Anna Gliszczyńska-Świglo
email: a.gliszczynska-swiglo@ae.poznan.pl

Henryk Szymusiak

Paulina Malinowska

Anna Gliszczyńska-Świgło

Betanin, the main pigment of red beet -molecular origin of its exceptionally high free radical scavenging activity

Keywords: Betalains, betanin, pH dependent free radical scavenging activity, TEAC value, 13 DFT calculations

In the present study, the pH-dependent free radical scavenging activity of betanin in the TEAC 2 (Trolox equivalent antioxidant capacity) assay was determined. It was found that at pH > 4 3 betanin is about 1.5-2-fold more active than some anthocyanins considered very good free 4 radical scavengers as determined in the TEAC assay. The increase in the TEAC values of 5 betanin with increasing pH is discussed in terms of its calculated phenolic OH homolytic bond 6 dissociation energy (BDE) and ionization potential (IP). The results suggest that the 7 exceptionally high antioxidant activity of betanin is associated with increasing of its H-8 donation and electron-donation ability when going from cationic state to mono-, di-and tri-9 deprotonated states present at basic solutions.

as additives for food, cosmetic and drug products, is an ancient practice but currently there is 5 an increasing interest from consumers in the use of naturally derived colourants. This interest 6 is associated with consumer perception of synthetic dyes as harmful, whereas pigments 7 naturally occurring in edible plants are usually considered to be rather safe. A variety of 8 different pigments are produced by nature and a number of them have found practical 9 application in colouring of food e.g. water-soluble anthocyanins and betalains and fat-soluble 10 carotenoids and curcuminoids. Moreover, there is growing evidence suggesting that some 11 natural colourants may be nutritionally important antioxidants and that their presence in the 12 diet may reduce the risk of cardiovascular disease, cancer, and other diseases associated with 13 aging (Cai et al. 2003).

14 15

Betalains occur in plants of most families of the plant order Caryophyllales (with the 16 exception of Caryophyllaceae and Moluginaceae) and in some higher fungi (Frank et al. 17 2005). Among the numerous natural sources of betalains, red and yellow beet, prickly pear, 18 coloured Swiss chard, grain amaranth and cactus fruits are the only foods containing these 19 compounds (Kanner et al. 2001, Stintzing et al. 2004, Frank et al. 2005). Red beet (Beta 20 vulgaris L.) is consumed in the form of lactofermented juice, pickled preserves or as a cooked 21 vegetable. The pigment mixture in the form of beet juice concentrate or beet powder are 22 approved additives for use in food, drugs and cosmetic products (Dornenburg et al. 1996).

23

Red beet betalains are composed of two main groups: the red-violet betacyanins (e.g. betanin 24 and isobetanin) and the yellow betaxanhins (e.g. vulgaxanthin I and II). The betacyanins 25 (betanin and isobetanin) are water-soluble immonium conjugates of betalamic acid with 3,4-26 dihydroxyphenylalanine (cyclo-DOPA), which may be glucosilated. The most important 27 betacyanin in red beet is betanin, which is a betanidin 5-O-β-glucoside (Figure 1) containing a 28 phenolic and a cyclic amine groups, both shown to be very good electron donors, acting as 29 antioxidants (Kanner et al. 2001). Betanin makes up 75-95% of the total colouring matter 30 found in the beet. The isobetanin is C15-epimer of betanin (Figure 1) however it is present in 31 fresh beet juice in small amounts. In food processing, betalains are less commonly used than 32 water-soluble anthocyanins, although the colour of betalains is more stable between pH 3 and In several studies it was shown that betalains are effective free radical scavengers and that 12 they prevent active oxygen-induced and free radical-mediated oxidation of biological 13 molecules (Escribaño et al. 1998, Zakharova et al. 1998, Pedreno et al. 2000, 2001, Kanner et 14 al. 2001, Butera et al. 2002, Pavlov et al. 2002, Wettasinghe et al. 2002, Cai et al. 2003, 15 Tesoriere et al. 2004, 2005, Allegra et al. 2005, Frank et al. 2005, Stintzing et al. 2005) The TEAC assay is based on the ability of the antioxidant to scavenge the blue-green coloured and anthocyanins suggest that possible beneficial health effects of these natural dyes will vary 13 with the tissue under investigation and that betanin at pH higher than 4 could be even better 3C) is also more acidic than the C2-COOH group and the 12 acidity of the C2-COOH group is comparable to that of C15-COOH (Figure 3B) and C6-OH 13 phenolic groups (Figure 3E). In fact, our gas-phase calculations do not take into account 14 solvation effects, which tend to stabilize rather more polar structures. with pH increasing free radical scavenging activity of betanin observed in the TEAC assay.

6

Clear dependence between decreasing BDE and IP parameters and degree of betanin In the present study it was found that free radical scavenging activity of betanin, measured in 26 the TEAC assay, is exceptionally high at pH higher than 4. This natural colourant is 1.5-2-fold The high antiradical activity of betacyanin pigments at wide pH range encourages wider 8 application of these natural dyes in food, cosmetic and pharmaceutical products.

2

  Betanin, the main pigment of red beet -molecular origin of its exceptionally high free radical scavenging activity Introduction 1 Colour is one of the most important factors indicating the quality of food. It plays significant 3 role in consumer acceptance and preference of products. The use of natural and synthetic dyes, 4

  Figure1

(

  azinobis(3-ethylbenzothiozoline-6-sulphonic acid) diammonium salt) radical 17 cation relative to the ABTS •+ scavenging ability of the water-soluble vitamin E analogue, 18Trolox (Miller et al. 1993). The antioxidant activity of betanin was measured by the modified 19 TEAC assay performed essentially as described previously(Miller et al. 1993), with some 20 modifications(Tyrakowska et al. 1999). In the present study microperoxidase-8 (MP8) instead 21 of metmyoglobin, was used to generate the ABTS •+ in PBS (0.01 M phosphate buffer, 0.14 M 22 NaCl, 0.002 M KCl) pH 7.4. MP8 (final concentration 0.2 µM) and ABTS (final 23 concentration 3.0 mM) in PBS were mixed and the reaction was initiated by the addition of 24 hydrogen peroxide (final concentration 0.1 mM). The major advantage of the modified TEAC 25 assay is that it permits studies of radical scavenging activity over a wide pH range (2 solution thus obtained was diluted 1:1 (v/v) using 0.2 M potassium phosphate 29 buffers of various pH values to give ABTS •+ solutions at pH values varying between 2 and 9 30 (an absorption was about 0.6 at 734 nm). The ABTS •+ solutions thus obtained were used for 31 determination of the TEAC values. Antioxidants (Trolox or betanin) were added as 1% (v/v) concentrated stock solutions in methanol (Trolox) or water (betanin) to give the 1 final concentration required. The molar concentration of betanin in aqueous stock solution 2 was determined spectrophotometrically at 536 nm using molar absorption coefficient ε = 3 65.000 M -1 cm -1 (Kanner et al. 2001, Allegra et al., 2005). The decrease in absorption caused 4 by the antioxidant compound, measured at 6 min, is reflecting the ABTS •+ radical scavenging 5 capacity and was plotted against the concentration of the antioxidant. The TEAC value is 6 defined as the concentration of Trolox solution, used as an antioxidant standard, with 7 equivalent antioxidant potential to a 1 mM concentration of the compound under investigation 8 Miller et al. 1993, Rice-Evans et al. 1994). The TEAC value was calculated as the ratio of the 9 slope of the plot for scavenging of ABTS •+ by the antioxidant under investigation, to the 10 slope of the plot for ABTS •+ scavenging by Trolox. The free radical scavenging activity of 11 Trolox was previously shown to be unaffected over the whole pH range tested (optimization of molecules studied (betanin, cyclo-DOPA-5-O-β-D-glucoside 16 -product of betanin decomposition under high pH) was performed using B3LYP functional 17 with 6-31G** basis set. In order to obtain some useful thermochemical parameters, such as 18 deprotonation energy (DE), phenolic OH bond dissociation energy (BDE) and ionization 19 potentials (IP) values, the compounds were studied in various protonation/deprotonation states

(

  Figure2

  Figure3

7Figure 4 .

