J Cancer Res Clin Oncol (1990) 116:475-479
Mutagenic and genotoxic activities of extracts derived from the cooked and raw edible mushroom Agaricus bisporus * B.L. Pool-Zobel 1, p. Schmezer 1, y. Sinrachatanant 2, F. Kliagasioglu 3, K. Reinhart 1, R. Martin 4, p. Klein 1, and A.R. Tricker 1 1 Institute for Toxicology and Chemotherapy, German Cancer Research Center, Ira Neuenheimer Feld 280, and * Institute for Pharmaceutical Biology, University of Heidelberg, Im Neuenheimer Feld 364, D-6900 Heidelberg, Federal Republic of Germany, 2 Department of Pharmacology, Mahidol University, Rama VI Road, Bangkok, Thailand 3 Section of Medical Oncology, Cerrahpasa Medical School, Istanbul, Turkey Received 18 June 1990/Accepted 25 June 1990
Summary. A. bisporus has been reported to be carcinogenic to mice [Toth et al. (1986) Cancer Res 38:177 180] and mutagenic in Salmonella typhimurium [Sterner et al. (1982) Mutat Res 101:269-281]. The effects of different heat treatments on the mutagenicity of raw, cooked (boiled) and fried A.bisporus extracts in the S. typhirnurium test is reported. The spectrum of potential mutagenic activity ofA. bisporus extracts was tested in vitro in Syrian hamster embryo cells for selective D N A amplification and in primary rat hepatocytes for D N A singlestrand breaks. D N A single-strand breaks were also determined in liver cells of rats and micronuclei were measured in bone marrow cells of mice in vivo following oral application ofA. bisporus extracts. It was shown that the complex A. bisporus extracts per se are not detectably mutagenic in S. typhimurium and that the previously observed increase in number of colonies per plate is probably due to a histidine artefact. No indication of genotoxicity was seen in the two in vitro assays with primary mammalian cells with two different end points. No evidence of in vivo genotoxic effects was observed in the rat liver cells. Finally, A. bisporus was not genotoxic in the micronucleus assay of mouse bone marrow cells in contrast t o i t s previously reported carcinogenicity in mice.
Key words: Agaricus bisporus Histidine artefacts Genotoxic effects Salmonella typhimurium - Primary rat hepatocytes - D N A amplification Short-term in vivo tests micronuclei - D N A single-strand breaks
Introduction Previous feeding studies with the common edible mushroom Agaricus bisporus to 6-week-old Swiss mice show * Dedicated to Professor Dr. D. Schm/ihl on the occasion of his 65th birthday Abbreviation: AAV, adeno-associated virus Offprint requests to: B. L. Pool-Zobel
an increased tumor frequency in bone, forestomach, liver and lung (Toth and Erickson 1986). Suspect compounds, which may be responsible for this carcinogenic effect, include hydrazines such as agaritine, a major component present in A. bisporus. Ross et al. (1982) have shown that 1 kg raw A. bisporus contains 400-700 mg agaritine. Fischer et al. (1984) identified 94~629 mg/kg agaritine and noted that the content decreases by heat treatment and with storage. Agaritine has not been shown to be carcinogenic to mice after oral application (Toth et al. 1981 a), whereas several model compounds that decompose to liberate its potential metabolites have induced tumors in mice (Toth 1975, 1980). N'-Acetyl-4-(hydroxymethyl)phenylhydrazine induces tumors in the lung and blood vessels (Toth et al. 1978), 4-methylphenylhydrazine hydrochloride is carcinogenic in the lung, blood vessels and subcutis (Toth et al. 1977), whereas the tumors induced by 4-(hydroxymethyl)benzene diazonium tetrafluorborate are localized in the subcutis and skin (Toth et al. 1981b). Owing to the different patterns of afflicted tissues in which tumors are induced by the above compounds and the mushroom itself, and since agaritine is not carcinogenic, it seems probable that A. bisporus contains other carcinogenic compounds. One feasible mechanism for the carcinogenic activity may be via genotoxicity. Therefore, we investigated extracts of A. bisporus in various genotoxicity tests to determine whether mutagens are identifiable, and how this mutagenicity is detectable in the target organs suspectible to carcinogenicity following in vivo application. Finally, it was also an aim of this study to determine to what degree the potential mutagens are destroyed by cooking. One possibility in studying these aims was to assess the mutagenicity of A. bisporus extracts with the S. typhimurium assay (Ames et al. 1975; M a r o n and Ames 1983). Accordingly, Sterner et al. (1982) have shown 37 of 48 investigated extracts of larger fungi to be mutagenic in this test system. These studies were performed within a large screening program, but also included an aqueous extract of A. bisporus, which was suggested to contain mutagens.
476 W e repeated these d e t e r m i n a t i o n s with raw a n d heated A. bisporus samples to s t u d y the effect o f heat t r e a t m e n t o n the m u t a g e n c o n t e n t . A d d i t i o n a l l y , we used other genotoxicity assays to elucidate whether the m u s h r o o m c o n t a i n s c o m p o n e n t s also genotoxic in m a m m a l i a n cells. The following systems were applied. I n vitro selective a m plification o f a d e n o associated virus ( A A V ) D N A was assessed in Syrian h a m s t e r e m b r y o cells, a n d p r i m a r y rat hepatocytes were used as i n d i c a t o r cells to detect D N A single-strand breaks. Ex vivo D N A single-strand breaks were also assessed i n rat hepatocytes a n d micronuclei were detected in b o n e m a r r o w cells o f mice.
Materials and methods
ml with suspension medium (Spinner MEM, Gibco) for further use in the genotoxicity assays. The hepatocytes were either treated in vitro (1-ml suspension cultures containing 5-20 gl extract solution, 1 h incubation in shaking water bath at 37~ C) or they were obtained from treated animals (ex vivo assay).
