Agaritine Content of 53 Agaricus Species Collected from Nature Vera Schulzova, Jana Hajslova, R

Agaritine content of 53 Agaricus species collected from nature Vera Schulzova, Jana Hajslova, R. Peroutka, Jiri Hlavasek, Jørn Gry, Hans Christer Andersson

To cite this version:

Vera Schulzova, Jana Hajslova, R. Peroutka, Jiri Hlavasek, Jørn Gry, et al.. Agaritine content of 53 Agaricus species collected from nature. Food Additives and Contaminants, 2009, 26 (01), pp.82-93. 10.1080/02652030802039903. hal-00577390

HAL Id: hal-00577390 https://hal.archives-ouvertes.fr/hal-00577390 Submitted on 17 Mar 2011

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Agaritine content of 53 Agaricus species collected from nature

Journal: Food Additives and Contaminants

Manuscript ID: TFAC-2008-057

Manuscript Type: Original Research Paper

Date Submitted by the 22-Feb-2008 Author:

Complete List of Authors: Schulzova, Vera; Institute of Chemical Technology, Department of Food Chemistry and Analysis Hajslova, Jana; Institute of Chemical Technology, Department of Food Chemistry and Analysis Peroutka, R.; Institute of Chemical Technology, Department of Food Chemistry and Analysis Hlavasek, Jiri; Czech Mycological Society Gry, Jørn; Copenhagen Technical University, Fødevareinstituttet Andersson, Hans; National Food Administration, Research and Development, Toxicology Division

Methods/Techniques: HPLC

Additives/Contaminants: Agaratine

Food Types: Mushrooms

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1 2 Agaritine content of 53 Agaricus species collected from nature 3 4 5 6 Abstract 7 8 Fifty-three different species of the genus Agaricus were collected in the Czech Republic 9 10 11 during the period 1998-2001 and identified by an experienced mycologist. The samples 12 13 were analysed for agaritine (N 2-(γ-L-glutamyl)-4-hydroxymethylphenylhydrazine) content, 14 15 16 a precursor toFor a suspected Peer rodent carcinogen. Review There was a hugeOnly variation in agaritine 17 18 content between species, less variation between samples of a species. Whereas the 19 20 21 cultivated mushroom Agaricus bisporus commonly contain 200-500 mg agaritine/kg fresh 22 23 weight, no less than 24 of the 53 species contained agaritine levels above 1000 mg/kg fresh 24 25 26 weight. The highest level was found in A. elvensis containing up to 10,000 mg/kg fresh 27 28 weight. Seventeen species contained intermediate levels (125-1000 mg/kg), and twelve 29 30 species below 125 mg/kg. Some of the species producing low levels of agaritine might be 31 32 33 candidates for future strain-development of Agaricus mushrooms for cultivation. No 34 35 correlation could be observed between the agaritine content and size of the mushroom, 36 37 38 week of the year when collected, year of collection, or site of collection. Besides occurring 39 40 in the genus Agaricus , some species of the genera Leucoagaricus and Macrolepiota were 41 42 43 also shown to contain agaritine. 44 45 46 47 Introduction 48 49 50 The commonly eaten cultivated mushroom, Agaricus bisporus , belongs to the Agaricaceae 51 52 family. For a long time A. bisporus was believed to have been derived from several 53 54 55 different wild Agaricus species (Toth 2000), and its continued propagation over many 56 57 centuries to have resulted in several commercial strains which have no exact counterpart in 58 59 60 nature. This hypothesis has now been proven incorrect as wild A. bisporus populations

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1 2 have been identified in various parts of the world (Mozina et al. 1993; Kerrigan 1995). In 3 4 5 some countries, strains of other species, such as A. bitorquis, are the preferred cultivated 6 7 varieties (Schulzová et al. 2002). 8 9 10 11 12 In 1961 Levenberg isolated a characteristic glutamine-containing compound from the 13 14 press-juice of A. bisporus. He characterised the compound physically and elucidated its 15 16 For Peer Review Only 2 17 chemical structure as N -(γ-L-glutamyl)-4-hydroxymethylphenylhydrazine (Figure 1), and 18 19 gave it the trivial name agaritine. As shown in Table I, the level of agaritine in the 20 21 22 cultivated mushroom is fairly high, usually in the range 200 to 500 mg/kg fresh weight 23 24 (Andersson and Gry 2004), but levels as high as 1700 mg/kg fresh weight have been 25 26 27 reported (Liu et al. 1982). 28 29 30 31 [Fig. 1 inserted about here] 32 33 34 [Table I inserted about here] 35 36 37 38 39 Agaritine is the predominant, although not the only phenylhydrazine derivative in A. 40 41 bisporus . The compounds 4-carboxyphenylhydrazine, N 2-(γ-L-glutamyl)-4-carboxy- 42 43 44 phenylhydrazine, and 4-hydroxymethylbenzenediazonium ion make up around 10% of the 45 46 total phenylhydrazines in the mushroom (Levenberg 1962; Chauhan et al. 1984, 1985; Ross 47 48 et al. 1982). 49 50 51 52 53 The potential carcinogenicity of A. bisporus , and the possible role of agaritine and the other 54 55 56 phenylhydrazine derivatives in inducing this severe adverse effect, has been a matter of 57 58 controversy for a number of years (Gry and Pilegaard 1991; Gry and Andersson 1998; Toth 59 60

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1 2 2000; Andersson and Gry 2004). In long-term in vivo studies, a diet of fresh or baked A. 3 4 5 bisporus for 3 days per week over 2 years produced tumours in various tissues of both male 6 7 and female outbred Swiss mice (Toth and Ericsson 1986; Toth et al. 1997a). An elevated 8 9 tumour incidence, although not statistically significant, was observed when baked 10 11 12 mushrooms were fed to mice according to a more balanced feeding schedule - 12 hours 13 14 each day for 5 days each week during life-time (Toth et al. 1997b). Similarly, oral 15 16 For Peer Review Only 2 17 administration of 4-carboxyphenylhydrazine or N -(γ-L-glutamyl)-4- 18 19 carboxyphenylhydrazine, or the 4-hydroxymethylbenzenediazonium ion resulted in tumour 20 21 22 induction in Swiss mice (Toth 2000). The main phenylhydrazine in the mushroom, 23 24 agaritine (98% pure), however, did not result in tumour induction when administered in 25 26 27 drinking water in a long-term carcinogenicity study (Toth et al. 1981). This observation is 28 29 unexpected, as agaritine has been shown to bind covalently to mouse DNA (Shephard and 30 31 Schlatter 1998) and to increase the mutation frequency in forestomach and kidney tissue of 32 33 34 lacI transgenic mice in vivo (Shephard et al. 1995). As it was recently shown that agaritine 35 36 is unstable in oxygenated aqueous solution (Hajslova et al. 2002), it cannot be ruled out 37 38 39 that the exposure to agaritine was lower than expected in the long-term carcinogenicity 40 41 experiment. 42 43 44 45 46 Until it has been established whether consumption of the cultivated mushroom constitutes 47 48 a risk for adverse health effects or not, it seems advisable to abstain from exaggerated 49 50 51 exposures to phenylhydrazine derivatives (Schulzová et al. 2002). This could be done by 52 53 growers substituting the strains of the cultivated mushroom producing high levels of 54 55 56 agaritine with strains or other Agaricus species having comparable organoleptic properties 57 58 but producing lower levels of agaritine. Sources for such strains may be available from the 59 60