 4 Figure 4. Altogether, the calculated molecular parameters clearly show why betanin becomes

Page

  27 more active than popular anthocyanins, especially at neutral and basic solutions. The result of 28 the present study indicate that pH dependent increasing of free radical scavenging activity of 29 betanin can be attributed to the formation of its different mono-, di-and tri-deprotonated 30 forms. The contribution of cyclo-DOPA-5-O-β-D-glucoside to the antiradical activity of 31 betanin at basic solution can not be also excluded. The calculated OH BDE and IP values of 32 different mono-, di-and tri-deprotonated forms of betanin show that with the increasing 33 degree of deprotonation of betanin molecule, the BDE and IP values significantly decrease. It 34 higher pH betanin becomes better hydrogen and electron donator what 1 results in the increase in its free radical scavenging activity. Analysis of resonance structures 2 of betanin reveals strong electronic conjugation between betalamic acid and cyclo-DOPA-5-3 O-β-D-glucoside moieties what is an additional factor contributing to the observed high 4 antiradical activity of betanin. Altogether, the calculated electronic parameters give more 5 insight into the mechanism of action of betanin as a exceptionally good free radical scavenger.6 7

Figure 1 .

 1 Figure 1. Chemical structures and atom numbering system of betanidin, betanidin 5-O-β-D-

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 3. Structures (A, B, C, D, E) of deprotonated betanin molecule and calculated

Figure 1 .

 1 Figure 1. Chemical structures and atom numbering system of betanidin, betanidin 5-O-β-D-

Figure 4 .

 4 Figure 3. Structures (A, B, C, D, E) of deprotonated betanin molecule and calculated

Figure 5 .

 5 Figure 5. Comparison of bond dissociation energies (BDE, kcal/mol) calculated for betanin in

  ). Assuming that betanin in cationic state

	1 1	resonance structure is neutral amine molecule stabilized by two hydrogen bonds formed by theoretical calculations performed without taking into account the solvation phenomena give
	2 2	adjacent COOH groups (Figure 4D). However, the existence of such a structure in aqueous overestimated results, in spite of using time lasting extended basis sets. Nevertheless, very
	3 3	solution seems to be less probable, remembering that betanin is very well soluble in water and careful interpretation of the data presented in Table I still gives some support for explanation
	4 4	worse in other solvents. Probably in real solution, depending on pH value, betanin can exist in of observed extraordinary high antiradical activity of betanin with increasing pH. Both
	5 5	various deprotonated forms with different contributions. Therefore, we have calculated set of calculated parameters, the BDE and IP values of C6-OH and H-N16, are nicely consistent
	6	thermochemical parameters describing ability of hydrogen and electron donation by betanin in
	7	various possible deprotonation states. These electronic parameters include OH bond
	8 9 10 11	F o r dissociation energy (BDE), representing the ease of hydrogen atom donation and IP, representing the ease of electron donation. The calculated BDE values for C6-OH and, if possible, for H-N16 of betanin in cationic and in all mono-deprotonation states are presented in Figure 5. We have found that each mono-deprotonated form of betanin is better H donor F o r
	12	P (lower BDE values) than betanin in cationic form. In the case of C2-COO -form, the BDE P
	13	e drops by about 10 kcal/mol in comparison to cationic form. Similar decrease in H-N16 BDE e
	14 15	value is observed.	e r e r
	16 17	R e v i e Figure 4 and 5 R e Table I shows the calculated thermochemical data extended to various di-and tri-deprotonated v i e
			w w
	23 24 25	O n l when C6-OH phenolic group is deprotonated (carboxylate anions themselves have very high ionization potentials that are unrealistic for antioxidant action). From results presented in O n l
	26	y Table I it follows that each step of betanin deprotonation (mono, di-and tri-deprotonation) y
	27	leads to the decrease of the BDE values of these forms. The higher degree of betanin
	28	deprotonation, the lower BDE values and the more easily betanin donates hydrogen atom. At
	29	higher pH, where betanin occurs in tri-deprotonated forms, the BDE of C6-OH reaches the
		value nearly 60 kcal/mol. The second parameter, i.e. IP, that describes ability of a molecule to
	33
	33
			10

30

exists in two structures of immonium and ammonium salts it seems reasonable that betanin at 31 H-N16 could be partly dissociated at less acidic conditions than other groups. Analysis of 32 betanin with H-N16 deprotonated group (Figure

4C

) reveals, however that its second 18 forms that may be expected at higher pH. The most stable structures, denoted as "0.0" 19 (calculated as a structure with minimum energy), may be expected in solution in relatively 20 high amounts. It is 16N -for monoanions, 16N -/C6O -for dianions and C2-COO -/16N -/C6O - 21 for trianions. The relative stability of other mono-, di-and tri-deprotonated forms is calculated 22 with respect to appropriate the most stable structure. The IP values are generally calculable 30 donate electron decreases even more dramatically when going from cationic form of betanin 31 (219.3 kcal/mol) through mono-deprotonated (140-160 kcal/mol), and di-deprotonated (84-89 32 kcal/mol) to tri-deprotonated forms (28-35 kcal/mol). However, it should be pointed out that

  Tyrakowska, B., Soffers, A. E. M. F., Szymusiak, H., Boeren, S., Boersma, M. G., Lemańska,