Detection ofAA VDNA amplification. The rationale and methods of detecting DNA amplification have been specified previously (Klein et al. 1990; Pool et al. 1989). For this assay 2 x 1087Syrian hamster embryo cells, infected with AAV, are seeded into the wells ofa 6-well microtiter plate. After 24 h, the appropriate dilutions of the A. bisporus extract are added (each concentration in triplicate) and the plate is further incubated for 4 days. After this period, the cells are counted, and cell viability is determined. The DNA is lysed, hybridized with 87 AAV DNA and then the total AAV content per cell is determined by scintillation counting. A reproducible, AAV DNA content greater than twice that of the untreated control is taken as an indication of selective DNA amplification.
Preparation ofA. bisporus. The extracts were prepared according to the procedures described by Sterner et al. (1982). For this, 50 g raw, cooked (in 100 ml water for 10 min) or fried (in 50 g butter for 10 min) A. bisporus samples, obtained from the local food store, were homogenized and extracted either with ethanol or with ethyl acetate. Extracts from the total water and butter residues, following heat treatment of the mushrooms were extracted in the same way. The extracts were concentrated and the residues were dissolved in 8 ml ethanol/dimethylsulfoxide (1 : 1). Samples of 0-100 p.1 were used in the in vitro assays. The 150-~tland 200-~tlextract equivalents referred to in some ofthe tables are actually 75 ~tland 100 I~1doublestrength extracts obtained by extracting 100 g A. bisporus and dissolving the residues also in 8 ml solvent. The sample quantities used in the in vivo assays are described in the tables. For the micro.nucleus test, aqueous extracts were prepared from freeze-dried samples.
Determination of mutagenicity with S. typhimurium. The determination of his + reversion in histidine auxotrophic S. typhimurium strains was carried out according to standard methods (Ames et al. 1975; Maron and Ames 1983). The tests with A.bisporus samples were performed with and without metaholic activation by 9000 g fractions obtained from Aroclor-pretreated Sprague-Dawley rats. Mutagenicity was determined both with the preincubation modification as well as with the plate incorporation assay. All detailed specifications are noted in the tables. In some cases the assays were performed in the presence of y-glutamyltransferase, in order to stimulate activation of agaritine (Friederich et al. 1986). An increased mutagenicity of the A. bisporus extracts in the presence of this enzyme would be expected, if agaritine was the natural ingredient responsible for the mutagenicity of the extract.
Experimental animals. Maie Sprague-Dawley rats, C57BL/6J mice and Syrian hamsters were obtained from Charles River Wiga, Sulzfeld. Male Sprague-Dawley rats weighing approximately 200 g were used as donor animals for the subcellular liver fractions and for the primary rat hepatocytes. Micronuclei were determined in C57BL/6J mice weighing 25-30 g. Male and female Syrian hamsters were mated at the rate of 1 : 3 for the generation of emhryos as donors of cells.
Syrian hamster embryo cells. The Syrian hamster embryo cells were isolated from embryos on the 13th day of gestation using the saine procedure as described for the cells used to detect morphological transformation (Klein et al. 1990; Pienta et al. 1979). Isolation/treatment ofhepatoeytes. Primary rat hepatocytes were obtained following a two-step in situ perfusion method described by Berry and Friend (1969) and Bradley et al. (1984). The liberated cells were centrifuged at 30 g for purification, and the pellet was resuspended in a Ca 2+-, Mg 2+-free buffer for counting and determination of viability. The cell suspensions were adjusted to 2 x 106 cells/
Determination of DNA single-strand breaks. DNA single-strand breaks were determined with the alkaline filter elution method according to Kohn et al. (1981). Aliquots of 0.75 ml of the 1-ml suspension cultures were lysed on polycarbonate filters. The DNA was eluted at alkaline pli and the DNA in the resulting fractions determined fluorimetrically (Pool et al. 1990; Schmezer et al. 1990). The results are calculated as C - T , which is the percentage DNA retained on filters in the control - the percentage DNA retained on filters in the test groups.
Micronuclei detection. The procedures closely followed the original protocol of Schmid (1975), including modifications introduced by Ashby and Mohammed (1986). Male C57BL/6J mice were given a single oral application of an aqueous A. bisporus extract equivalent to 10 mg mushroom/kg mouse. A 65-mg/kg sample of cyclophosphamide was applied as the positive control; after 36 h the animals were sacrificed by Metofane anesthesia and the prepared bone marrow cells were streaked on microscope slides and stained with Giemsa. A total of 1000 polychromatic erythrocytes per mouse were evaluated for the occurrence of micronuclei and the data were statistically analyzed by a one-sided Student's t-test.