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1 2 nature. The present study reports on the quantity of agaritine detected in 53 different 3 4 5 Agaricus species collected in the Czech Republic. The data may also be of interest for 6 7 mushroom pickers when assessing the edibility of Agaricus species to be collected. 8 9 10 11 12 13 14 Materials and Methods 15 16 For Peer Review Only 17 Samples and chemicals 18 19 Different Agaricus species were picked at various locations in the Czech Republic (most of 20 21 22 them in the central part of Bohemia, close to Prague) during the mushroom growing 23 24 seasons 1998-2001. All species were determined by one of the authors (Jiri Hlavá ček), an 25 26 experienced mycologist specialized on the taxonomy of the genus Agaricus . The Agaricus 27 28 29 taxonomy remains far from clear, despite the contributions of many authors over the years, 30 31 especially Pilát (1951), Möller (1952), Essette (1964), Heinemann (1977), Wasser (1980), 32 33 34 Cappelli (1984), Knudsen (1992), Bohus (1995), Nauta (2001) and Parra (2003). As a 35 36 consequence, mycologists advocate different schools of Agaricus taxonomy. We have 37 38 39 chosen the practical approach of presenting both the taxonomic names as given by the 40 41 mycologist who determined the mushroom species, J. Hlavá ček, who used more narrow 42 43 concepts identifying a species, and the taxonomic names commonly used in the Western 44 45 46 and Northern European countries where broader concepts identifying the various Agaricus 47 48 species are prevailing (Knudsen 1992; Nauta 2001). 49 50 51 52 53 Most mushroom samples delivered to the Institute of Chemical Technology (Prague) for 54 55 56 chemical analysis of agaritine content were clearly identified and did not require further 57 58 consultation. In a few cases where the delivered samples were not adequately identified, 59 60

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1 2 Hlavá ček was consulted again. As he unfortunately died during the present study, his 3 4 č 5 colleagues Borovi ka and Burel took over the confirmation step of a few samples. 6 7 8 9 Mushrooms were stored in the refrigerator until analysed (Schulzová et al. 2002), which 10 11 12 generally was within three days from being collected. We recently showed that the 13 14 agaritine content of cultivated Agaricus purchased on the open market remained 15 16 For Peer Review Only o 17 unchanged for the first three days in the refrigerator (5 C), after which the agaritine content 18 19 started to decrease (Schulzová et al. 2002). 20 21 22 23 24 Water used for preparation of solutions was distilled and further purified using the Milli Q 25 26 RG purification system (Millipore, Germany). Methanol used for extraction was supplied 27 28 29 by Penta (Chrudim, Czech Republic), and gradient grade methanol for the HPLC mobile 30 31 phase was purchased from Merck (Darmstadt, Germany). The agaritine standard was 32 33 34 synthesised by Henrik Frandsen, Institute of Food and Veterinary Research, according to 35 36 the method of Wallcave et al. (1979), but with some important modifications being 37 38 39 introduced (Frandsen 1998). The synthesised agaritine was more than 85% pure as shown 40 41 at wavelength 200, 237 and 280 nm by high performance liquid chromatography (HPLC). 42 43 The purity factor of the agaritine peak reported by the Hewlett Packard ChemStation peak 44 45 46 purity software was 999.9. The main impurity (11.9-13.6%) in the standard originated from 47 48 the agaritine synthesis and was stable during the experiments reported here. The standard 49 50 51 was protected from light and stored under oxygen-free nitrogen in the freezer. 52 53 54 55 56 Sample preparation 57 58 Agaritine standard for identification: a stock solution of agaritine in methanol (0.25 mg/ml) 59 60

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1 2 was prepared. From this 100 l were transferred into vials and evaporated to dryness by a 3 4 5 stream of oxygen-free nitrogen. The vials were stored sealed in the freezer. Stability tests 6 7 showed the dry agaritine standard in the freezer to be stable (Schulzová et al. 2002). Before 8 9 10 analysis, the agaritine standard was dissolved in 1 ml of Milli Q water and analysed within 11 12 6 hours (Schulzová et al. 2003). 13 14 15 16 For Peer Review Only 17 Approximately 20 gram of fresh whole mushroom was mixed with 100 ml methanol and 18 19 homogenised for 10 minutes in an Ultra Turax (Janke a Kunkel, IKA-Werk). The 20 21 22 homogenate was shaken for 30 minutes and a crude extract prepared by filtration. The 23 24 volume of the filtrate was adjusted to 200 ml with methanol, 10 ml of the extract was 25 26 27 evaporated to dryness and the residue dissolved in 2 ml Milli Q water. This solution was 28 29 filtered through a microfilter (25 mm Filter Unit, 5.0 m PTFE, PP, ThermoQuest) into a 30 31 32 vial and an 20 l aliquot injected (always within 6 hours) onto the HPLC column. 33 34 35 36 Identification and Quantification 37 38 39 HPLC was performed using a Hewlett-Packard HP 1100 liquid chromatograph (Wallbron, 40 41 Germany) equipped with diode array detector (DAD) and autosampler. A sample amount 42 43 44 of 20 l was applied to the separating column (250x4 mm), LiChrospher 100 RP-18 (5 m), 45 46 Merck, with precolumn (4x4 mm), Li Chrospher 100 RP-18 (5 m), Merck. Optimization 47 48 49 studies showed a methanol-water mixture to be suitable as mobile phase for separation; the 50 51 mobile phase gradient being 10% methanol held for 5 min, changed to 80% over 15 min, 52 53 54 and held at 80% methanol for 5 min. Total run time was 25 min, post time was 5 min. The 55 56 flow rate was 1 ml/min, the column temperature being 35 oC. All analyses were run in 57 58 duplicate. For identification of agaritine the retention time, DAD spectra and peak purity 59 60

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1 2 software function were used. Agaritine was detected at 237 nm and quantified by 3 4 5 comparing the peak area of the analysed sample with the peak area for known amounts of 6 7 the pure standard. Free amino acids that strongly absorb in the UV were shown not to 8 9 interfere with the agaritine peak. The correlation coefficient of the UV spectra (normalized 10 11 12 absorbance) of agaritine in standard solution and in a real sample was 0.999. The 13 14 chromatograms and UV spectra of the agaritine standard and an Agaricus campestris 15 16 For Peer Review Only 17 sample are shown in Figure 2. The detector response was linear in the range 0.2 g/ml – 18 19 2.0 mg/ml, the equation of the linear regression of the calibration curve being y=31.325x + 20 21 2 22 17.584, with a correlation coefficient of R =0.9999.. Under the experimental conditions 23 24 described for sample preparation, the limit of detection for the method was 0.02 g/ml 25 26 27 (based on a signal/noise of 3:1), corresponding to 0.2 mg/kg fresh weight of mushroom, 28 29 limit of quantification was 0.6 mg/kg. The repeatability of measurements at agaritine levels 30 31 commonly found in fresh mushroom samples (200 mg/kg), expressed as the relative 32 33 34 standard deviation, was 4.5% for six replicates. Method validation has been presented 35 36 elsewhere (Schulzová et al. 2002). 37 38 39 [Fig. 2 inserted about here] 40 41 42 43 44 The data was analysed by Student’s T-test ( α=0.05), using the software of Microsoft Excel 45 46 2003. 47 48 49 50 51 Results 52 53 Mushrooms of the genus Agaricus were collected in various regions of the Czech Republic 54 55 56 during different periods of the growing seasons 1998-2001, and identified by an 57 58 experienced mycologist. Fifty-three different species were found. Table II shows the 59 60