				Food Additives and Contaminants
	1 1 1	Escribaño, J., Pedreno, M. A., Garcia-Carmona, F., Munoz, R., 1998, Characterization of the
	2 2 2			antiradical activity of betalains from Beta vulgaris L. roots. Phytochemical Analysis, K., Vervoort, J., Rietjens, I. M. C. M., 1999, TEAC antioxidant activity of 4-
	3 3 3			9, 124-127. hydroxybenzoates. Free Radical Biology and Medicine, 27, 1427-1436.
	4 4	Francis, F. J., 2000, Anthocyanins and betalains: composition and application. Cereal Foods Wettasinghe, M., Bolling, B., Plhak, L., Xiao, H., Parkin, K., 2002,. Phase II enzyme-inducing O H H
	5 5	H	O	World, 45, 208-213. and antioxidant activities of beetroot (Beta vulgaris L.) extracts from phenotypes of N + COOH
	6 6	Frank, T., Stintzing, F. C., Carle, R., Bitsch, I., Quas, D., Straβ, G., Bitsch, R., Netzel, M., different pigmentation. Journal of Agriculture and Food Chemistry, 50, 6704-6709.
	7 7	2005, Urinary pharmacokinetics of betalains following consumption of red beet juice Zakharova, N. S., Petrova, T. A., 1998, Relationship between the structure and antioxidant
	9 10 11 8 9 10 11 8	References in healthy humans. Pharmacological Research, 52, 290-297. F o r Gliszczyńska-Świgło, A., 2006, Antioxidant activity of water soluble vitamins in the TEAC (trolox equivalent antioxidant capacity) and the FRAP (ferric reducing antioxidant power) assays, Food Chemistry, 96, 131-136. F o r F o r activity of various betalains. Priklady Biokhimczeskoj. Mikrobiologii, 34, 199-202. H F r H o N COOH HOOC
	12 12	P Grzymisławski, M., 2000, Physique of systems related to food bioavailability. Human P P P
	13	Allegra, M., Furtmueller, P. G., Jantschko, W., Zederbauer, M., Tesoriere, L., Livrea, M. A., e Nutrition. Principles of Nutritional Science (in Polish), edited by J. Gawęcki, L. e e e
	14 15	e r Hryniewiecki (Warszawa Poland: PWN), pp. 56-72. Kanner, J., Harel, S., Granit, R., 2001, Betalains -a new class of dietary cationized e r e r e r
	16 17 18 19 20	R e v i e antioxidants. Journal of Agriculture and Food Chemistry, 49, 5178-5185. Lapidot , T., Harel, S., Akiri, B., Granit, R., Kanner, J., 1999, pH-dependent forms of red R R R wine anthocyanins as antioxidants. Journal of Agricultural and Food Chemistry, 47, e e e 67-70. Lemańska, K., Szymusiak, H., Tyrakowska, B., Zieliński, R., Soffers, A. E. M. F., Rietjens, I. v i e v i e v i e
	22 21 22			w M. C. M., 2001. The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones. Free Radical Biology and Medicine, 31, 869-w w w
	23 24 25 23 24 25	indica) fruit extracts and reducing properties of its betalains: betanin and indicaxanthin. Journal of Agriculture and Food Chemistry, 50, 6895-6901. Cai, Y., Sun, M., Corke, H., 2001, Identification and distribution of simple and acylated O n l 881. Lemańska, K., Van der Woude, H., Szymusiak, H., Boersma, M. G., Gliszczyńska-Świgło, A., Rietjens, I. M. C. M., Tyrakowska, B., 2004, The effect of catechol O-methylation on O n l O n l O n l
	26 26			betacyanins in the Amaranthaceae., Journal of Agricultural and Food Chemistry, 49; y radical scavenging characteristic of quercetin and luteolin -a mechanistic insight. y y y
	27 27			1971-1978. Free Radical Research, 38, 639-647.
	28 28	Cai, Y., Sun, M., Corke, H., 2003, Antioxidant activity of betalains from plants of the Miller, N. J., Rice-Evans, C. A., Davies, M. J., Gopinathan, V., Milner, A., 1993, A novel
	29 29			Amaranthaceae. Journal of Agriculture and Food Chemistry, 51, 2288-2294. method for measuring antioxidant capacity and its application to monitoring the
	30 30	Dornenburg, H., Knorr, D., 1996, Generation of colours and flavors in plant cell and tissue antioxidant status in premature neonate. Clinical Science, 84, 407-412.
	31	cultures. Critical Review in Plant Science, 15, 141-168. Nilsson, T., 1970. Studies into the pigments in beetroot (Beta vulgaris L. vulgaris var. rubra
	32			L.). Lantbrukshoegskolans Annaler, 36, 179-219.
				13 16

13 Obinger, Ch., 2005, Mechanism of interaction of betanin and indicaxanthin with 14 human myeloperoxidase and hypochlorous acid. Biochemical and Biophysical 15 Research Communications 332, 837-844. 16 Belitz, H., Grosch, W., 1999, Food Chemistry. (Berlin, Germany: Springer-Verlag), p. 209. 17 Borkowski, T., Szymusiak, H., Gliszczyńska-Świgło, A., Rietjens, I. M. C. M., Tyrakowska, 18 B., 2005, Radical scavenging capacity of wine anthocyanins is strongly pH-19 dependent. Journal of Agricultural and Food Chemistry, 53, 5526-5534. 20 Butera, D., Tesoriere, L., Di Gaudio, F., Bongiorno, A., Allegra, M., Pintaudi, A. M., Kohen, 21 R., Livrea, M. A., 2002, Antioxidant activities of sicilian prickly pear (Opuntia ficus 31 Page 12 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

List of Tables:

1 2

Table I. Calculated thermochemical parameters for betanin in different deprotonation states:


James Throne

Dr Lise Stengard Hansen

Molecular techniques for detection of granary weevil (Sitophilus

Keywords: Sitophilus granarius, detection, real-time PCR 21 22 23

The granary weevil (Sitophilus granarius L.) is a stored grain pest, which causes major 5 economic losses. It reduces the quantity and quality of the grain by its feeding and 6 excretion. We have analyzed sequences of S. granarius mitochondrial cytochrome oxidase 7 subunits genes: mtCOI and mtCOII and compared them with mtCOI/II sequences available 8 in GenBank. The analyzed genes displayed a high level of homology between 9 corresponding subunits. We have undertaken attempts to develop detection methods for 10 contamination by S. granarius in wheat and wheat flour based on the molecular biology 11 techniques: standard and real-time PCR with a TaqMan molecular probe. We applied 12 specific primers designed on the basis of available sequences for mtCOI and mtCOII genes, 13 and established optimal reaction conditions. The specificity of both methods was studied by 14 using a species closely related to S. granarius -S. oryzae and S. zeamais. We showed that 15 the sensitivity threshold was very high -we were able to detect the equivalent of one beetle 16 per 100 kg of flour when the real-time PCR with TaqMan probe method was applied to 17 model samples. The primer sets used turned out to be species specific, and technique was 18 rapid, reliable and very sensitive.

12 oryzae and S. zeamais were used to classify both species to two distinct gene pools using [START_REF] Barcenas | DNA diagnostics to identify internal feeders 14 (Lepidoptera: Tortricidae) of pome fruit quarantine importance[END_REF] PCR-RAPD and PCR-RFLP approaches (Hidayat et al. 1996). These pests also were 14 analyzed using DNA amplification fingerprinting with primers for aldolase, prolactin 15 receptor, and interleukin-beta genes and using PCR for ITS1/2 fragments of rDNA genes 16 [START_REF] Peng | DNA identification of two laboratory 20 colonies of the weevils, Sitophilus oryzae (L.) and S. zeamays Motschulsky (Coleoptera: 21 Curculionidae) in Taiwan[END_REF]. The ITS2 fragment of rDNA was also used to develop marker-assisted ). The major advantage of this approach is their occurrence of mtDNA in many copies 1 in each cell. Copies exist either in tandem, as rDNA genes or in organelles like 2 mitochondria that are numerous in every cell. This feature enables detection or 3 identification of organisms even if a limited quantity of material is available for analysis. [START_REF] Madani | Quantitative detection of the potato cyst 5 nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye[END_REF] The objective of our study was to analyze the sequences of mitochondrial oxidase 5 subunits (COI and COII), and to design specific primers using this information and to 6 determine the possibility of detection of S. granarius infestations in food by using the First, primers complementary to the sequence encoding I and II subunits of 14 mitochondrial cytochrome oxidase were applied (Table I). We performed amplification of 15 COI and COII in triplicate. The obtained sequences (see DNA cloning, sequencing and 16 sequence analyses section) were compared with their counterparts from GenBank and 17 together used to design other specific primer sets (giving shorter products) for detection 18 purposes. These primers were utilized for detection of S. granarius larvaes in wheat flour 19 and wheat using standard and real-time PCR (Table II To achieve our goal, we used the sequences encoding subunits of mitochondrial 10 cytochrome oxidase (COI and COII). We compared DNA sequences encoding I and II 11 subunits of mitochondrial cytochrome oxidases by using PCR amplification with 12 appropriate primer sets (Table I). We analyzed the sequences of DNA of S. granarius 13 beetles available in our laboratory and compared them with sequences already deposited in 14 GenBank (AY131101 and M83970). We assumed that there could have been nucleotide 15 changes in these genes for S. granarius populations that have been geographically isolated. 16 Therefore, the amplified fragments were cloned, multiplied and sequenced. We performed The species specificity of the the PCR methods were evaluated using samples of 1 Sitophilus oryzae (L.) and Sitophilus zemais (Motsch.) a close relatives of S. granarius L., 2 as well as Tribolium confusum (Duv.) and Tribolium castaneum (Herbst.) as the negative 3 controls. We established the optimal conditions for the PCR method, and the sensitivity and The sensitivity of the reaction was analyzed using several decreasing concentration of S. 9 granarius in flour and then their dilutions in water (as described in Materials and Methods 10 section). We found that sensitivity of detection is very high, and even very small quantities 11 of genetic material of granary weevil are possible to detect using the real-time PCR 12 technique. The analysis revealed that it is possible to detect S. granarius contamination 13 even when DNA isolated from contaminated flour at the concentration of 1 beetle/1 kg was 14 diluted 100 times in water (Figure 4). The lower concentrations were not detectable. Quality is determined by not only meeting contamination limits for various organic and 2 inorganic substances, but also by presence of microorganisms or insect pests. In the case of 3 raw commodities, some stored-product insect pests and other contaminants are removed by 4 cleaning prior to processing. [START_REF] Sheppard | Detection 6 of secondary predation by PCR analyses of the gut contents of invertebrate generalist 7 predators[END_REF] The traditional method -so called 'filth test' is simple and currently used for 6 detection of insect fragments in grain. It is based on flotation and separation of extraneous 7 matter from grain followed by counting. This is useful for detection of contamination in 8 grain, but for detection of insect fragments in processed food is not applicable. 9