Results and discussion I n n u m e r o u s initial assays, a slight e n h a n c e m e n t o f his + revertants u p to d o u b l e the c o n t r o l was observed for the S. typhimurium strains T A 1537, T A 9 7 , T A 100, a n d T A 98. This e n h a n c e m e n t was within the range o f 2 5 200 ~1 A. bisporus extract a n d occurred b o t h with a n d w i t h o u t m e t a b o l i c a c t i v a t i o n for b o t h raw a n d cooked m u s h r o o m s ; it was also seen in the water a n d b u t t e r residues o f heat-treated A. bisporus samples. T a b l e 1 shows the results o b t a i n e d in strain T A 97 as a n example o f the observed activities. Three positive factors c o u l d be responsible for the observed e n h a n c e m e n t : (a) growth o f h i s - colonies as a result of excessive A. bisporus histidine in the ethanolic extracts, (b) a m u t a g e n i c effect o f a g a r i t i n e , a n d (c) the presence o f u n i d e n t i f i e d m u t a g e n s . Y a m a s a k i et al. (1977) have s h o w n t h a t certain o r g a n i c materials m a y c o n t a i n histidine, which m a y n o t be r e m o v e d by the extraction m e t h o d s e m p l o y e d here. These m i n u t e a m o u n t s o f histidine m a y evoke a growth o f h i s - bacteria, which in the Salmonella typhimurium assay m a y be falsely interpreted as a n i n d u c t i o n o f his + revertants. C u l t i v a t e d m u s h r o o m s o f the Agaricus types m a y c o n t a i n t 40-80 m g his-
477 Table 1. Studies on the mutagenic activities of ethanolic Agaricus bisporus extracts in Salmonella typhimurium TA97a: plate incorporation assay. Mean values and standard deviations of three plates per concentration are shown
A. bisporus extract per plate (gl)
Raw -S9 a
+$9
-$9"
+$9
0 18.75 37.5 70 100 150b 200 b
116_+ 5 129_+10 141_+12 134_+ 6 160+ 3 175_+12 211_+17
161_+16 196_+ 6 195_+ 7 168_+ 7 197-+21 237_+11 232_+14
116_+ 5 136_+ 8 150_+12 158-+15 187-+14 219_+ 9 246_+23
161_+16 190_+ 5 192_+ 3 191+_10 222_+12 246_+12 237_+41
Table 2. Studies on the mutagenic activities of XAD-purified A. bisporus extracts in S. typhimurium TA97a, 16h preincubation in 8 ml volume (bacteria, A. bisporussample, enzyme, buffer). The high number of spontaneous revertants is due to the long period of preincubation
Boiled
A. bisporus extract per plate (gl)
Raw
Cooked
- Enzyme + Enzyme
-- Enzyme + Enzyme
0 25 50 75 100 150 200
427+39 423_+25 369_+30 349_+42 371_+30 332_+119 348_+12
427_+39 360_+14 369_+15 423_+13 427_+24 414_+23 294_+32
297_+51 311_+43 314_+21 318_+ 0 373_+17 343_+28 373_+ 8
297_+51 359_+17 371 _+16 374_+ 2 355_+16 342_+ 6 383_+10
" $9, supernatant 9000g b Aliquot values, double concentrate in 100 gl solvent
tidine/100 g mushroom. Based on our extraction method using 50 g mushroom and a solution of the residue in 8 ml solvent, the highest expected histidine content would be approximately 0.25-0.5 mg/100 gl extract. This concentration is, however, likely to be considerably lower, since the total amount of histidine is probably not extractable by organic solvents because of its greater water solubility. Also, if 250-500 lag had been present in 100 gl A. bisporus extract, toxic effects should have been seen in Salmonella. Pure histidine, tested at concentrations of 93.8-125 gg/plate, was toxic to all strains of Salmonella. This toxicity was apparent as a decrease in microscopically and macroscopically visible colonies. The colony score, for example, of S. typhimurium TA 97 in the presence of increasing histidine concentrations was 162 + 13, 250+31, 235+_29, 236+_35, 307+21, 213_+26, 2 4 + 4 , and 8+-1 a t 0 , 3.9, 7.8, 15.6, 31.8, 62.5, 93.8, and 125 gg histidine/plate, respectively (plate incorporation assay, without $9). The increase of colonies at the lower histidine concentrations was similar to the pattern of increase observed after A. bisporus treatment. The artefactual his- growth was further studied by repurifying the extract over X A D to remove the histidine (Yamasaki and Ames 1977) and then assessing the mutagenic activity of the histidine-free extract. The purified X A D extract was confirmed to be histidine-free by thin-layer chromatography. This sample also resulted in a lower score of colonies per plate, which never reached 1.5 or 2 times that of the untreated control in rive S. typhimurium strains (results not shown). Accordingly, we conclude that the enhancement of colonies seen in Table 1 is hOt necessarily a result of induced his + revertants, but due to an artefact resulting from co-extracted histidine in the ethanolic A. bisporus extracts. Similar conclusions were drawn in a study by Griiter (1988) with the fungal species Lactarius helvas. Another contributing factor, however, could be agaritine, which is present in high amounts in A. bisporus and has been shown to act as a mutagen. According to an assumed content of 100-600 mg agaritine/kg mushroom (Fischer et al. 1984; Ross et al. 1982), our extracts could contain agaritine at a concentration of 0.25-1.25 gmol/
100 ~tl. In order to investigate the contribution of this hydrazine to the observed enhancement of Salmonella colonies, we selected conditions for which agaritine has been shown to be mutagenic (Friederich et al. 1986) and tested A. bisporus extracts in the presence of the activating enzyme y-glutamyltransferase. Table 2 shows the results obtained with a histidine-free sample purified over XAD. On the basis of the assumed amount of agaritine present in the extracts, the colony score should well exceed 1000 colonies/plate (Friederich et al. 1986). However, as can be seen from the results, an enhancement in colony number that would indicate agaritine mutagenicity was not evident. This may be due to the fact that the levels of agaritine present in the tested sample are less than those calculated above and/or that the presence of other compounds in the test sample inhibits the mutagenic effect of agaritine (Pool 1988). Finally, the possibility can not be excluded that other factors may be contained in A. bisporus that are genotoxic and, therefore, highly likely to be responsible for the carcinogenic effects seen in mice. These factors are not readily detectable by the S. typhimurium assay, since mainly histidine artefacts were observed (see above)~ However, they may be readily detectable by other shortterm assays utilizing mammalian cells. Accordingly, we investigated A. bisporus extracts for some other genetic end points in vitro using Syrian hamster embryo cells and primary rat hepatocytes as indicator organisms. In order to study the influences of the whole organism (Pool and Schm/ihl 1987) in vivo assays were also performed. Table 3 shows results obtained for the analysis of D N A amplification in Syrian hamster embryo cells. This genetic end point is an important indicator of a cell's answer to toxic stress by chemical compounds. It is associated both with the induction of resistance and with the processes of carcinogenesis (Pool et al. 1989). The detection of AAV amplification in Syrian hamster embryo cells is a new system and the first demonstration of this end point in primary cells. The extract was tested at up to 25 ~tl/incubation mixture in which A. bisporus did not induce D N A amplification. Higher solvent amounts were toxic to the cells.