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1 2 average agaritine contents of the various Agaricus species identified, and, where 3 4 5 appropriate, the range in agaritine content, without controlling for mushroom size. When 6 7 the average agaritine content was calculated, a value of 50% of the detection limit was used 8 9 for the samples in which no agaritine could be detected. Only a few samples were available 10 11 12 for many of the more uncommon Agaricus species. 13 14 [Table II inserted about here.] 15 16 For Peer Review Only 17 18 19 A significant difference in agaritine content was observed between species. The amounts 20 21 22 detected in individual species varied from non-detectable levels up to several g per kg fresh 23 24 weight. One sample of A. elvensis contained 10 g/kg fresh weight. Some species were 25 26 characterized by low agaritine content (<100 mg/kg fresh weight), others by intermediate 27 28 29 (100-1000 mg/kg fresh weight) or high ( >1000 mg/kg fresh weight) levels of agaritine. It 30 31 should be noted that the allocation of species to groups having these arbitrarily set boarders 32 33 34 is hampered by the low number of samples analysed for many species, resulting in less 35 36 reliable values of average agaritine content. 37 38 39 [Table III inserted about here.] 40 41 42 43 Nine of the 53 species analysed in our study contained less than 100 mg agaritine/kg fresh 44 45 46 weight (Table III), which was the level chosen defining mushrooms with a low agaritine 47 48 content. Of these nine species with low agaritine content only the relatively rare A. maskae , 49 50 51 A. silvaticus var. pallidus , and possibly also A. caroli are sought after by mushroom 52 53 connoisseurs. None of them are common mushrooms. 54 55 56 57 58 59 60

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1 2 Twenty-four of the 53 analysed Agaricus species contained high levels of agaritine, more 3 4 5 than 1000 mg/kg fresh weight (Table III). In 20 species intermediate levels of agaritine 6 7 were found, 100-1000 mg/kg fresh weight (Table III), not deviating substantially from the 8 9 levels reported to occur in the cultivated mushroom, A. bisporus . 10 11 12 13 14 For the seven species for which more than 10 mushroom samples were analysed ( A. 15 16 For Peer Review Only 17 arvensis , A. bitorquis , A. bisporus var. bisporus ( A. brunnescens ), A. campestris, A. 18 19 maleolens , A. silvaticus , and A. xanthoderma ) it was studied whether a correlation could be 20 21 22 found between the agaritine content and size of the mushroom, phenology, year of 23 24 collection, or site of collection. 25 26 27 28 29 No correlation could be found between diameter of the mushroom cap and its agaritine 30 31 content. A typical example of the poor correlation between cup size and agaritine content is 32 33 34 demonstrated by A. arvensis (r = 0.033; n.s.), Figure 3. These observations contradict the 35 36 findings of Stijve et al. (1986), that smaller fruit bodies of A. arvensis and A. augustus 37 38 39 contain higher amounts of agaritine than bigger mushrooms of the same species. There was 40 41 also no correlation in our studies between week of the year when the mushrooms were 42 43 collected and their agaritine content (data not shown). 44 45 46 47 48 [Figure 3 inserted about here.] 49 50 51 52 53 Among the 13 Agaricus species of which enough mushroom samples had been collected 54 55 56 over more than one year, there was in general no statistical difference in the average 57 58 agaritine content between years (shown by students t-test). The only data that reached 59 60

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1 2 statistical significance were those of A. maleolens . In the years 1998 and 1999 A. maleolens 3 4 5 contained significantly lower levels of agaritine (51±90 and 25±54 mg/kg fresh weight, 6 7 respectively), than in year 2000 (165±50 mg/kg fresh weight), t-test, p=0.023. During the 8 9 first two years the agaritine content was below the detection limit of the analytical method 10 11 12 in the majority of the samples, whereas none of the samples collected in year 2000 13 14 contained below 100 mg/kg fresh weight. It is our interpretation that this significant 15 16 For Peer Review Only 17 difference might have disappeared had larger number of mushroom samples been analysed. 18 19 20 21 22 No correlation could be found between agaritine content and geographical region where the 23 24 mushrooms were collected (data not shown). 25 26 27 28 29 In addition to analysing for agaritine in the different Agaricus species, we also analysed for 30 31 agaritine in four species belonging to each of the related genera Leucoagaricus and 32 33 34 Macrolepiota. If agaritine occurs in mushroom species outside the genus Agaricus , 35 36 Leucoagaricus and Macrolepiota are the genera where agaritine might be found. As shown in 37 38 39 Table IV, one of the four Macrolepiota species, M. konradii , contained low amounts of 40 41 agaritine, 42 mg/kg fresh weight. Agaritine was also detected in three of the Leucoagaricus 42 43 species – L. bresadolae , L. cretaceus , and L. naucinus - but here the contents were higher, up 44 45 46 to 838 mg/kg fresh weight (Table IV). As agaritine has been reported to occur in the 47 48 cultivated Oyster mushroom Pleurotus ostreatus (Burini et al., 1999), we also analysed for 49 50 51 agaritine in this mushroom but found none. 52 53 [Table IV inserted about here.] 54 55 56 57 58 59 60

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1 2 3 4 5 Discussion 6 7 Two research groups, one from the USA and the other from Switzerland, have previously 8 9 analysed wild-growing Agaricus and non-Agaricus species for agaritine. Levenberg (1964) 10 11 12 examined boiled press-juice extracts from as many as 45 representative genera of wild 13 14 basidiomycetes collected in the USA. As part of this survey, fruit bodies of 15 different 15 16 For Peer Review Only 17 species belonging to the genus Agaricus were analysed. Unfortunately, Levenberg used a 18 19 nomenclature for the mushroom that hardly fit with that used in Europe today. Of the 15 20 21 22 Agaricus species, ten were reported to contain agaritine in quantities comparable to those 23 24 found in A. bisporus , whereas five species were devoid of the compound (Table V). The 25 26 limit of detection for the analytical method (spectrophotometric determination of agaritine 27 28 29 by measurement of its rate of enzymatic hydrolysis) used by Levenberg (1964) was not 30 31 reported. The single sample of fresh A. bisporus analysed for its agaritine content was 32 33 34 reported to contain 3300 mg agaritine/kg dry weight, which corresponds to approximately 35 36 330 mg/kg fresh weight. 37 38 39 [Table V inserted about here.] 40 41 42 43 Subsequently, Swiss investigators reported HPLC (with a limit of detection in the region 44 45 46 20-25 mg/kg) and thin layer chromatography data on freeze-dried mushrooms, indicating 47 48 that some wild-growing Agaricus species contain substantially higher amounts of agaritine 49 50 51 than the cultivated A. bisporus (Stijve et al. 1986; Stijve and Pittet 2000). Because a 52 53 considerable time had elapsed between picking of some mushroom samples and freeze- 54 55 56 drying, the latter investigators were unsure that the amounts detected were the 57 58 59 60

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1 2 characteristic levels of freshly harvested wild mushrooms and, therefore, did not give 3 4 5 specific information on the quantity of agaritine in the various species. 6 7 8 9 The agaritine contents given for the various Agaricus species in the present study should 10 11 12 fairly accurately describe the levels in freshly collected mushroom samples, at least when 13 14 the number of samples studied for a species have been large enough. Several factors may 15 16 For Peer Review Only 17 influence the agaritine content of a mushroom. Studies performed with the cultivated 18 19 Agaricus bisporus have revealed that, in addition to hereditary factors, cultural practice 20 21 22 employed in production, stage of cropping-cycle (flush), and maturity of mushrooms at 23 24 harvest may influence the agaritine level in the mushroom (Andersson and Gry 2004). 25 26 Some of these factors are likely to influence the agaritine content also of wild-growing 27 28 29 species. The length of storage after harvest and the conditions during storage are other 30 31 factors that may influence the agaritine content (Andersson and Gry 2004). This is 32 33 34 exemplified by the data given in Table I. The average level of agaritine in A. bisporus 35 36 obtained directly from the growers was 723 ± 276 mg/kg fresh weight, whereas it was 475 37 38 39 ± 221 mg/kg fresh weight in mushrooms purchased on the open market. We have 40 41 minimized the potential influence of storage on the agaritine content in the present study by 42 43 analysing samples within three days after being collected. 44 45 46 47 48 Already in 1983, Speroni and co-workers suggested that wild-growing Agaricus 49 50 51 mushrooms contain higher amounts of agaritine than the corresponding cultivated 52 53 mushrooms. These investigators had noted that a cultivated strain of A. bisporus with 54 55 56 brown colour phenotype, PSU-351, which was only recently isolated from nature, 57 58 contained higher agaritine levels than other cultivated A. bisporus strains. It was 59 60