In our study, we have evaluated the usefulness of molecular biology techniques 10 (standard and real-time PCR) for detection of one of the most common stored-product 11 pests, S. granarius. After establishing optimal conditions of detection reactions we then 12 analyzed the specificity of the investigated methods using species closely related to S. The number of mitochondria depends upon the metabolic requirements of the cell and may 20 range from a few up to a few thousand.

21

Our earlier analysis of COI and COII genes showed that differences in sequences 22 between S. granarius populations (our sequences and those previously deposited in 23 GenBank) are not a concern, so we may assume that our primers will be useful for detection of any populations of S. granarius. However, both primers and reaction 1 conditions have to be designed very carefully to ensure their species specificity. As 2 previous analyses had shown [START_REF] Peng | DNA identification of two laboratory 20 colonies of the weevils, Sitophilus oryzae (L.) and S. zeamays Motschulsky (Coleoptera: 21 Curculionidae) in Taiwan[END_REF], even relatively variable sequences, serving 3 for phylogenetic or identification studies, such as ITS1 and ITS2 share a very high level of 4 homology (96-97%) between closely related species of laboratory colonies of S. oryzae (L) 5 and S. zeamais Motschulsky. The application of more conservative genes, such as aldolase, 6 prolactine receptor, or interleukin-beta genes using the DNA fingerprinting approach did 7 not result in the ability to distinguish between these species. In the reaction, we applied a TaqMan probe in addition to the specific primer pairs. 9 Using a specific probe allows one to avoid the problem of an unspecific fluorescence signal 10 arising from competitive reactions, such as primer-dimer formation. The presence of such a 11 probe enables direct reading of results because only the specific product of reaction arising 12 de novo is detected, and no melting point analysis is needed. In the present work we are not presenting a ready-to-use protocol for quantitative anaysis of 3 S. granarius. We describe the method, appriopriate sequences, specific primers, and a 4 molecular probe, as well as optimized reaction conditions. The method is good for 5 detection, but for quantitative assessment we suggest preparing a standard curve with 6 material that has been stored for a comparable time with known amounts of granary weevil Nonetheless, the methods have great potential for application in the future. We think that 12 these results will be useful for rapid and sensitive detection of live or dead stored-product 13 pests.

14

A very important aspect of appropriate assessment of the infestation level is the 15 method of sampling as well as sample size. To obtain credible results it is crucial to obtain 16 a sample with maximum of homogeneity in the analyzed material. Therefore, flour should 17 be mixed thoroughly, or in the case of grain this should be crushed beforehand. Afterwards, 18 several random-chosen samples should be taken for analysis. In our case we were limited 19 by small-scale laboratory capacity and our tests were carried out on a small sample size of 20 50 mg that in turn was limited by applied kit possibilities. In our experience, however, the 21 sample size though small was sufficient to detect infestation level of the equivalent of 1 22 beetle per kg (as it was allowed in some countries), or lower, from the 50 ng of DNA taken 23 out from that 50 mg sample. The results were comparable when tested in triplicate. To enhance the reliability of detection and infestation assessment it is suggested to use more 1 samples to analyzes as well as increase the sample size, when possible. Then statistically 2 the obtained results will be more accurate. [START_REF] Russel | Evaluation of four methods to detect weevils in wheat: granary weevil, 4 Sitophilus granarius (L.), in soft white wheat[END_REF] Based on our results, we can assume that the real-time reaction should be 4 sufficiently sensitive to meet even the most restrictive detection requirements. In addition, 5 standard and real-time PCR reactions significantly reduce the time required for detection 6 without compromising sensitivity and reproducibility. Our approach has shown that both 7 the primers and protocols used for amplification of subunits of mitochondrial cytochrome 8 oxidase result in reliable, sensitive, and species-specific detection of insects.

  weevil, Sitophilus granarius (L.), and other stored-product insect pests cause 3 extensive economic losses in stored grain. Each year in the USA, stored-product insects are employed. Examples are the Cauliflower Mosaic Virus 35S promoter 1 (CaMV35S) (Akiyama et al. 2005), the α-glucuronidase gene (GUS) (Ding et al. 2005), 2 and the 5'-transgene integration sequence (Yang et al. 2005). 3 Substantial progress has been made in molecular genetic analysis of insects in the 4 areas of identification, gene structure-function relationships, and use of molecular markers 5 to track insect populations (Handler and Beeman 2003). There has been much research 6 conducted in the field of stored-product pests. Molecular tools have been used to determine 7 the distribution of species (Fields and Philips 2002) and in determination of genetic 8 relationships and molecular diversity between strains and populations of stored-product 9 insect pests using the PCR-RAPD aproach (Fields and Philips 2002, Fleurat-Lessard and 10 Pronier 2006). Also diversity of Sitophilus spp. was analyzed genetically with RAPD 11 fingerprints (Fleurat-Lessard and Pronier 2006), and molecular markers for diagnosis of S.

Food

  17 identification method of S. oryzae (Jeong et al. 2006) 18 Sequences of mitochondrial DNA (mtDNA) and DNA encoding ribosomal RNAs 19 (rDNA) are commonly used for detection, identification or phylogeny studies (Sheppard et 20 al. 2005, Horn et al. 2006). Among insects, these genes were used to identify internal 21 feeders of pome fruits by both standard and real-time PCR reaction (Barcenas et al. 2005). 22 Also, multiplex PCR reaction based on mtCOI and mtCOII was applied for differentiation 23 of western and northern corn rootworm larvae from the genus Diabrotica sp. (Roehrdanz 24 Page 4 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

7 molecular

 7 biology techniques: standard PCR and real-time PCR. The specificity of both 8 methods as well as sensitivity and reactions conditions are reported.

  of the granary weevil used in experiments came from our laboratory 13 colony maintained on whole wheat kernels (winter variety KANCLER) at 24˚C and 65-14 70% relative humidity. Adults were collected every three days, and larvae were extracted 15 from kernels by manually dissecting them at different stages of larval development. Also 16 three populations from Germany, France and Denmark kindly provided by Dr Cornel 17 Adler, Dr Patrick Ducom and Dr Lise Stengard Hansen, respectively, were tested. In 18 detection studies, samples of DNA isolated from S. granarius L., S. oryzae L., S. zeamais 19 Motsch., and secondary stored-products pests: Tribolium confusum Duv. and T. castaneum 20 Herbst. as well as uncontaminated flour or wheat, and contaminated with S. granarius flour 21 and wheat were analyzed. The flour and wheat samples were purposely contaminated (with 22 beetles and larvae of S. granarius, respectively) with appropriate amount of insect material, case of flour mixed thoroughly in homogenizer for equal distribution. Later, 1 several of random-chosen samples of 50 mg were taken for analyzes. 2 DNA isolation 3 Insects were ground under liquid nitrogen with a mortar and pestle. The DNA from adult or 4 larval insects was isolated with DNeasy Tissue Kit (Qiagen, Wroclaw, Poland) and from 5 wheat or flour with DNeasy Plant Mini kit (Qiagen, Wroclaw, Poland) according to the 6 manufacturer's instructions. We used a prolonged incubation time of 3 hours for lysis of the 7 cells. Efficiency and integrity of isolated DNA were checked by electrophoresis in agarose 8 gels, and its purity was analyzed spectrophotometrically by measuring A 260 /A 280 ratio. 9 PCR amplification 10 The genomic DNA was amplified in vitro with specific primers. All primers were 11 designed with PrimerSelect Software from DNASTAR Lasergene 7.1 package (Madison, 12 USA).