478 A n o t h e r efficient a n d r e l e v a n t s h o r t - t e r m assay for g e n o t o x i c i t y is the h e p a t o c y t e D N A s i n g l e - s t r a n d b r e a k test ( S c h m e z e r et al. 1990; Sina et al. 1983). I n this sensitive o n e - c o m p a r t m e n t assay in vitro, D N A d a m a g e is m o n i t o r e d w i t h i n m e t a b o l i c a l l y c o m p e t e n t r a t liver cells. A. bisporus was tested using u p to 20 gl e x t r a c t / m l incub a t i o n m i x t u r e , since the solvent was h o t toxic u n d e r the test c o n d i t i o n s . T a b l e 4 shows no D N A - d a m a g i n g activity b y A. bisporus e x t r a c t d e r i v e d f r o m either c o o k e d o r r a w m u s h r o o m s . Similarly, i n c u b a t i n g the s a m p l e s with the cells in tissue-culture flasks for l o n g e r p e r i o d s o f l i m e (8-24 h) d i d h o t p r o d u c e g e n o t o x i c effects (results n o t shown). Table 3. Detection of adeno-associated virus amplification in Syrian hamster embryo celts by extracts of A. bisporus. Each "~alue is the mean of three determinalions per concentration Extract" (~tl)
Number of cells x 10 87 viable/dead
0 625 12.5 25 DMBA 0.6 gg/ml 0 6.25 12.5 25 DMBA i gg/ml
AF b
Evaluation
t 2.6/0 12.8/0.6 14.2/0.6 12.0/0.6 3.0/0.6
1.6 1.1 1.1 7.8
+
6.2/0 6.4/0.6 5.4/0.6 3.8/0.6 4.2/0.6
1 1.4 1.3 1.6 5.1
--+
A. bisporus samples (in vitro, 1 h incubation at 37~C) a Concen- Viability DNA retained on tration abs/rel filter b (~tl extract 10 ~ cells) (%) (%)
C-T
0
57/100
(73,77,73) 74_+ 2
-
5 10 20
57/107 57/109 57/102
(81,80,79) 80_+ 1 (72,74,75) 74_+ 2 (79,73,69) 74_+ 5
-6 0 0
Cooked
5 10 20
57/9I 57/68 57/25
(70, 69, 66) 68 _+ 2 (80,78,79) 79+ 1 (80,76,49) 68_+17
6 5 6
5 i0 20
57/95 57/104 57/89
(62, 73, 78) 71 • 8 (73,66) 70+ 3 (73, 52, 77) 67 • 13
3 4 7
(33,37) 35+ 2
39
2.5gmol.es 57/86
Individual animal data V b DNA on filter (%) (%) 76 65 60 69 85 86 89 74
(82, 85, 88, 88, 89, 86) (90, 89, 93, 92, 92, 92) (94, 94, 95, 94, 96, 95) (88,87,91,87,91,85) (97,97,96,95,95,95) (92, 90, 96, 89, 86, 83) (93, 98, 94, 95, 95, 95) (97,95,94,94,95,93)
Mean Group C - T mean • • 86• 91• 95• 88• 96• 89• 95_+2 91+_4 95•
A. bisporus raw 87 11 85 110 79 22O
(88,94,93,95,95,100) 94_+4 (73,85,85,94,82,91) 85• (92,90,96,89, 86,83) 894-5 89_+5
NDMA ™ 0.5 0.5
(42,38,38,39,53) (60,50,56,56,61,53)
71 85
42+_6 56-+4 49
2
42
a Individual animal data show the single filter values and means with SD derived therefrom b V, Vitality NDMA, N-nitrosodimethylamine, positive control compound; other explanations see legend of Table 4
(%)
Raw
NHB 87
Compound (mg/kg)
Bidist. H20 NaCt
Table 4. Induction of DNA single-strand breaks in hepatocytes with
10ml DMSO
Table 5. Assay for induction of DNA single-strand breaks in livers of maie SD rats treated l b p.o. with the ethanol extract of A. bisporus ~
NaCI
" DMBA, 7,12-dimethylbenzanthracene was the positive control b AF, amplification factor (counts test/counts control), AF > 2 is positive
Compound in solvent
A s s e s s m e n t o f D N A d a m a g e f o l l o w i n g oral e x p o s u r e o f S p r a g u e - D a w l e y rats to the m u s h r o o m e x t r a c t s conf i r m e d t h a t the results o b t a i n e d f r o m in vitro test assays were h o t artefactuM. T a b l e 5 s h o w s the ex vivo results o b t a i n e d in the liver cells after 1 h t r e a t m e n t . F i n a l l y , in o r d e r to exclude species or t a r g e t - o r g a n specificity as the cause o f the n o n - d e t e c t i o n o f A. bisporus g e n o t o x i c i t y , the extracts were i n v e s t i g a t e d in the m o u s e b o n e m a r r o w m i c r o n u c l e u s assay. T h e results o f two i n d e p e n d e n t assays are s h o w n in T a b l e 6. M i c r o n u c l e i were n o t i n d u c e d b y A. bisporus.
