
Mutagenicity of edible mushroom 2000 151)0 > 1It(1(I
f
(a)
T/T/T
.~_ 51111 7~ 0 ~/ 11
I
I
I
I
I
!
21)
40
60
NO
ll)O
120
Concentration of extract (pd/plate) o
"~
101)0
U~> 800 ~u
(b)
9(R) /
700 600
4oo
I I J I I i I I 1) l 2 3 4 6 7 S 9 I() Tyrosinase concn (units/plate x 1000)
Fig. 5. Mutagenicity of Agaricus bisporus ethanolic extracts by tyrosinase. (at Mushroom extracts were preincubated with tyrosinase (8000 units) for 25 min at 37C in a shaking water-bath: ( + ) with tyrosinase and (Q) without tyrosinase. The spontaneous reversion rate of 353 +_9 has already been subtracted. (b) Mushroom extracts (75/~1) were preincubated with S. o'phimurium TAI04 and tyrosinase for 25 min at 37"C in a shaking water-bath. Each point represents the mean _+SD of a triplicate assay. Incorporation of purified mushroom tyrosinase as an activation system increased the mutagenicity of mushroom extracts (Fig. 5at. The increase in mutagenie response was dependent on tyrosinase concentration up to a level of 2000 units/plate (Fig. 5b). Increasing the pH from 7.4 to 8.0 increased the mutagenicity of the mushroom extracts in the presence of tyrosinase (Table 6). Finally, in the presence of cytosol, tyrosine (1 mM) failed to influence the mushroom mutagenicity in TAI04 (results not shown). DISCUSSION Agaritine is the mushroom constituent that was implicated in the mutagenictty and carcinogenicity of A. bisporus despite the fact that it was not carcinogenic in mice (Toth et al., 1981; Toth and Sornson, 1984). In the present study, we provided direct evidence complementing our previous observations (Papaparaskeva et al., 1991) that agaritine is not the major mutagenic component of this mushroom. The mutagenicity of the mushroom ethanolic extracts was compared with that of the equivalent amount of agaritine, calculated on the basis of a content of 50mg agaritine/kg fresh mushroom (Stijve et al.,
565
1986), in eight different S. typhimurium strains. The mutagenic profile induced by agaritine was markedly different from that induced by the mushroom ethanolic extracts, in that bacterial strains reverted by the extracts were not reverted by agaritine itself. Moreover, incorporation of hepatic cytosolic fractions as an activation system increased the direct mutagenic potential of mushroom extracts but not that of agaritine. These observations provide unequivocal evidence that the contribution of agaritine to the mutagenicity of the ethanolic extracts of A. bisporus is minimal and that this compound does not constitute the major mutagenic mushroom component. Our initial observation (Papaparaskeva et al., 1991) that by far the most susceptible bacterial strain to the mutagenicity of mushroom was TA104, a bacterial strain particularly sensitive to reactive oxygen-generating species such as hydroperoxides and quinones (Levin et al., 1982), led us to consider the possibility that such structures, rather than hydrazincs, may be responsible for the mutagenicity/ carcinogenicity of mushroom. As A. bisporus contains such phenolic and quinonoid compounds (Boekelheide et al., 1980) associated with the pink-tobrown pigmentation change in the gill tissue, the role of these components in mushroom mutagenicity was considered. Such compounds may be converted enzymically through one- or two-electron transfers to yield the reactive semiquinone radical or hydroquinone, respectively. Quinones are capable of inducing DNA damage with A : T sequences (Lee et al., 1991), the base pairs present at the critical sites of TA 104. The presence of rat hepatic cytosolic fractions potentiated the mutagenicity of the mushroom extracts TAI04; the fact that these fractions did not increase the mutagenic potential in all bacterial strains mirrors the multiplicity of mutagenic compounds present in the mushroom. The potentiation of the mutagenicity by cytosolic fractions required NADPH, but could not be supported by NADH. In the presence of NADPH, NADH decreased the cytosol-mediated mutagenicity of the mushroom extracts, presumably by diverting electron flow. The cytosolic potentiation of the mutagenicity of the mushroom extract was effectively inhibited by menadione and dicoumarol, an excellent substrate and a potent inhibitor, respectively, of the cytosolic enzyme DTdiaphorase. Although DT-diaphorase is largely linked with the deactivation of quinones (Prochaska and Talalay, 1986), it has been associated with activation in certain cases (Lee et al., 1991; Siegel et al., 1990; Talcott et al., 1983). In order to confirm the capacity of mushroom to convert phenols to quinones in the mushroom itself, mushroom tyrosinase was incorporated into the Ames assay. This enzyme is responsible for the metabolism of phenols, such as 7-L-glutaminyl'4-hydroxybenzene (GHB) to 7-L-glutaminyl-3,4-benzoquinone through the formation of 7-L-glutaminyl-
566
C. PAPAPARASKEVA-PETRIDESet al.
3,4-dihydroxybenzene (agaridoxin). The benzoquinone undergoes a base-catalysed non-enzymic conversion to the 2-hydroxy-4-iminoquinone also known as 490 quinone (Boekelheide et al., 1980). Furthermore, raising the pH to 8, the optimum for the first metabolic step (i.e. conversion of G H B to agaridoxin), stimulated the tyros(he-mediated mutagenic response. These data show that m u s h r o o m tyrosinase can mediate the formation of mutagens in the Ames test. The level of tyrosinase in the ethanolic extracts is likely to be low as a result of denaturation during the extraction, thus necessitating the addition of an exogenous supply. However, the possibility that some of the m u s h r o o m phenol quinones were activated to genotoxic species by endogenous tyrosinase during the extraction procedure c a n n o t be excluded. A m a m m a l i a n tyrosinase is unlikely to be involved in the cytosolic potentiation of the mutagenicity of m u s h r o o m extracts since tyrosine failed to inhibit the mutagenic response. Semiquinones may interact with molecular oxygen to generate reactive oxygen species which are themselves genotoxic (Totter, 1980). In the present study, superoxide dismutase caused a decrease in the directacting and particularly in the cytosol-mediated mutagenicity of the m u s h r o o m extracts, thus implicating superoxide anions in m u s h r o o m mutagenicity. Superoxide anions in m u s h r o o m may also dismutate to yield hydrogen peroxide, which itself possesses mutagenic capacity (Levin et al., 1982). Such a mechanism is supported by our observation that catalase and reduced glutathione inhibited the mutagenicity of m u s h r o o m extracts, especially in the presence of cytosolic fractions. The observation that oxidized glutathione was effective in decreasing the cytosolmediated, but not the direct-acting mutagenicity of m u s h r o o m , may be attributed to the reduction of glutathione by glutathione reductase in the cytosol. The most reactive oxygen species is the hydroxyl radical (Hsie et al., 1986), which may be formed t¥om hydrogen peroxide and superoxide anion in metalcatalysed reactions; in the present study, its scavenger D M S O inhibited the mutagenicity of the m u s h r o o m ethanolic extracts, establishing its role in the mutagenic mechanism. In s u m m a r y , the present study provides direct evidence that agaritine is not the m a j o r m u t a g e n in the ethanolic extracts of A. bisporus and that phenolic and q u i n o n o i d c o m p o u n d s , t h r o u g h generation of genotoxic oxygen species, may play a significant role in the induction of the mutagenic response. Acknowledgements The authors thank the Food Science Group, Ministry of Agriculture, Fisheries and Food, for supporting this work (grant no. 501).
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