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1 2 hypothesised that this strain had not yet adapted to the less stressing environmental 3 4 5 conditions during cultivation and not yet lost or reduced its inherited ability to produce 6 7 compounds inhibiting growth of certain competing fungi. A similar observation is made in 8 9 the present study. In the Czech Republic, it is strains of A. bitorquis and ‘ A. hortensis’ that 10 11 12 are cultivated. Samples of these cultivated strains have been reported to contain 13 14 comparable levels of agaritine, varying between 165 and 457 mg/kg fresh weight, with an 15 16 For Peer Review Only 17 average content of 272 mg/kg fresh weight (Schulzová et al. 2002). As shown in Table II, 18 19 A. bitorquis and A. bisporus collected from nature contain significantly higher amounts of 20 21 22 agaritine, 1 473 and 746 mg/kg fresh weight, respectively. 23 24 25 26 Although too few samples were analysed for some Agaricus species to prove their normal 27 28 29 agaritine content, enough samples were analysed to conclude on the agaritine content for 30 31 other species. As shown in Table II and III, no less than 24 of the studied species contained 32 33 34 agaritine levels above 1000 mg/kg fresh weight. This amount should be compared with the 35 36 levels commonly found in A. bisporus , 200-500 mg/kg fresh weight. The highest amounts 37 38 39 were observed in A. elvensis (mean 4445 mg/kg), A. purpurellus (4100 mg/kg), A. augustus 40 41 (3995 mg/kg), A. fissuratus (3526 mg/kg) and A. sagatus (3479 mg/kg). As there is an 42 43 ongoing discussion whether it is advisable or not to consume fresh cultivated mushroom 44 45 46 (Toth 2000; Andersson and Gry 2004), it seems highly questionable to advise consumption 47 48 of wild-growing Agaricus species with even higher agaritine contents than the cultivated 49 50 51 species. Some of the species with a high agaritine content, however, have a firm reputation 52 53 as being good or even excellent culinary delicacies, e.g. A. augustus , A. fissuratus , A. 54 55 56 essettei and A. substramineus . On the other hand, as indicated in Table II, some of them are 57 58 59 60

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1 2 recognised as bio-accumulating cadmium and other heavy metals from the soil ( Řanda and 3 4 č 5 Ku era 2004) 6 7 8 9 The present study aimed at identifying tasteful, non-toxic Agaricus species with a low 10 11 12 agaritine content. Such species would be potential sources for strain development for the 13 14 mushroom cultivation industry, could it be shown they are devoid of other constituents 15 16 For Peer Review Only 17 with potentially harmful effects. Only nine of the analysed Agaricus species had an 18 19 agaritine content bellow 100 mg/kg fresh weight. Of these at least five should be classified 20 21 22 as non-edible as they may give rise to gastrointestinal symptoms due to formation of 23 24 phenol. Based on the agaritine data presented in the present report, the most promising 25 26 species for strain development seem to be A. caroli , A. maskae , A. silvaticus var. pallidus , 27 28 29 and possibly A. squamulifer . Additional studies are required to establish whether these 30 31 species fulfil the criteria necessary to be good substitutes for A. bisporus as cultivated 32 33 34 mushrooms. 35 36 37 38 39 We have not addressed the question whether the presence of agaritine within the genus 40 41 Agaricus may have chemotaxonomic significance. However, we have noted that the 42 43 various sections of the genus Agaricus seem to be characterized by different abilities to 44 45 46 produce agaritine (Table IV). Stijve et al. (1986) have made a similar observation. 47 48 49 50 51 The question whether agaritine may be found in mushrooms outside the genus Agaricus 52 53 has been addressed also by other investigators. Levenberg (1964) was unable to detect 54 55 56 agaritine in any of the 30 non-Agaricus species analysed (detection limit for the analytical 57 58 technique not mentioned), and Stijve and co-workers in any of 39 wild-growing and 30 59 60

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1 2 cultivated non-Agaricus species (Stijve et al. 1986; Stijve and Pittett 2000). Two reports on 3 4 5 popular cultivated mushrooms are in conflict with these data. Hashida et al. (1990) found 6 7 very low levels of agaritine in shiitake ( Lentinus edodes ), 0.82 mg/kg fresh weight, and 8 9 Burini et al. (1999) very high levels (average level 680 mg/kg fresh weight; range 478-905 10 11 12 mg/kg) in the Oyster mushroom ( Pleurotus ostreatus ). With such low quantities of 13 14 agaritine in fresh shiitake, it came as no surprise that Andersson et al. (1999) were unable 15 16 For Peer Review Only 17 to detect agaritine in a Swedish sample of canned shiitake, and that Stijve et al. (1986) 18 19 were unable to detect agaritine in fresh shiitake using an analytical technique having a limit 20 21 22 of detection around 20-25 mg/kg fresh weight. Further studies are needed to determine 23 24 whether fresh shiitake mushrooms contain low levels of agaritine. The report that agaritine 25 26 occurs in the Oyster mushroom, is, however, most likely an artefact due to the use of an 27 28 29 unspecific analytical method. The only other explanation, that the cultivated strains of 30 31 Pleurotus ostreatus may vary substantially in their ability to produce agaritine seems less 32 33 34 likely. Neither Swiss investigators (Stijve et al. 1986; Stijve and Pittett 2000), nor we in the 35 36 present study could identify agaritine in this species, although the limit of detection was as 37 38 39 low as 0.2 mg/kg fresh weight in the present study. Furthermore, there is no indication that 40 41 agaritine production can be suppressed to negligible levels by the method of cultivation. 42 43 44 45 46 However, the data in the present study clearly show that agaritine is present in some 47 48 species of the genera Leucoagaricus and Macrolepiota , which both are saprotrophic 49 50 51 mushrooms closely related to the genus Agaricus (Singer 1986). The compound was 52 53 detected in Leucoagaricus bresadolae , L. cretaceus , L. naucinus , and Macrolepiota 54 55 56 konradii (detection limit 0.1 mg/kg), but not in one other species of Leucoagaricus and 57 58 59 60