OEabs, absolute percentage of viable cells; rel, relative, percentage viable cells based on 100% viable in control; C, DNA retained on tirer (%) in control groups; T, DNA retained on filter in (%) in treated groups; ( C - T) > 20 is evaluated as positive b Mean and standard deviation of three duplicate determinations/ concentration NHB3A, N-nitrosomethylbenzylamine, positive control compound
Table 6. Incidence of micronucleated cells per 1000 polychromatic erythrocytes (mMe C57BL/J6 mice; oral dosing; 24 h sampling time) Compounds" (dose level)
No. of an•
MPE/1000 PE r ind. animal values
Group mean+_ SD
Experiment 1 Water dist.(10 ml/kg) 5 A. bisporus(lOml/kg) b 7 CP (65 mg/kg) 4
1,3, 6, 4, 5 8,5,8,5,6,3,8 19,15,31,29
3.8 • 1.9 6.1• 23.5_+4*
Experiment 2 Water dist.(10 ml/kg) 5 A. bisporus (10 ml/kg) b 5 CP (65 mg/kg) 1
6,4,5,4,5 6, 6, 5, 5, 5 26
4.8_+0.89 5.4 • 0.5 26
CP, cyclophosphamide, positive control compound b 10ml is equivalent to 13.25 g A. bisporus c MPE, micronucIea~ed polychromatic erythrocyte; PE, polychromatic erythrocyte * P
Mutagenic and genotoxic activities of extracts derived from the cooked and raw edible mushroom Agaricus bisporus * B.L. Pool-Zobel 1, p. Schmezer 1, y. Sinrachatanant 2, F. Kliagasioglu 3, K. Reinhart 1, R. Martin 4, p. Klein 1, and A.R. Tricker 1 1 Institute for Toxicology and Chemotherapy, German Cancer Research Center, Ira Neuenheimer Feld 280, and * Institute for Pharmaceutical Biology, University of Heidelberg, Im Neuenheimer Feld 364, D-6900 Heidelberg, Federal Republic of Germany, 2 Department of Pharmacology, Mahidol University, Rama VI Road, Bangkok, Thailand 3 Section of Medical Oncology, Cerrahpasa Medical School, Istanbul, Turkey Received 18 June 1990/Accepted 25 June 1990
Summary. A. bisporus has been reported to be carcinogenic to mice [Toth et al. (1986) Cancer Res 38:177 180] and mutagenic in Salmonella typhimurium [Sterner et al. (1982) Mutat Res 101:269-281]. The effects of different heat treatments on the mutagenicity of raw, cooked (boiled) and fried A.bisporus extracts in the S. typhirnurium test is reported. The spectrum of potential mutagenic activity ofA. bisporus extracts was tested in vitro in Syrian hamster embryo cells for selective D N A amplification and in primary rat hepatocytes for D N A singlestrand breaks. D N A single-strand breaks were also determined in liver cells of rats and micronuclei were measured in bone marrow cells of mice in vivo following oral application ofA. bisporus extracts. It was shown that the complex A. bisporus extracts per se are not detectably mutagenic in S. typhimurium and that the previously observed increase in number of colonies per plate is probably due to a histidine artefact. No indication of genotoxicity was seen in the two in vitro assays with primary mammalian cells with two different end points. No evidence of in vivo genotoxic effects was observed in the rat liver cells. Finally, A. bisporus was not genotoxic in the micronucleus assay of mouse bone marrow cells in contrast t o i t s previously reported carcinogenicity in mice.
Key words: Agaricus bisporus Histidine artefacts Genotoxic effects Salmonella typhimurium - Primary rat hepatocytes - D N A amplification Short-term in vivo tests micronuclei - D N A single-strand breaks
Introduction Previous feeding studies with the common edible mushroom Agaricus bisporus to 6-week-old Swiss mice show * Dedicated to Professor Dr. D. Schm/ihl on the occasion of his 65th birthday Abbreviation: AAV, adeno-associated virus Offprint requests to: B. L. Pool-Zobel
an increased tumor frequency in bone, forestomach, liver and lung (Toth and Erickson 1986). Suspect compounds, which may be responsible for this carcinogenic effect, include hydrazines such as agaritine, a major component present in A. bisporus. Ross et al. (1982) have shown that 1 kg raw A. bisporus contains 400-700 mg agaritine. Fischer et al. (1984) identified 94~629 mg/kg agaritine and noted that the content decreases by heat treatment and with storage. Agaritine has not been shown to be carcinogenic to mice after oral application (Toth et al. 1981 a), whereas several model compounds that decompose to liberate its potential metabolites have induced tumors in mice (Toth 1975, 1980). N'-Acetyl-4-(hydroxymethyl)phenylhydrazine induces tumors in the lung and blood vessels (Toth et al. 1978), 4-methylphenylhydrazine hydrochloride is carcinogenic in the lung, blood vessels and subcutis (Toth et al. 1977), whereas the tumors induced by 4-(hydroxymethyl)benzene diazonium tetrafluorborate are localized in the subcutis and skin (Toth et al. 1981b). Owing to the different patterns of afflicted tissues in which tumors are induced by the above compounds and the mushroom itself, and since agaritine is not carcinogenic, it seems probable that A. bisporus contains other carcinogenic compounds. One feasible mechanism for the carcinogenic activity may be via genotoxicity. Therefore, we investigated extracts of A. bisporus in various genotoxicity tests to determine whether mutagens are identifiable, and how this mutagenicity is detectable in the target organs suspectible to carcinogenicity following in vivo application. Finally, it was also an aim of this study to determine to what degree the potential mutagens are destroyed by cooking. One possibility in studying these aims was to assess the mutagenicity of A. bisporus extracts with the S. typhimurium assay (Ames et al. 1975; M a r o n and Ames 1983). Accordingly, Sterner et al. (1982) have shown 37 of 48 investigated extracts of larger fungi to be mutagenic in this test system. These studies were performed within a large screening program, but also included an aqueous extract of A. bisporus, which was suggested to contain mutagens.
476 W e repeated these d e t e r m i n a t i o n s with raw a n d heated A. bisporus samples to s t u d y the effect o f heat t r e a t m e n t o n the m u t a g e n c o n t e n t . A d d i t i o n a l l y , we used other genotoxicity assays to elucidate whether the m u s h r o o m c o n t a i n s c o m p o n e n t s also genotoxic in m a m m a l i a n cells. The following systems were applied. I n vitro selective a m plification o f a d e n o associated virus ( A A V ) D N A was assessed in Syrian h a m s t e r e m b r y o cells, a n d p r i m a r y rat hepatocytes were used as i n d i c a t o r cells to detect D N A single-strand breaks. Ex vivo D N A single-strand breaks were also assessed i n rat hepatocytes a n d micronuclei were detected in b o n e m a r r o w cells o f mice.
Materials and methods
ml with suspension medium (Spinner MEM, Gibco) for further use in the genotoxicity assays. The hepatocytes were either treated in vitro (1-ml suspension cultures containing 5-20 gl extract solution, 1 h incubation in shaking water bath at 37~ C) or they were obtained from treated animals (ex vivo assay).