13

 13

2 11 ( 5 ' 13 (Food 8

 115138 were neither due to polymerase error nor a result of sequencing of one of the alleles from a 3 gene in heterozygous state. The obtained sequences were subsequently analyzed and 4 compared with appropriate sequences from GenBank by using blastn tool 5 (www.ncbi.nlm.nih.gov/blast ). 6 Real-time PCR 7 Qualitative real-time PCR reactions were performed using MxPro 3005P (Stratagene, 8 Warsaw, Poland). Analysis and interpretation of results were done with the software 9 provided by the manufacturer. Reactions were carried out in a 10 µl mixture containing: 10 AmpliLight Mix (Novazym, Poznan, Poland), 0.5 µM primers, 1 pmol of Taqman probe FAM-TGACGGAACACCTGGTC-TMR-3') designed in Beacon Desinger, PREMIER 12 Biosoft International, linked with FAM as a fluorophore and TAMRA as a quencher TibMolBiol, Poznan, Poland) and DNA template. 14 The real-time PCR reactions were performed under the following conditions: initial 15 denaturation at 95°C for 2 minutes followed by 40 cycles consisting of denaturation at 16 95°C for 20 sec., and annealing at 60°C for 30 sec., with single measurement of 17 fluorescence emitted by FAM dye released from TaqMan probe hydrolysis due to 5' 18 exonuclease activity of DNA polymerase. 19 Analysis of the sensitivity and specificity of real-time PCR 20 We evaluated also the absolute sensitivity of the real-time PCR reaction. We 21 prepared several concentrations of contaminated flour: 100 beetles/1 kg, 10 beetles/1 kg, 1 22 beetle/1 kg of flour. From the last concentration, samples were taken and diluted with 23 Page 8 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk obtain concentrations corresponding to equivalent of 1 beetle/10 kg (=0,1 1 beetle/kg, see Figure 4) and 1 beetle/100 kg of flour (=0,01 beetle/kg). 2 To assess the specificity of the reaction as negative control beetles two closely 3 related species belonging to primary stored-products pests: S. oryzae and S. zeamais, and 4 two species belonging to secondary stored-products pests: T. confusum and T. castaneum Analysis of the DNA sequences for mtCOI and mtCOII9

Food

  17 amplification of COI and COII in triplicate. These obtained sequences are available in 18 GenBank under accession numbers: DQ453486 for mtCOI and DQ462235 for mtCOII. The 19 comparison of sequences originating from our laboratory with those already deposited in 20 GenBank showed a very high level of sequence similarity (about 99%). The comparison of 21 mtCOII sequence from S. granarius with sequences derived from other Sitophilus species 22 (S. oryzae: AY014880, S. zeamais: AY014881) (Fig. 1) allowed us to design distinct primer 23 pair that specifically detect only S. granarius in standard and real-time PCR reaction. 24 Page 9 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

4 reaction 7 For

 47 conditions of the real-time PCR reaction for the primers and biochemicals also 5 detection of S. granarius, we used a primer pair specific for the sequence of II subunit 8 of mitochondrial cytochrome oxidase (COIISG1/2). The following samples were used: 9 DNA from uncontaminated flour and wheat, from contaminated flour and wheat, from 10 S. granarius as a positive control and from S. oryzae as a negative control. S. oryzae was 11 also a control of the specificity of primers and method. PCR reaction under conditions 12 described in the materials and methods and TableIIgave single and distinct products in the 13 positive control and contaminated samples (Figure2). There was no reaction product in 14 either the uncontaminated samples nor in the sample containing DNA from S. oryzae. 15 Therefore, the primers specificity for only S. granarius was confirmed and we can conclude 16 that standard PCR reaction works properly for detection of infestation with granary weevil.

  17

[Food 11 oryzae 3 granarius

 113 Insert Figure 2 about here] 18 Real-time PCR detection 19 For real-time PCR, the primers qCOIISG1/2 (Table II) and TaqMan probe were used. 20 Detection of contamination with S. granarius with real-time PCR was performed using 21 DNA samples isolated from contaminated with insect beetles flour and from contaminated 22 with insect larvae wheat, from uncontaminated flour, and from uncontaminated wheat. 23 DNA isolated from S. granarius was used as a positive control of reaction. DNA from S. 24 Page 10 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk was used as a negative control and, simultaneously, as control of detection 1 specificity. The control sample and contaminated samples gave positive results entering the 2 logarithmic phase with fewer than 40 cycles of amplification. The sample containg S. DNA exceeded the Ct value after 16 cycle. The contaminated flour and grain 4 exceeded the threshold after 31 and 36 cycle, respectively. The rest of samples did not enter 5 logarithmic phase before 40 cycle (Figure 3, Table 3) 6 [Insert Figure 3 about here] 7 Analysis of the sensitivity of real-time PCR reaction 8

15[

  Insert Figure4about here]16 To check the specificity of real-time PCR method we analyzed samples containing DNA 17 isolated from two closely related species of S. granarius -S. oryzae and S. zeamais as well 18 as two secondary stored-products pests: T. confusum and T. castaneum. Only samples of 19 four populations of S. granarius were detected, confirming specificity of the reaction (Fig.

  20

13 granarius

 13 -S. oryzae and S. zeamais as well as secondary stored-products pests: Tribolium 14 castaneum and T. confusum as negative controls. The threshold sensitivity of real-time 15 reaction under the described conditions is corresponding to DNA concentration in the 16 sample equivalent to 1 beetle per 100 kg of flour. 17 Detection methods using sequences of genes occuring in numerous copies in each 18 cell increases the sensitivity (Hsu et al. 2003, Cheng et al. 2003, Guha and Kashyap 2005).

19

 19

8

 8

13 MolecularFood 14 PCR

 1314 techniques have very big advantages over other methods used at present 14 for detection of stored-product pests. They are very rapid, sensitive, and species specific. 15 Our results have shown that is possible to readily distinguish between infestations of 16 closely related species, such as S. granarius and S. oryzae. However, these methods also 17 have disadvantages. The price of appropriate kits for PCR and equipment, especially for 18 real-time PCR, are still relatively high, but tend to decrease along with rising popularity and 19 accessibility of real-time PCR systems. Because DNA is the target for detection, the 20 reaction is very specific however, DNA can degrade under very long storage, which can be 21 a drawback, especially because in the case of stored products where problems occur either 22 because of larvae or remains of adults inside the grains. However, the primers designed for 23 Page 13 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk and real-time PCR give short product and hybridize to the genes existing in many 1 copies per cell, which may overcome these obstacles.2

7 remains.

 7 Then, one should analyze randomly chosen samples and compare them to the 8 standard curve. Our concentration curve was prepared using genetic material from 9 relatively fresh S. granarius beetles and served to present the sensitivity of real-time PCR 10 with TaqMan probe under the described reaction conditions.