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1 2 three other species of Macrolepiota (Table IV). Stijve et al. (1986) found no agaritine in 3 4 5 Leucoagaricus pudicus , Macrolepiota procera and Macrolepiota rachodes . 6 7 8 9 Acknowledgements 10 11 12 The synthesis of agaritine was supported by a grant from the Nordic Council of Ministers, 13 14 and the chemical analysis by grant MSM 6046137305 given by the Ministry of Education, 15 16 For Peer Review Only 17 Youth and Sports of the Czech Republic. We are very much indepted to Jan Borovi čka, Ji ří 18 19 Burel and Henning Knudsen for very valuable information on Agaricus taxonomy. 20 21 22 23 24 25 26 References 27 28 Andersson HC, Gry J. 2004. Phenylhydrazines in the cultivated mushroom ( Agaricus 29 bisporus ) - occurrence, biological properties, risk assessment and recommendations. 30 31 TemaNord 2004:558, 123 p. Also at: 32 33 http://www.norden.org/pub/sk/showpub.asp?pubnr=2004:558 34 35 Andersson HC, Hajslová J, Schulzová V, Panovská Z, Hajková L, Gry J.1999. Agaritine 36 37 content in processed foods containing the cultivated mushroom ( Agaricus bisporus) 38 39 on the Nordic and the Czech market. Food Additives and Contaminants 16:439-446. 40 Berkeley MJ, Broome CE. 1865: Notices of British Fungi 34, The Annals and Magazine of 41 42 Natural History Vol. 15, 3 rd series, London, p. 316, n o 1009. 43 44 Bohus G. 1995. Agaricus studies XIII. Key to subgenus Agaricus from Europe. Mikológiai 45 46 Közlemények 34: 5-35. 47 48 Bon M. 1983. Documents Mycoligiques 13(49):56. 49 Bon M, Cappelli A. 1983. Documents Mycoligiques 13(52):16. 50 51 Burini G, Curini M, Marcotullio MC, Policicchio P. 1999. Determination of agaritine in 52 53 cultivated mushrooms using high performance liquid chromatography with 54 55 fluorometric detection. Italian Journal of Food Science 11:39-46. 56 57 Cappelli A. 1984a. Agaricus . Fungi Europaei 1. Saronno, 560 p. 58 59 60

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1 2 Chauhan YS, Toth B. 1984. Synthesis of N 2-[γ-L-(+)-glutamyl]-4-carboxyphenylhydrazine, 3 4 a postulated precursor of agaritine of Agaricus bisporus . Journal of Agricultural and 5 6 Food Chemistry 32:676-678. 7 8 Chauhan Y, Nagel D, Issenberg P, Toth B. 1984. Identification of p-hydrazinobenzoic acid 9 10 in the commercial mushroom Agaricus bisporus . Journal of Agricultural and Food 11 Chemistry 32:1067-1069. 12 13 Chauhan Y, Nagel D, Gross M, Cerny R, Toth B. 1985. Isolation of N 2-[γ-L-(+)-glutamyl]- 14 15 4-carboxyphenylhydrazine in the cultivated mushroom Agaricus bisporus. Journal of 16 For Peer Review Only 17 Agricultural and Food Chemistry 33:817-820. 18 19 Chilton WS, Hsu CP. 1975. N-Nitrosamines of Agaricus silvaticus . Phytochemistry 20 21 14:2291-2292. 22 Courtecuisse R. 1985. Documents Mycoligiques 16(61):49. 23 24 Essette H. 1964. Les Psalliotes. Lechevalier. Paris. 25 26 Frandsen H. 1998. Synthesis of L-glutamic acid 5-(2-(hydroxymethyl)phenylhydrazine) 27 28 (Agaritine). Nordic Seminar of Phenylhydrazines in the Cultivated Mushrooms 29 30 (Agaricus bisporus ), edited by J. Gry and H.C. Andersson. TemaNord 539: 38. 31 Freeman AE. 1979. Mycotaxon 8:90. 32 33 Gigliotti HJ, Levenberg B. 1964. Studies on the γ-glutamyltransferase of Agaricus 34 35 bisporus . Journal of Biological Chemistry 239:2274-2284. 36 37 Gry J, Andersson HC. 1998. Nordic seminar on phenylhydrazines in the cultivated 38 39 mushroom ( Agaricus bisporus ). TemaNord 1998:539. 48 pp. 40 41 Gry J, Pilegaard K. 1991. Hydrazines in the Cultivated Mushroom ( Agaricus bisporus ). 42 Vår Föda 43S:1-38. 43 44 Hajšlová J, Hájková L, Schulzová V, Frandsen H, Gry J, Andersson HC. 2001. Stability of 45 46 agaritine. Food Additives and Contaminants 19:1028-1033. 47 48 Hashida C, Hayashi K, Jie L, Haga S, Sakurai M, Shimizu H. 1990. Quantities of agaritine 49 50 in mushrooms ( Agaricus bisporus ) and the carcinogenicity of mushroom methanol 51 52 extracts on the mouse bladder epithelium. Nippon-Koshu-Eisei-Zasshi 37:400-405. 53 Heinemann P. 1977. Les Psalliotes. Les Naturalistes Belges. Bruxelles. 54 55 Hlavá ček J. 1983. Systém rodu Agaricus L. - pe čárka - I. Mykologický Sborník 60:129- 56 57 131. 58 59 Hlavá ček J. 1984a. Systém rodu Agaricus L. - pe čárrk - II. Mykologický Sborník 61:4-12. 60

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1 2 Hlavá ček J. 1984b. Systém rodu Agaricus L. - pe čárka - III. Mykologický Sborník 61:65- 3 4 68. 5 6 Hlavá ček J. 1984c. Systém rodu Agaricus L. - pe čárrk - IV. Mykologický Sborník 61:145- 7 8 148. 9 Hlavá ček J. 1999. Poznámky ke studiu žampion ů ( Agaricus ) III. Pe čárky rostoucí trsnat ĕ 2. 10 11 Mykologický Sborník 76:49-53. 12 13 Hlavá ček J. 2000. Poznámky ke studiu žampion ů ( Agaricus ) III. Pe čárky rostoucí trsnat ĕ 3. 14 15 Mykologický Sborník 77:97-102. 16 For Peer Review Only 17 Hlavá ček J. 2001. Vzácné, kritické a nové taxony pe čárek ( Agaricus ) I. Mykologický 18 19 Sborník 78:113-120. 20 Hlavá ček J. 2002. Vzácné, kritické a nové taxony pe čárek ( Agaricus ) II. Mykologický 21 22 Sborník 79:3-8. 23 24 Kerrigan RW. 1995. Global genetic resources of Agaricus breeding and cultivation. 25 26 Canadian Journal of Botany 73:S973-S979. 27 28 Knudsen H. 1992. Agaricaceae Fr. In: Nordic Macromycetes, Vol. 2, L Hansen, H Knudsen 29 (Eds.), Nordsvamp, Copenhagen, p. 205-214. 30 31 Levenberg B. 1961. Structure and enzymatic cleavage of agaritine, a phenylhydrazine of L- 32 33 glutamic acid isolated from Agaricaceae . Journal of the American Chemical Society 34 35 83:503-504. 36 37 Levenberg B. 1962. An aromatic diazonium compound in the mushroom Agaricus 38 39 bisporus . Biochimica et Biophysica Acta 63:212-214. 40 Levenberg B. 1964. Isolation and structure of agaritine, a γ-glutamyl-substituted 41 42 arylhydrazine derivative from Agaricaceae . Journal of Biological Chemistry 43 44 239:2267-2273. 45 46 Liu J-W, Beelman RB, Lineback DR, Speroni JJ. 1982. Agaritine content of fresh and 47 48 processed mushrooms ( Agaricus bisporus (Lange) Imbach). Journal of Food Sciience 49 50 47:1542-1544 and 1548. 51 Mozina IA, Belyakova GA, Fireal MS and Dyakov YA. 1993. Marking of cultivars and 52 53 wild strains of cultivated mushroom Agaricus bisporus with esterase allozymes. 54 55 Biologicheskie Nauki 1:131-140. (In Russian with English abstract) 56 57 Möller F. 1952. Danish Psalliota species. II. Friesia IV, 135-220. 58 59 60