Detection ofAA VDNA amplification. The rationale and methods of detecting DNA amplification have been specified previously (Klein et al. 1990; Pool et al. 1989). For this assay 2 x 1087Syrian hamster embryo cells, infected with AAV, are seeded into the wells ofa 6-well microtiter plate. After 24 h, the appropriate dilutions of the A. bisporus extract are added (each concentration in triplicate) and the plate is further incubated for 4 days. After this period, the cells are counted, and cell viability is determined. The DNA is lysed, hybridized with 87 AAV DNA and then the total AAV content per cell is determined by scintillation counting. A reproducible, AAV DNA content greater than twice that of the untreated control is taken as an indication of selective DNA amplification.
Preparation ofA. bisporus. The extracts were prepared according to the procedures described by Sterner et al. (1982). For this, 50 g raw, cooked (in 100 ml water for 10 min) or fried (in 50 g butter for 10 min) A. bisporus samples, obtained from the local food store, were homogenized and extracted either with ethanol or with ethyl acetate. Extracts from the total water and butter residues, following heat treatment of the mushrooms were extracted in the same way. The extracts were concentrated and the residues were dissolved in 8 ml ethanol/dimethylsulfoxide (1 : 1). Samples of 0-100 p.1 were used in the in vitro assays. The 150-~tland 200-~tlextract equivalents referred to in some ofthe tables are actually 75 ~tland 100 I~1doublestrength extracts obtained by extracting 100 g A. bisporus and dissolving the residues also in 8 ml solvent. The sample quantities used in the in vivo assays are described in the tables. For the micro.nucleus test, aqueous extracts were prepared from freeze-dried samples.
Determination of mutagenicity with S. typhimurium. The determination of his + reversion in histidine auxotrophic S. typhimurium strains was carried out according to standard methods (Ames et al. 1975; Maron and Ames 1983). The tests with A.bisporus samples were performed with and without metaholic activation by 9000 g fractions obtained from Aroclor-pretreated Sprague-Dawley rats. Mutagenicity was determined both with the preincubation modification as well as with the plate incorporation assay. All detailed specifications are noted in the tables. In some cases the assays were performed in the presence of y-glutamyltransferase, in order to stimulate activation of agaritine (Friederich et al. 1986). An increased mutagenicity of the A. bisporus extracts in the presence of this enzyme would be expected, if agaritine was the natural ingredient responsible for the mutagenicity of the extract.
Experimental animals. Maie Sprague-Dawley rats, C57BL/6J mice and Syrian hamsters were obtained from Charles River Wiga, Sulzfeld. Male Sprague-Dawley rats weighing approximately 200 g were used as donor animals for the subcellular liver fractions and for the primary rat hepatocytes. Micronuclei were determined in C57BL/6J mice weighing 25-30 g. Male and female Syrian hamsters were mated at the rate of 1 : 3 for the generation of emhryos as donors of cells.
Syrian hamster embryo cells. The Syrian hamster embryo cells were isolated from embryos on the 13th day of gestation using the saine procedure as described for the cells used to detect morphological transformation (Klein et al. 1990; Pienta et al. 1979). Isolation/treatment ofhepatoeytes. Primary rat hepatocytes were obtained following a two-step in situ perfusion method described by Berry and Friend (1969) and Bradley et al. (1984). The liberated cells were centrifuged at 30 g for purification, and the pellet was resuspended in a Ca 2+-, Mg 2+-free buffer for counting and determination of viability. The cell suspensions were adjusted to 2 x 106 cells/
Determination of DNA single-strand breaks. DNA single-strand breaks were determined with the alkaline filter elution method according to Kohn et al. (1981). Aliquots of 0.75 ml of the 1-ml suspension cultures were lysed on polycarbonate filters. The DNA was eluted at alkaline pli and the DNA in the resulting fractions determined fluorimetrically (Pool et al. 1990; Schmezer et al. 1990). The results are calculated as C - T , which is the percentage DNA retained on filters in the control - the percentage DNA retained on filters in the test groups.
Micronuclei detection. The procedures closely followed the original protocol of Schmid (1975), including modifications introduced by Ashby and Mohammed (1986). Male C57BL/6J mice were given a single oral application of an aqueous A. bisporus extract equivalent to 10 mg mushroom/kg mouse. A 65-mg/kg sample of cyclophosphamide was applied as the positive control; after 36 h the animals were sacrificed by Metofane anesthesia and the prepared bone marrow cells were streaked on microscope slides and stained with Giemsa. A total of 1000 polychromatic erythrocytes per mouse were evaluated for the occurrence of micronuclei and the data were statistically analyzed by a one-sided Student's t-test.