11

 11

Roche

  cycle threshold -parameter defined as the cycle at which PCR enters the 4 exponential phase and the fluorescence emission exceeds the assigned threshold. 5TaqMan® probes are dual labeled hydrolysis probes and are a registered trademark of the 6 feeds by species-specific PCR. Journal of Animal and Feed Science 12L 851-Krischik V. 1995. Why stored product integrated pest management is needed.

4 In:FoodFood 19 Food

 419 Figures

20Food 3 oryzae, 3 -Figure 3 7 12 Figure 4 13Food

 333124 Figure 2

24 Page 6 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

	1	mM Tris-HCL, 16 mM (NH 4 ) 2 SO 4 and 2.5 mM MgCl 2 ]. The samples were amplified for 35
	2	cycles. Each cycle consisted of the following steps: denaturation at 95°C for 1 min,
	3	annealing at temperatures listed in Tables I or II (depending on the primers used) for 30
	4	sec., and primer extension at 72°C for 1 min. Each food or insect sample was tested in
	5	triplicate. Products of amplifications were analysed in 2% agarose gel stained with
	6 7	ethidium bromide. F o [Insert Table I about here]
	8	r [Insert Table II about here]
	9 10 11	P [Insert Figure I about here] e DNA cloning, sequencing and sequence analyses e r DNA fragments obtained in PCR reactions with primers listed in Table I (three samples for
	12 13 14 15 16	each mitochondrial cytochrome oxidase subunit) were separated on 2% agarose gels, and R then extracted and purified with QiaExII Gel Extraction Kit (Qiagen, Wroclaw, Poland). e v Purified fragments were cloned in pGEM®T-Easy Vector System (Promega, Straszyn, i e Poland) according to the manufacturer's instruction. The obtained recombined plasmids w were added to 50 µl of DH5α competent cells and transformed by electroporation into
	17 18	using Micro Pulser electroporation system (BIO-RAD, Warsaw, Poland). Then bacterial O n cells were incubated with shaking in LB medium at 37ºC for one hour. Next,
	19	l y transformation cultures were plated onto LB/ampicilin (100 µg/ml)/IPTG (0,5 mM)/X-gal
	20 20	, primers' hybrydization positions are (80 µg/ml) plates and incubated overnight at 37 ºC. The obtained bacterial colonies were
	21 21	shown in Figure 1). screened for the presence of inserted DNA sequence in vector by using PCR amplification
	22 22	PCR reactions were carried out in an Eppendorf Mastercycler in a final volume of with primers from Table 1 as described before (PCR amplification section). Colonies with
	23 23	10 µl. The reaction mixtures contained 50 ng DNA, 1 µM of each primer, 200 µM dNTP, confirmed recombinant plasmids were grown again in 3 ml of LB/ ampicilin medium and
		0.2 U of Allegro Taq polymerase (Novazym, Poznan, Poland) in 1x polymerase buffer [70 eventually plasmids were isolated using Qiaprep Spin Miniprep Kit (Qiagen, Wroclaw,
		7

24 Page 7 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

  Poland). Then, 8 clones from each sample were automatically sequenced (DNA Sequencing 1 and Oligonucleotides Synthesis Service, IBB Warsaw) to confirm that nucleotide changes

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	8

Table I

 I List of primers used for comparative analysis of DNA sequences encoding subunits of mitochondrial cytochrome oxidases.Table IIList of specific primers used for detection S. granarius in standard and real-time PCR

	Page 27 of 31 Page 29 of 31 Page 31 of 31	Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants		Page 28 of 31 Page 30 of 31
	Fig. 4 Fig. 5
	A) A)
	Primer Primer	Sequence (5'→3') Sequence (5'→3')	Region of Region of	Product Product	Temperature Temperature
			amplification amplification	length length	of annealing of annealing
	SgCOI1 AAACCACAAAGATATCGGCA COIISG1 TGATAACCGAACACCAATTCCC	mtCOI gene mtCOII gene	299 bp 53ºC 125 bp 68ºC
	Sg SgCOIIa ATTCCTGCTATTATTCTTATTT P P P P F o r F o r F o r SgCOI2 TTAAAGATGGGGGAAGTAGT SgCOI3 GAGCCCCAGATATAGCCTTC SgCOI4 CTCCGGTTAGTCCTCCAATA COIISG2 TTAGACGACCAGGTGTTCCGTC F qCOISG2 GATTTCTGAGCATTGACCAAAG r o qCOIISG1 TCCTGAACAATCCCAAGAATAAG	FlSG mtCOII gene of S. granarius mtCOI gene of S. granarius of S. granarius of S. granarius mtCOII gene	450 bp 50ºC GrSG 803 bp 57ºC 114 bp 60ºC
	e e r e e r e e r SgCOIIb AGTTGGGAGTGATTCTTTCTA e e r	100 SgDe SgFr SgPo SgGe of S. granarius 10 1 *0.1 *0.01
		R R R R
		e e e e
	Sample B) Fl Gr Sg So FlSG Flour contaminated with S. granarius (1 insect/kg) Sample content v i e v i e v i e v i e Pure flour Pure grain Sitophilus granarius Sitophilus oryzae GrSG Grain contaminated with S. granarius H2O Water w O n Ct value --16.11 -31.79 36.79 -l y Sample Number of S. granarius beetles Ct value 100 100 beetles/ kg w B) w w 26.00 10 10 beetles/kg 29.60 1 1 beetle/kg 32.39 0.1 Corresponds to 1 beetle/ 10 kg 35.13 0.01 Corresponds to 1 beetle/ 100 kg 37.88 O n l y Sample Sample content Ct value SgPo Sitophilus granarius from Poland 18.72 SgGe Sitophilus granarius from Germany 18.07 SgFr Sitophilus granarius from France 18.39 SgDe Sitophilus granarius from Denmark 18.02 So Sitophilus oryzae O n l O n l -Sz Sitophilus zeamais -y y
	Tcas	Tribolium castaneum		-
	Tc	Tribolium confusum		-
	H2O	water		-

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

Acknowledgements

This study was supported by a grant from Polish Ministry of Science and Education no


Vera Schulzova

Jana Hajslova

R Peroutka

Jiri Hlavasek

Jørn Gry

Hans Christer Andersson

Hans ; Andersson

Agaritine content of 53 Agaricus species collected from nature

come

Introduction

The commonly eaten cultivated mushroom, Agaricus bisporus, belongs to the Agaricaceae family. For a long time A. bisporus was believed to have been derived from several different wild Agaricus species [START_REF] Toth | Hydrazines and Cancer. A Guidebook on the Carcinogenic Activities of Hydrazines, Related Chemicals, and Hydrazine-Containing Natural Products[END_REF], and its continued propagation over many centuries to have resulted in several commercial strains which have no exact counterpart in nature. This hypothesis has now been proven incorrect as wild A. bisporus populations 2 have been identified in various parts of the world [START_REF] Mozina | Marking of cultivars and wild strains of cultivated mushroom Agaricus bisporus with esterase allozymes[END_REF][START_REF] Kerrigan | Global genetic resources of Agaricus breeding and cultivation[END_REF]. In some countries, strains of other species, such as A. bitorquis, are the preferred cultivated varieties [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF].

In 1961 Levenberg isolated a characteristic glutamine-containing compound from the press-juice of A. bisporus. He characterised the compound physically and elucidated its chemical structure as N 2 -(γ-L-glutamyl)-4-hydroxymethylphenylhydrazine (Figure 1), and gave it the trivial name agaritine. As shown in Table I, the level of agaritine in the cultivated mushroom is fairly high, usually in the range 200 to 500 mg/kg fresh weight [START_REF] Andersson | Phenylhydrazines in the cultivated mushroom (Agaricus bisporus) -occurrence, biological properties, risk assessment and recommendations[END_REF]), but levels as high as 1700 mg/kg fresh weight have been reported [START_REF] Liu | Agaritine content of fresh and processed mushrooms (Agaricus bisporus (Lange) Imbach)[END_REF]).