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1 2 Nauta MM. 2001. 1. Agaricus L. In: Flora Agaricina Neerlandica. Critical monographs on 3 4 families of agarics and boleti occurring in the Netherlands. Volume 5, ME 5 6 Noordeloos, TW Kuyper, EC Vellinga, J van Os (Eds.), AA Balkema Publishers, 7 8 Lisse, p. 23-61. 9 Parra LA. 2003. Contribution to the knowledge of genus Agaricus . Fungi non delineati 10 11 XXIV. Massimo Candusso, Italia, 108 p. 12 13 Pilát A. 1951. The Bohemian species of the genus Agaricus . Acta Musei Nationalis Pragae, 14 15 VII B (1). 16 For Peer Review Only 17 Pilat A. 1954. Pe čárka Moškova. Agaricus Maskae Pilat, nový druh z blizkého 18 ř č Č č 19 p íbuzenstva pe áry velkovytrusé. eská Mykologická spole nost 8:165. 20 Řanda Z, Ku čera J. 2004. Trace elements in higher fungi (mushrooms) determined 21 22 by activation analysis. Journal of Radioanalytical and Nuclear Chemistry 21:99-107. 23 24 Ross AE, Nagel DL, Toth B. 1982a. Evidence for the occurrence and formation of 25 26 diazonium ions in the Agaricus bisporus mushroom and its extracts. Journal of 27 28 Agricultural and Food Chemistry 30:521-525. 29 Schulzová V, Hajšlová J, Peroutka R, Gry J, Andersson HC. 2002. Influence of storage and 30 31 household processing on the agaritine content of the cultivated Agaricus mushroom. 32 33 Food Additives and Contaminants 19:853-862. 34 35 Shepard SE, Schlatter C. 1998. Covalent binding of agaritine to DNA in vivo. Food and 36 37 Chemical Toxicology 36:971-974. 38 39 Shepard SE, Gunz D, Schlatter C. 1995. Genotoxicity of agaritine in the lacI transgenic 40 mouse mutation assay: Evaluation of the health risk of mushroom consumption. Food 41 42 and Chemical Toxicology 33:257-264. 43 44 Southcott RV. 1974. Notes on some poisonings and other clinical effects following 45 46 ingestion of Australian fungi. South Australian Clinics 6:441-478. 47 48 Stijve T, Pillet A. 2000. Absence of agaritine in Pleurotus species and in other cultivated 49 and wild-growing mushrooms not belonging to the genus Agaricus . Deutsche 50 51 Lebensmittel-Rundschau 96:251-254. 52 53 Stijve T, Fumeaux R, Philippossian G. 1986. Agaritine, a p-hydroxymethyl- 54 55 phenylhydrazine derivative in cultivated mushrooms ( Agaricus bisporus ), and in 56 57 some of its wild-growing relatives. Deutsche Lebensmittel-Rundschau 82:243-248. 58 59 60

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1 2 Toth B. 1983. Carcinogens in edible mushrooms. In: Carcinogens and Mutagens in the 3 4 Environment. Vol. III Naturally Occurring Compounds: Epidemiology and 5 6 Distribution. HF Stich (Ed.), CRC Press, Inc., Boca Raton, Florida, pp. 99-108. 7 8 Toth B. 2000. Hydrazines and Cancer. A Guidebook on the Carcinogenic Activities of 9 Hydrazines, Related Chemicals, and Hydrazine-Containing Natural Products. 10 11 Harwood academic publications, Australia. 243 pp. 12 13 Toth B, Erickson J. 1986. Cancer induction in mice feeding of the uncooked cultivated 14 15 mushroom of commerce Agaricus bisporus . Cancer Research 46:4007-4011. 16 For Peer Review Only 17 Toth B, Raha CR, Wallcave L, Nagel D. 1981a. Attempted tumor induction with agaritine 18 19 in mice. Anticancer Research 1:255-258. 20 Toth B, Erickson J, Gannett PM, Patil K. 1997a. Carcinogenesis by the cultivated baked 21 22 Agaricus bisporus mushroom in mice. Oncology Reports 4:931-936. 23 24 Toth B, Erickson J, Gannett P. 1997b. Lack of carcinogenesis by the baked mushroom 25 26 Agaricus bisporus in mice: different feeding regimen. in vivo 11:227-232. 27 28 Toth B, Gannett P, Visek WJ, Patil K. 1998. Carcinogenesis studies with the lyophilized 29 mushroom Agaricus bisporus in mice. in vivo 12:239-244. 30 31 Wallcave L, Nagel DL, Raha CR, Jae H-S, Bronczyk S, Kupper R, Toth B. 1979. An 32 33 improved synthesis of agaritine. Journal of Organic Chemistry 44:3752-3755. 34 35 Wasser SP. 1977. Ukrainian Botanical Journal 34:85. 36 37 Wasser SP. 1980. Flora fungorum RSS Ucrainicae. Basidiomycetes, Agaricaceae Cohn. 38 39 Academia Scientianum RSS UCR. Institutum botanicum nomine N.G. Cholodnii, 40 Kiev. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 Agaritine content of 53 Agaricus 20 21 22 species collected from nature 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 Table I. Agaritine content (mg/kg fresh weight) of methanolic extracts of fresh 5 6 Agaricus bisporus as determined by HPLC. If samples were not freeze-dried 7 8 (which does not influence the agaritine level) shortly after being obtained, 9 they were analysed within 24 hours. 10 11 12 13 Reference Number Diameter of Content 14 of cap (cm) Average Range 15 samples 16 Samples directlyFor from growersPeer Review Only 17 18 Kelly et al., 1962* 1 not given ~ 400 - 19 Daniels et al., 1961* 1 not given ~ 220 - 20 Levenberg, 1964* 1 not given ~ 3300*** - 21 22 Liu et al., 1982 14 ** 3.0-4.5 880 330-1730 23 Speroni and Beelman, 1982 1 not given 2 190*** - 24 Speroni et al., 1983 8 ** 2.5-5.0 - 1700-5100*** 25 Fischer et al., 1984 2 ** 5.0-6.0 304±6.0; - 26 27 438±2.5 - 28 Sharman et al., 1990 2 ** not given ~ 180 80-250 29 Andersson et al., 1994 1 not given 340 - 30 31 Samples from the market 32 Ross et al., 1982b 2 not given 440; 720 170-1170 33 Fischer et al., 1984 11 2.0-5.5 368±45 94-629 34 Hashida et al., 1990 1 not given 228 - 35 36 Stijve et al., 1986 5** not given 160-650 37 Burini et al., et al., 1999 5 not given 820 630-980 38 Andersson et al., 1999 2 not given 212; 229 - 39 40 * gravimetric method; ** different strains: *** mg/kg dry weight (approximately 10% dry 41 matter) 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 Table II. Agaritine content (average±S.D.) and usefulness as food (A = popular as food; B = seldom used as food; C = not suitable as food) of different 7 Agaricus species collected from the nature in the Czech Republic. 8 9 Agaricus Agaricus 10 Culi- speciesFor (preferred Peer speciesReview - preferred SectionOnly Number Mean content Range (% with 11 nary nomenclature according to nomenclature according to accord- of (mg/kg fresh undetectable levels) 12 useful- Hlavá ček (1983, 1984a, 1984b, Knudsen (1992) and Nauta (2001) ing to samples weight) (mg/kg fresh weight) 13 ness 1984c) Nauta 14 (2001)* 15 16 B A. aestivalis (F.H. Møller) Pilát same Ag 2 824±200 682 - 966 17 C A. altipes (F.H. Møller) Pilát same Ag 1 713 - 18 A# A. arvensis Schaeff.:Fr. same Ar 15 987±334 475 - 1 551 19 20 A# A. augustus Fr. same Ar 6 3 995±1 885 2 264 - 7 553 21 A# A. perrarus Schulzer A. augustus Fr. Ar 2 1 003±512 841- 1 164 22 B A. biberi Hlavá ček (1) same ? 1 524 - 23 A A. bisporus (J.E. Lange) Imbach var. same B 11 997±490 550 - 2 066 24 25 bisporus 26 A A. bisporus (J.E. Lange) Imbach var. same B 4 639±38 599 - 672 27 avellanea (J.E. Lange) Singer 28 A A. bisporus (J.E. Lange) Imbach var. same B 3 746±173 613 - 990 29 30 albidus (J.E. Lange) Singer 31 A A. bitorquis (Quél.) Sacc. same B 11 1 473±781 216 - 3 006 32 B A. bresadolianus Bohus same Sp 1 111 - 33 B A. caespitipes Hlavá ček (2) A. bohusii Bon 2 1 855±24 1 838 - 1 872 34 35 A A. campestris L.:Fr. same Ag 22 487±475 n.d. (14%) - 2 230 36 B A. cappellianus Hlavá ček (3) A. vaporarius (Pers.) Capelli 11 1 652±826 233 - 3 456 37 A A. caroli Pilát (4) A. benesii (Pilát) Pilát Sa 1 n.d. - 38 39 B A. devoniensis Orton same B 1 2 089 - 40 B A. elvensis Berk. & Broome (5, 6) A. bohusii Bon B 8 4 445±3 074 2 080 - 9 986 41 42 43 44 3 45 http://mc.manuscriptcentral.com/tfac Email: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Food Additives and Contaminants Page 24 of 33