Results and discussion I n n u m e r o u s initial assays, a slight e n h a n c e m e n t o f his + revertants u p to d o u b l e the c o n t r o l was observed for the S. typhimurium strains T A 1537, T A 9 7 , T A 100, a n d T A 98. This e n h a n c e m e n t was within the range o f 2 5 200 ~1 A. bisporus extract a n d occurred b o t h with a n d w i t h o u t m e t a b o l i c a c t i v a t i o n for b o t h raw a n d cooked m u s h r o o m s ; it was also seen in the water a n d b u t t e r residues o f heat-treated A. bisporus samples. T a b l e 1 shows the results o b t a i n e d in strain T A 97 as a n example o f the observed activities. Three positive factors c o u l d be responsible for the observed e n h a n c e m e n t : (a) growth o f h i s - colonies as a result of excessive A. bisporus histidine in the ethanolic extracts, (b) a m u t a g e n i c effect o f a g a r i t i n e , a n d (c) the presence o f u n i d e n t i f i e d m u t a g e n s . Y a m a s a k i et al. (1977) have s h o w n t h a t certain o r g a n i c materials m a y c o n t a i n histidine, which m a y n o t be r e m o v e d by the extraction m e t h o d s e m p l o y e d here. These m i n u t e a m o u n t s o f histidine m a y evoke a growth o f h i s - bacteria, which in the Salmonella typhimurium assay m a y be falsely interpreted as a n i n d u c t i o n o f his + revertants. C u l t i v a t e d m u s h r o o m s o f the Agaricus types m a y c o n t a i n t 40-80 m g his-
477 Table 1. Studies on the mutagenic activities of ethanolic Agaricus bisporus extracts in Salmonella typhimurium TA97a: plate incorporation assay. Mean values and standard deviations of three plates per concentration are shown
A. bisporus extract per plate (gl)
Raw -S9 a
+$9
-$9"
+$9
0 18.75 37.5 70 100 150b 200 b
116_+ 5 129_+10 141_+12 134_+ 6 160+ 3 175_+12 211_+17
161_+16 196_+ 6 195_+ 7 168_+ 7 197-+21 237_+11 232_+14
116_+ 5 136_+ 8 150_+12 158-+15 187-+14 219_+ 9 246_+23
161_+16 190_+ 5 192_+ 3 191+_10 222_+12 246_+12 237_+41
Table 2. Studies on the mutagenic activities of XAD-purified A. bisporus extracts in S. typhimurium TA97a, 16h preincubation in 8 ml volume (bacteria, A. bisporussample, enzyme, buffer). The high number of spontaneous revertants is due to the long period of preincubation
Boiled
A. bisporus extract per plate (gl)
Raw
Cooked
- Enzyme + Enzyme
-- Enzyme + Enzyme
0 25 50 75 100 150 200
427+39 423_+25 369_+30 349_+42 371_+30 332_+119 348_+12
427_+39 360_+14 369_+15 423_+13 427_+24 414_+23 294_+32
297_+51 311_+43 314_+21 318_+ 0 373_+17 343_+28 373_+ 8
297_+51 359_+17 371 _+16 374_+ 2 355_+16 342_+ 6 383_+10
" $9, supernatant 9000g b Aliquot values, double concentrate in 100 gl solvent
tidine/100 g mushroom. Based on our extraction method using 50 g mushroom and a solution of the residue in 8 ml solvent, the highest expected histidine content would be approximately 0.25-0.5 mg/100 gl extract. This concentration is, however, likely to be considerably lower, since the total amount of histidine is probably not extractable by organic solvents because of its greater water solubility. Also, if 250-500 lag had been present in 100 gl A. bisporus extract, toxic effects should have been seen in Salmonella. Pure histidine, tested at concentrations of 93.8-125 gg/plate, was toxic to all strains of Salmonella. This toxicity was apparent as a decrease in microscopically and macroscopically visible colonies. The colony score, for example, of S. typhimurium TA 97 in the presence of increasing histidine concentrations was 162 + 13, 250+31, 235+_29, 236+_35, 307+21, 213_+26, 2 4 + 4 , and 8+-1 a t 0 , 3.9, 7.8, 15.6, 31.8, 62.5, 93.8, and 125 gg histidine/plate, respectively (plate incorporation assay, without $9). The increase of colonies at the lower histidine concentrations was similar to the pattern of increase observed after A. bisporus treatment. The artefactual his- growth was further studied by repurifying the extract over X A D to remove the histidine (Yamasaki and Ames 1977) and then assessing the mutagenic activity of the histidine-free extract. The purified X A D extract was confirmed to be histidine-free by thin-layer chromatography. This sample also resulted in a lower score of colonies per plate, which never reached 1.5 or 2 times that of the untreated control in rive S. typhimurium strains (results not shown). Accordingly, we conclude that the enhancement of colonies seen in Table 1 is hOt necessarily a result of induced his + revertants, but due to an artefact resulting from co-extracted histidine in the ethanolic A. bisporus extracts. Similar conclusions were drawn in a study by Griiter (1988) with the fungal species Lactarius helvas. Another contributing factor, however, could be agaritine, which is present in high amounts in A. bisporus and has been shown to act as a mutagen. According to an assumed content of 100-600 mg agaritine/kg mushroom (Fischer et al. 1984; Ross et al. 1982), our extracts could contain agaritine at a concentration of 0.25-1.25 gmol/
100 ~tl. In order to investigate the contribution of this hydrazine to the observed enhancement of Salmonella colonies, we selected conditions for which agaritine has been shown to be mutagenic (Friederich et al. 1986) and tested A. bisporus extracts in the presence of the activating enzyme y-glutamyltransferase. Table 2 shows the results obtained with a histidine-free sample purified over XAD. On the basis of the assumed amount of agaritine present in the extracts, the colony score should well exceed 1000 colonies/plate (Friederich et al. 1986). However, as can be seen from the results, an enhancement in colony number that would indicate agaritine mutagenicity was not evident. This may be due to the fact that the levels of agaritine present in the tested sample are less than those calculated above and/or that the presence of other compounds in the test sample inhibits the mutagenic effect of agaritine (Pool 1988). Finally, the possibility can not be excluded that other factors may be contained in A. bisporus that are genotoxic and, therefore, highly likely to be responsible for the carcinogenic effects seen in mice. These factors are not readily detectable by the S. typhimurium assay, since mainly histidine artefacts were observed (see above)~ However, they may be readily detectable by other shortterm assays utilizing mammalian cells. Accordingly, we investigated A. bisporus extracts for some other genetic end points in vitro using Syrian hamster embryo cells and primary rat hepatocytes as indicator organisms. In order to study the influences of the whole organism (Pool and Schm/ihl 1987) in vivo assays were also performed. Table 3 shows results obtained for the analysis of D N A amplification in Syrian hamster embryo cells. This genetic end point is an important indicator of a cell's answer to toxic stress by chemical compounds. It is associated both with the induction of resistance and with the processes of carcinogenesis (Pool et al. 1989). The detection of AAV amplification in Syrian hamster embryo cells is a new system and the first demonstration of this end point in primary cells. The extract was tested at up to 25 ~tl/incubation mixture in which A. bisporus did not induce D N A amplification. Higher solvent amounts were toxic to the cells.