[Fig. 1

inserted about here] [Table I inserted about here]

Agaritine is the predominant, although not the only phenylhydrazine derivative in A.

bisporus. The compounds 4-carboxyphenylhydrazine, N 2 -(γ-L-glutamyl)-4-carboxyphenylhydrazine, and 4-hydroxymethylbenzenediazonium ion make up around 10% of the total phenylhydrazines in the mushroom [START_REF] Levenberg | An aromatic diazonium compound in the mushroom Agaricus bisporus[END_REF]Chauhan et al. 1984[START_REF] Chauhan | Isolation of N 2 -[γ-L-(+)-glutamyl]-4-carboxyphenylhydrazine in the cultivated mushroom Agaricus bisporus[END_REF][START_REF] Ross | Evidence for the occurrence and formation of diazonium ions in the Agaricus bisporus mushroom and its extracts[END_REF].

The potential carcinogenicity of A. bisporus, and the possible role of agaritine and the other phenylhydrazine derivatives in inducing this severe adverse effect, has been a matter of controversy for a number of years [START_REF] Gry | Nordic seminar on phenylhydrazines in the cultivated mushroom (Agaricus bisporus)[END_REF][START_REF] Gry | Nordic seminar on phenylhydrazines in the cultivated mushroom (Agaricus bisporus)[END_REF]; Toth [START_REF] Andersson | Phenylhydrazines in the cultivated mushroom (Agaricus bisporus) -occurrence, biological properties, risk assessment and recommendations[END_REF]. In long-term in vivo studies, a diet of fresh or baked A. bisporus for 3 days per week over 2 years produced tumours in various tissues of both male and female outbred Swiss mice (Toth and Ericsson 1986;Toth et al. 1997a). An elevated tumour incidence, although not statistically significant, was observed when baked mushrooms were fed to mice according to a more balanced feeding schedule -12 hours each day for 5 days each week during life-time (Toth et al. 1997b). Similarly, oral administration of 4-carboxyphenylhydrazine or N 2 -(γ-L-glutamyl)-4carboxyphenylhydrazine, or the 4-hydroxymethylbenzenediazonium ion resulted in tumour induction in Swiss mice [START_REF] Toth | Hydrazines and Cancer. A Guidebook on the Carcinogenic Activities of Hydrazines, Related Chemicals, and Hydrazine-Containing Natural Products[END_REF]. The main phenylhydrazine in the mushroom, agaritine (98% pure), however, did not result in tumour induction when administered in drinking water in a long-term carcinogenicity study [START_REF] Toth | Attempted tumor induction with agaritine in mice[END_REF]). This observation is unexpected, as agaritine has been shown to bind covalently to mouse DNA (Shephard and Schlatter 1998) and to increase the mutation frequency in forestomach and kidney tissue of lacI transgenic mice in vivo (Shephard et al. 1995). As it was recently shown that agaritine is unstable in oxygenated aqueous solution (Hajslova et al. 2002), it cannot be ruled out that the exposure to agaritine was lower than expected in the long-term carcinogenicity experiment.

Until it has been established whether consumption of the cultivated mushroom constitutes a risk for adverse health effects or not, it seems advisable to abstain from exaggerated exposures to phenylhydrazine derivatives [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF]. This could be done by growers substituting the strains of the cultivated mushroom producing high levels of agaritine with strains or other Agaricus species having comparable organoleptic properties but producing lower levels of agaritine. Sources for such strains may be available from the Agaricus species collected in the Czech Republic. The data may also be of interest for mushroom pickers when assessing the edibility of Agaricus species to be collected.

Materials and Methods

Samples and chemicals

Different Agaricus species were picked at various locations in the Czech Republic (most of them in the central part of Bohemia, close to Prague) during the mushroom growing seasons 1998-2001. All species were determined by one of the authors (Jiri Hlaváček), an experienced mycologist specialized on the taxonomy of the genus Agaricus. The Agaricus taxonomy remains far from clear, despite the contributions of many authors over the years, especially [START_REF] Pilát | The Bohemian species of the genus Agaricus[END_REF], [START_REF] Möller | Danish Psalliota species[END_REF], [START_REF] Essette | Les Psalliotes[END_REF][START_REF] Heinemann | Les Psalliotes[END_REF], [START_REF] Wasser | Flora fungorum RSS Ucrainicae. Basidiomycetes, Agaricaceae Cohn. Academia Scientianum RSS UCR[END_REF], Cappelli (1984), [START_REF] Knudsen | Agaricaceae Fr[END_REF], [START_REF] Bohus | Agaricus studies XIII. Key to subgenus Agaricus from Europe[END_REF], Nauta (2001) and [START_REF] Parra | Contribution to the knowledge of genus Agaricus[END_REF]. As a consequence, mycologists advocate different schools of Agaricus taxonomy. We have chosen the practical approach of presenting both the taxonomic names as given by the mycologist who determined the mushroom species, J. Hlaváček, who used more narrow concepts identifying a species, and the taxonomic names commonly used in the Western and Northern European countries where broader concepts identifying the various Agaricus species are prevailing [START_REF] Knudsen | Agaricaceae Fr[END_REF]Nauta 2001).

Most mushroom samples delivered to the Institute of Chemical Technology (Prague) for chemical analysis of agaritine content were clearly identified and did not require further consultation. In a few cases where the delivered samples were not adequately identified, Mushrooms were stored in the refrigerator until analysed [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF], which generally was within three days from being collected. We recently showed that the agaritine content of cultivated Agaricus purchased on the open market remained unchanged for the first three days in the refrigerator (5 o C), after which the agaritine content started to decrease [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF].

Water used for preparation of solutions was distilled and further purified using the Milli Q RG purification system (Millipore, Germany). Methanol used for extraction was supplied by Penta (Chrudim, Czech Republic), and gradient grade methanol for the HPLC mobile phase was purchased from Merck (Darmstadt, Germany). The agaritine standard was synthesised by Henrik Frandsen, Institute of Food and Veterinary Research, according to the method of [START_REF] Wallcave | An improved synthesis of agaritine[END_REF], but with some important modifications being introduced [START_REF] Frandsen | Synthesis of L-glutamic acid 5-(2-(hydroxymethyl)phenylhydrazine) (Agaritine). Nordic Seminar of Phenylhydrazines in the Cultivated Mushrooms (Agaricus bisporus)[END_REF]). The synthesised agaritine was more than 85% pure as shown at wavelength 200, 237 and 280 nm by high performance liquid chromatography (HPLC).

The purity factor of the agaritine peak reported by the Hewlett Packard ChemStation peak purity software was 999.9. The main impurity (11.9-13.6%) in the standard originated from the agaritine synthesis and was stable during the experiments reported here. The standard was protected from light and stored under oxygen-free nitrogen in the freezer.

Sample preparation

Agaritine standard for identification: a stock solution of agaritine in methanol (0.25 mg/ml) showed the dry agaritine standard in the freezer to be stable [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF]. Before analysis, the agaritine standard was dissolved in 1 ml of Milli Q water and analysed within 6 hours (Schulzová et al. 2003).

Approximately 20 gram of fresh whole mushroom was mixed with 100 ml methanol and homogenised for 10 minutes in an Ultra Turax (Janke a Kunkel, IKA-Werk). The homogenate was shaken for 30 minutes and a crude extract prepared by filtration. The volume of the filtrate was adjusted to 200 ml with methanol, 10 ml of the extract was evaporated to dryness and the residue dissolved in 2 ml Milli Q water. This solution was filtered through a microfilter (25 mm Filter Unit, 5.0 µm PTFE, PP, ThermoQuest) into a vial and an 20 µl aliquot injected (always within 6 hours) onto the HPLC column.