1 2 3 4 5 A# A. essetteii Bon (7) A. sylvicola (Vittad.) Peck Ar 7 2 605±702 1 744 - 3 958 6 A# A. excellens (F.H. Møller) F.H. A. urinascens (F.H. Møller & Jul. Ar 3 1 539±70 1 481 - 1 617 7 Møller (8) Schäff.) Singer var. excellens (F.H. 8 Møller) Nauta 9 10 A# A. fissuratus For(F.H. Møller) F.H.Peer A. arvensis Review Schaeff. Only Ar 4 3 526±858 2 430 - 4 553 11 Møller (8) 12 B A. fuscofibrillosus (F.H. Møller) same Sa 3 597±521 52 152 -1 170 13 Pilát 14 15 ? A. heimii Bon ? 5 1 521±574 1 024 - 2 317 16 B A. impudicus (Rea) Pilát same Sa 2 253±358 n.d. (50%) - 506 17 A A. kotlabae Hlavá ček (9) same ? 7 1 451±510 963 - 2 341 18 A A. langei (F.H. Møller) F.H. Møller same Sa 1 978 - 19 20 A# A. leucotrichus (F.H. Møller) F.H. A. arvensis Schaeff. Ar 5 1 156±912 383 - 2 534 21 Møller 22 B A. lutosus (F.H. Møller) F.H. Møller same M 1 2 269 - 23 A# A. macrocarpus (F.H. Møller) F.H. same Ar 1 1 501 - 24 25 Møller 26 A# A. macrosporus (F.H. Møller & Jul. A. urinascens (F.H. Møller & Jul. Ar 1 1 049 - 27 Schäff.) Pilát (4) Schäff.) Singer var. urinascens 28 B A. maleolens F.H . Møller (8) A. bernardii (Quél.) Sacc. 19 52±76 n.d. (64%) - 207 29 30 B A. maleolens F.H. Møller var. A. bernardii (Quel.) Sacc. B 7 186±246 n.d. (29%) - 693 31 bernardioides Hlavá ček (A) 32 A A. maskae Pilát (10) A. litoralis (Wakef. & A. Pearson) Sp 2 63±89 n.d. (50%) - 127 33 Pilát 34 35 A# A. nivescens (F.H. Møller) F.H. A. osecanus Pilát Ar 3 584±281 355 - 898 36 Møller (8) 37 C A. pearsonianus Hlavá ček (9) A. xanthodermus Genevier ? 6 103±63 n.d. (17%) - 185 38 C A. phaeolepidotus (F.H. Møller) same X 2 285±207 139 - 431 39 40 F.H. Møller 41 C A. pilatianus Bohus same X 4 40±46 n.d. (50%) - 82 42 43 44 4 45 http://mc.manuscriptcentral.com/tfac Email: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 25 of 33 Food Additives and Contaminants

1 2 3 4 5 C A. pilatianus Bohus var. same X 1 89 - 6 iodoformicus Hlavá ček (1) 7 B A. porphyrizon P.D. Orton same M 1 2 283 - 8 B A. porphyrocephallus F.H. Møller same Ag 1 354 - 9 10 C A. praeclaresquamosusFor A.E. Peer A. moelleri Review Wasser OnlyX 4 8±16 n.d. (75%) - 31 11 Freeman (11) 12 C A. praeclaresquamosus Freeman A. moelleri Wasser ? 3 386±276 72 - 589 13 var. terricolor (Møll.) Bon & 14 15 Cappelli (12) 16 B A. purpurellus (F.H. Møller) F.H. same M 1 4 100 - 17 Møller 18 ? A. romagnesii Wasser (13) A. bresadolianus Bohus Sp 1 60 - 19 20 B A. sagatus Fr. M 1 3 479 - 21 B A. semotus Fr. same B 3 2 755±396 2 299 - 3 021 22 A A. silvaticus (Schaeff.) Fr. same Sa 14 210±305 n.d. (57%) - 920 23 A A. silvaticus (Schaeff.) Fr. var. same Sa 1 n.d. - 24 25 pallidus (F.H. Møller) F.H. Møller 26 B A. slovenicus Hlavá ček nom. inval. B 4 1 955±841 1 072 - 3 327 27 A A. squamulifer (F.H. Møller) Pilát A. benesii (Pilát) Pilát Sa 2 110±156 n.d. (50%) - 220 28 (4) 29 30 A# A. substramineus Courtec. (14) A. urinascens (F.H. Møller & Jul. Ar 3 2 570±66 2 502 - 2 634 31 Schäff.) Singer 32 A# A. sylvicola (Vittad.) Peck same Ar 6 1 592±446 733 - 1 940 33 A A. tenuivolvatus (F.H. Møller) F.H. ? 4 1 400±1 361 231 - 3 309 34 35 Møller 36 C A. xanthodermus Genev. same X 12 12±31 n.d. (67%) - 108 37 (1) Hlavá ček, 2001; (2) Hlavá ček, 1999; (3) Hlavá ček, 1987; (4) Pilat, 1951; (5) Berkeley and Broome, 1865; (6) Hlavá ček, 2000; (7) Bon, 38 1983; (8) Møller, 1952; (9) Hlavá ček, 2002; (10) Pilat, 1954; (11) Freeman, 1979; (12) Bon and Cappelli, 1983; (13) Wasser, 1977; (14) 39 40 Courtecuisse, 1985. *Ag=Agaricus; Ar=Arvenses; B=Bitorques; M=Minores; Sa=Sanguinolenti; Sp=Spissicaules; X=Xxanthodermi. #Advice 41 not to consume this species due to high heavy metal content may exist. 42 43 44 5 45 http://mc.manuscriptcentral.com/tfac Email: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Food Additives and Contaminants Page 26 of 33