478 A n o t h e r efficient a n d r e l e v a n t s h o r t - t e r m assay for g e n o t o x i c i t y is the h e p a t o c y t e D N A s i n g l e - s t r a n d b r e a k test ( S c h m e z e r et al. 1990; Sina et al. 1983). I n this sensitive o n e - c o m p a r t m e n t assay in vitro, D N A d a m a g e is m o n i t o r e d w i t h i n m e t a b o l i c a l l y c o m p e t e n t r a t liver cells. A. bisporus was tested using u p to 20 gl e x t r a c t / m l incub a t i o n m i x t u r e , since the solvent was h o t toxic u n d e r the test c o n d i t i o n s . T a b l e 4 shows no D N A - d a m a g i n g activity b y A. bisporus e x t r a c t d e r i v e d f r o m either c o o k e d o r r a w m u s h r o o m s . Similarly, i n c u b a t i n g the s a m p l e s with the cells in tissue-culture flasks for l o n g e r p e r i o d s o f l i m e (8-24 h) d i d h o t p r o d u c e g e n o t o x i c effects (results n o t shown). Table 3. Detection of adeno-associated virus amplification in Syrian hamster embryo celts by extracts of A. bisporus. Each "~alue is the mean of three determinalions per concentration Extract" (~tl)
Number of cells x 10 87 viable/dead
0 625 12.5 25 DMBA 0.6 gg/ml 0 6.25 12.5 25 DMBA i gg/ml
AF b
Evaluation
t 2.6/0 12.8/0.6 14.2/0.6 12.0/0.6 3.0/0.6
1.6 1.1 1.1 7.8
+
6.2/0 6.4/0.6 5.4/0.6 3.8/0.6 4.2/0.6
1 1.4 1.3 1.6 5.1
--+
A. bisporus samples (in vitro, 1 h incubation at 37~C) a Concen- Viability DNA retained on tration abs/rel filter b (~tl extract 10 ~ cells) (%) (%)
C-T
0
57/100
(73,77,73) 74_+ 2
-
5 10 20
57/107 57/109 57/102
(81,80,79) 80_+ 1 (72,74,75) 74_+ 2 (79,73,69) 74_+ 5
-6 0 0
Cooked
5 10 20
57/9I 57/68 57/25
(70, 69, 66) 68 _+ 2 (80,78,79) 79+ 1 (80,76,49) 68_+17
6 5 6
5 i0 20
57/95 57/104 57/89
(62, 73, 78) 71 • 8 (73,66) 70+ 3 (73, 52, 77) 67 • 13
3 4 7
(33,37) 35+ 2
39
2.5gmol.es 57/86
Individual animal data V b DNA on filter (%) (%) 76 65 60 69 85 86 89 74
(82, 85, 88, 88, 89, 86) (90, 89, 93, 92, 92, 92) (94, 94, 95, 94, 96, 95) (88,87,91,87,91,85) (97,97,96,95,95,95) (92, 90, 96, 89, 86, 83) (93, 98, 94, 95, 95, 95) (97,95,94,94,95,93)
Mean Group C - T mean • • 86• 91• 95• 88• 96• 89• 95_+2 91+_4 95•
A. bisporus raw 87 11 85 110 79 22O
(88,94,93,95,95,100) 94_+4 (73,85,85,94,82,91) 85• (92,90,96,89, 86,83) 894-5 89_+5
NDMA ™ 0.5 0.5
(42,38,38,39,53) (60,50,56,56,61,53)
71 85
42+_6 56-+4 49
2
42
a Individual animal data show the single filter values and means with SD derived therefrom b V, Vitality NDMA, N-nitrosodimethylamine, positive control compound; other explanations see legend of Table 4
(%)
Raw
NHB 87
Compound (mg/kg)
Bidist. H20 NaCt
Table 4. Induction of DNA single-strand breaks in hepatocytes with
10ml DMSO
Table 5. Assay for induction of DNA single-strand breaks in livers of maie SD rats treated l b p.o. with the ethanol extract of A. bisporus ~
NaCI
" DMBA, 7,12-dimethylbenzanthracene was the positive control b AF, amplification factor (counts test/counts control), AF > 2 is positive
Compound in solvent
A s s e s s m e n t o f D N A d a m a g e f o l l o w i n g oral e x p o s u r e o f S p r a g u e - D a w l e y rats to the m u s h r o o m e x t r a c t s conf i r m e d t h a t the results o b t a i n e d f r o m in vitro test assays were h o t artefactuM. T a b l e 5 s h o w s the ex vivo results o b t a i n e d in the liver cells after 1 h t r e a t m e n t . F i n a l l y , in o r d e r to exclude species or t a r g e t - o r g a n specificity as the cause o f the n o n - d e t e c t i o n o f A. bisporus g e n o t o x i c i t y , the extracts were i n v e s t i g a t e d in the m o u s e b o n e m a r r o w m i c r o n u c l e u s assay. T h e results o f two i n d e p e n d e n t assays are s h o w n in T a b l e 6. M i c r o n u c l e i were n o t i n d u c e d b y A. bisporus.
OEabs, absolute percentage of viable cells; rel, relative, percentage viable cells based on 100% viable in control; C, DNA retained on tirer (%) in control groups; T, DNA retained on filter in (%) in treated groups; ( C - T) > 20 is evaluated as positive b Mean and standard deviation of three duplicate determinations/ concentration NHB3A, N-nitrosomethylbenzylamine, positive control compound
Table 6. Incidence of micronucleated cells per 1000 polychromatic erythrocytes (mMe C57BL/J6 mice; oral dosing; 24 h sampling time) Compounds" (dose level)
No. of an•
MPE/1000 PE r ind. animal values
Group mean+_ SD
Experiment 1 Water dist.(10 ml/kg) 5 A. bisporus(lOml/kg) b 7 CP (65 mg/kg) 4
1,3, 6, 4, 5 8,5,8,5,6,3,8 19,15,31,29
3.8 • 1.9 6.1• 23.5_+4*
Experiment 2 Water dist.(10 ml/kg) 5 A. bisporus (10 ml/kg) b 5 CP (65 mg/kg) 1
6,4,5,4,5 6, 6, 5, 5, 5 26
4.8_+0.89 5.4 • 0.5 26
CP, cyclophosphamide, positive control compound b 10ml is equivalent to 13.25 g A. bisporus c MPE, micronucIea~ed polychromatic erythrocyte; PE, polychromatic erythrocyte * P