1 2 3 4 5 Table III. Agaricus species with low, intermediate or high levels of agaritine. Section, 6 according to Nauta (2001) is given within brackets (Ag= Agaricus; 7 Ar=Arvenses; B=Bitorques; M=Minores; Sa=Sanguinolenti; 8 Sp=Spissicaules; X=Xanthodermi). 9 10 11 Agaritine content 12 low intermediate high 13 (<100 mg/kg f.w.) (100-1000 mg/kg f.w.) (>1 000 mg/kg f.w.) 14 A. bresadolianus (Sp) A. aestivalis (Ag) A. augustus (Ar) 15 16 A. caroli (Sa)For Peer A. altipes Review (Ag) Only A. augustus var. perrarus 17 (Ar) 18 A. maleolens (B) A. arvensis (Ar) A. bitorquis (B) 19 A. maskae (Sp) A. biberi A. caespitipes (B) 20 21 A. pearsonianus A. bisporus var. avellanea (B) A. devoniensis (B) 22 A. pilatianus (X) A. bisporus var. bisporus (B) A. elvensis (B) 23 A. pilatianus var. A. bisporus var. hortensis (B) A. essettei (Ar) 24 iodoformicus (X) 25 26 A. praeclaresquamosus (X) A. campestris (Ag) A. excellens (Ar) 27 A. romagnesii (Sp) A. fuscofibrillosus (Sa) A. fissuratus (Ar) 28 A. squamulifer (Sa) A. impudicus (Sa) A. heimii 29 A. silvaticus var. pallidus (Sa) A. langei (Sa) A. kotlabae 30 31 A. xanthoderma (X) A. maleolens var. bernardoides A. leucotrichus (Ar) 32 (B) 33 A. nivescens (Ar) A. lutosus (M) 34 A. phaeolepidotus (X) A. macrocarpus (Ar) 35 36 A. porphyrocephalus (Ag) A. macrosporus (Ar) 37 A. preclaresquamosus var. A. porphyrizon (M) 38 terricolor 39 A. silvaticus (Sa) A. purpurellus (M) 40 41 A. sagatus (M) 42 A. semotus (B) 43 A. slovenicus (B) 44 A. substramineus (Ar) 45 46 A. sylvicola (Ar) 47 A. tenuivolvatus 48 A. vaporarius (B) 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Table IV. Average agaritine content (mg/kg fresh weight) in some mushroom species of 4 5 the genera Leucoagaricus and Macrolepiota collected in the Czech Republic. 6 7 Species as determined by Species as interpreted by Content 8 Hlavácek Knudsen (1992) and (mg/kg fresh weight) 9 10 Noordeloos et al. (2001) (n=sample size) 11 Leucoagaricus bresadolae Leucoagaricus americanus 368 (n = 1) 12 (Schultz.) Bon (Peck) Vellinga 13 L. cinerascens Migl. & Coccia L. leucothites (Vitt.) Wasser n.d. (n = 3) 14 15 var. carneifolius (Gillet) 16 For PeerVellinga Review Only 17 L. cretaceus (Bull.:Fr.) Moser L. cretaceus (Bull.:Fr.) Moser 838 (n = 2) 18 L. naucinus (Fr.) Sing. L. leucothites (Vitt.) Wasser 68 (n = 2) 19 20 Macrolepiota rachodes var. same n.d. (n = 2) 21 bohemica (Wichansky) Bellù 22 & Lanzoni 23 M. konradii (Huijsman ex M. mastoidea (Fr.) Sing. 42 (n = 1) 24 25 P.D. Orton) Marchand 26 M. permixta (Barla) Pacioni same n.d. (n = 2) 27 M. rachodes (Vitt.) Sing. same n.d. (n = 2) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 Table V. Agaritine levels reported by other investigators in various Agaricus species 3 collected from nature. 4 5 6 Agaricus species Agaritine content 7 Levenberg Stijve and Stijve et al. (1986) Present study 8 (1964) Pittet (2000) 9 mg/kg d. w. mg/kg d.w. mg/kg dry weight mg/kg fresh weight 10 A. abruptibulbus 500 - 5 000 11 12 A. argentatus + 13 A. arvensis 15000-25000 6 500 (200 - 18 500) 987 (475 - 1 551) 14 A. augustus 15000-25000 8 000 (1 000 - 22 000) 3 995 (2 264 - 7 553) 15 A. benesi - 16 For Peer Review Only 17 A. bernardii doubtful 18 A. blazei 500 - 5 000 19 A. bisporus 5000 - 15 000 997 (550 - 2 066) 20 A. bitorquis 15000-25000 7 100 (500 - 20 000) 1 473 (216 - 3 006) 21 22 A. campestris + 5000 - 15 000 2 600 (200 - 10 000) 487 (n.d.-2 230) 23 A. comtulus + 500 - 5 000 24 A. crocodilinus + 25 A. cupreobrunneus n.d. 26 27 A. edulis + 28 A. excellens 4 600 (300 - 9 000) 1 539 (1 481 - 1617) 29 A. fuscofibrillosus n.d. 597 (152 - 1 170) 30 A. geesterani n.d. 31 32 A. haemorrhoidarius n.d. n.d. 33 A. hondensis n.d. 34 A. hortensis + 5000 - 15 000 746 (613 - 990) 35 A. langei n.d. n.d. 978 36 37 A. lilaceps 500 - 5 000 38 A. macrosporus 15000-25000 8 600 (700 - 25 000) 1 049 39 A. meleagris n.d. 40 A. micromegathus + 41 42 A. nivescens 500 - 5 000 584 (355 - 898) 43 A. niveolutescens 500 - 5 000 4 000 44 A. patersonii + 45 A. perrarus + 15000-25000 12 500 1 003 (841 - 1 164) 46 47 A. phaeleopidotus n.d. 285 (139 - 431) 48 A. placomyces n.d. 49 A. porphyrocephalus 500 - 5 000 354 50 A. semotus 500 - 5 000 2 755 (2 299 - 3 021) 51 52 A. sterlingii - 53 A. subperonatus 5000 - 15 000 2 500 2 788 (2 121 - 3 456) 54 A. subrutilescens - 55 A. silvaticus 56 - n.d. n.d. 210 (n.d. - 920) 57 A. sylvicola - 5000 - 15 000 5 200 (500 - 12 000) 1 592 (733 - 1 940) 58 A. vaporarius 500 - 5 000 1 000 1 382 (914 - 1851) 59 A. xanthoderma + n.d. n.d. 12 (n.d. - 108) 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 HN NH γ glutamyl 8 9 10 11 12 13 14 CH2OH 15 16 For Peer Review Only 2 17 Fig. 1 The molecular structure of agaritine (N -[γ-L-glutamyl]-4- 18 19 hydroxymethylphenylhydrazine). 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 mAU For Peer Review Only 11 A 12 80 13 14 60 15 40 16 20 17 18 0 19 0 1 2 3 4 5 6 7 min 20 mAU Agaritine 21 100 B

22 80 23 24 60 25 40 26 20 27 28 0 29 0 1 2 3 4 5 6 7 min 30 31 32 33 34 35 36 37 Figure 2. HPLC/UV (237 nm) chromatogram and UV spectrum of (A) agaritine standard - 25 µg/ml, and (B) sample of Agaricus 38 campestris with 270 mg agaritine per kg fresh weight. 39 40 41 42 43 44 3 45 http://mc.manuscriptcentral.com/tfac Email: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 33 Food Additives and Contaminants

1 2 3 4 5 6 7 8 9 10 Agaritine content of Agaricus arvensis 11 12 having various cap diameters 13 14 15 1800 16 1600 For Peer Review Only 17 18 1400 19 1200 20 21 1000 22 23 800 24 600 25 400

26 Agaritine(mg/kg f.w.) 27 200 28 29 0 30 0 2 4 6 8 10 12 31 Cap diameter (cm) 32 33 34 35 36 37 38 39 40 41 Fig. 3. Absence of a correlation between agaritine content (mg/kg fresh weight) and cap 42 43 diameter of Agaricus arvensis. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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