Identification and Quantification

HPLC was performed using a Hewlett-Packard HP 1100 liquid chromatograph (Wallbron, Germany) equipped with diode array detector (DAD) and autosampler. A sample amount of 20 µl was applied to the separating column (250x4 mm), LiChrospher 100 RP-18 (5µm), Merck, with precolumn (4x4 mm), Li Chrospher 100 RP-18 (5 µm), Merck. Optimization studies showed a methanol-water mixture to be suitable as mobile phase for separation; the mobile phase gradient being 10% methanol held for 5 min, changed to 80% over 15 min, and held at 80% methanol for 5 min. software function were used. Agaritine was detected at 237 nm and quantified by comparing the peak area of the analysed sample with the peak area for known amounts of the pure standard. Free amino acids that strongly absorb in the UV were shown not to interfere with the agaritine peak. The correlation coefficient of the UV spectra (normalized absorbance) of agaritine in standard solution and in a real sample was 0.999. The chromatograms and UV spectra of the agaritine standard and an Agaricus campestris sample are shown in Figure 2. The detector response was linear in the range 0.2 µg/ml -2.0 mg/ml, the equation of the linear regression of the calibration curve being y=31.325x + 17.584, with a correlation coefficient of R 2 =0.9999.. Under the experimental conditions described for sample preparation, the limit of detection for the method was 0.02 µg/ml (based on a signal/noise of 3:1), corresponding to 0.2 mg/kg fresh weight of mushroom, limit of quantification was 0.6 mg/kg. The repeatability of measurements at agaritine levels commonly found in fresh mushroom samples (200 mg/kg), expressed as the relative standard deviation, was 4.5% for six replicates. Method validation has been presented elsewhere [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF].

[Fig. 2

inserted about here]

The data was analysed by Student's T-test (α=0.05), using the software of Microsoft Excel 2003. average agaritine contents of the various Agaricus species identified, and, where appropriate, the range in agaritine content, without controlling for mushroom size. When the average agaritine content was calculated, a value of 50% of the detection limit was used for the samples in which no agaritine could be detected. Only a few samples were available for many of the more uncommon Agaricus species.

Results

Mushrooms of the genus

[Table II inserted about here.] A significant difference in agaritine content was observed between species. The amounts detected in individual species varied from non-detectable levels up to several g per kg fresh weight. One sample of A. elvensis contained 10 g/kg fresh weight. Some species were characterized by low agaritine content (<100 mg/kg fresh weight), others by intermediate (100-1000 mg/kg fresh weight) or high (>1000 mg/kg fresh weight) levels of agaritine. It should be noted that the allocation of species to groups having these arbitrarily set boarders is hampered by the low number of samples analysed for many species, resulting in less reliable values of average agaritine content.

[Table III inserted about here.] Nine of the 53 species analysed in our study contained less than 100 mg agaritine/kg fresh weight (Table III), which was the level chosen defining mushrooms with a low agaritine content. Of these nine species with low agaritine content only the relatively rare A. maskae, A. silvaticus var. pallidus, and possibly also A. caroli are sought after by mushroom connoisseurs. None of them are common mushrooms. III). In 20 species intermediate levels of agaritine were found, 100-1000 mg/kg fresh weight (Table III), not deviating substantially from the levels reported to occur in the cultivated mushroom, A. bisporus.

For the seven species for which more than 10 mushroom samples were analysed (A.

arvensis, A. bitorquis, A. bisporus var. bisporus (A. brunnescens), A. campestris, A.

maleolens, A. silvaticus, and A. xanthoderma) it was studied whether a correlation could be found between the agaritine content and size of the mushroom, phenology, year of collection, or site of collection.

No correlation could be found between diameter of the mushroom cap and its agaritine content. A typical example of the poor correlation between cup size and agaritine content is demonstrated by A. arvensis (r = 0.033; n.s.), Figure 3. These observations contradict the findings of [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF], that smaller fruit bodies of A. arvensis and A. augustus contain higher amounts of agaritine than bigger mushrooms of the same species. There was also no correlation in our studies between week of the year when the mushrooms were collected and their agaritine content (data not shown).

[Figure 3 inserted about here.] Among the 13 Agaricus species of which enough mushroom samples had been collected over more than one year, there was in general no statistical difference in the average agaritine content between years (shown by students t-test). The only data that reached No correlation could be found between agaritine content and geographical region where the mushrooms were collected (data not shown).

In addition to analysing for agaritine in the different Agaricus species, we also analysed for agaritine in four species belonging to each of the related genera Leucoagaricus and Macrolepiota. If agaritine occurs in mushroom species outside the genus Agaricus, Leucoagaricus and Macrolepiota are the genera where agaritine might be found. As shown in Table IV, one of the four Macrolepiota species, M. konradii, contained low amounts of agaritine, 42 mg/kg fresh weight. Agaritine was also detected in three of the Leucoagaricus species -L. bresadolae, L. cretaceus, and L. naucinus -but here the contents were higher, up to 838 mg/kg fresh weight (Table IV). As agaritine has been reported to occur in the cultivated Oyster mushroom Pleurotus ostreatus [START_REF] Burini | Determination of agaritine in cultivated mushrooms using high performance liquid chromatography with fluorometric detection[END_REF], we also analysed for agaritine in this mushroom but found none.

[Table IV

Discussion

Two research groups, one from the USA and the other from Switzerland, have previously analysed wild-growing Agaricus and non-Agaricus species for agaritine. [START_REF] Levenberg | Isolation and structure of agaritine, a γ-glutamyl-substituted arylhydrazine derivative from Agaricaceae[END_REF] examined boiled press-juice extracts from as many as 45 representative genera of wild basidiomycetes collected in the USA. As part of this survey, fruit bodies of 15 different species belonging to the genus Agaricus were analysed. Unfortunately, Levenberg used a nomenclature for the mushroom that hardly fit with that used in Europe today. Of the 15 Agaricus species, ten were reported to contain agaritine in quantities comparable to those found in A. bisporus, whereas five species were devoid of the compound (Table V). The limit of detection for the analytical method (spectrophotometric determination of agaritine by measurement of its rate of enzymatic hydrolysis) used by [START_REF] Levenberg | Isolation and structure of agaritine, a γ-glutamyl-substituted arylhydrazine derivative from Agaricaceae[END_REF] was not reported. The single sample of fresh A. bisporus analysed for its agaritine content was reported to contain 3300 mg agaritine/kg dry weight, which corresponds to approximately 330 mg/kg fresh weight.

[Table V inserted about here.] Subsequently, Swiss investigators reported HPLC (with a limit of detection in the region 20-25 mg/kg) and thin layer chromatography data on freeze-dried mushrooms, indicating that some wild-growing Agaricus species contain substantially higher amounts of agaritine than the cultivated A. bisporus [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF]Stijve and Pittet 2000). Because a considerable time had elapsed between picking of some mushroom samples and freezedrying, the latter investigators were unsure that the amounts detected were the Agaricus bisporus have revealed that, in addition to hereditary factors, cultural practice employed in production, stage of cropping-cycle (flush), and maturity of mushrooms at harvest may influence the agaritine level in the mushroom [START_REF] Andersson | Phenylhydrazines in the cultivated mushroom (Agaricus bisporus) -occurrence, biological properties, risk assessment and recommendations[END_REF]). Some of these factors are likely to influence the agaritine content also of wild-growing species. The length of storage after harvest and the conditions during storage are other factors that may influence the agaritine content [START_REF] Andersson | Phenylhydrazines in the cultivated mushroom (Agaricus bisporus) -occurrence, biological properties, risk assessment and recommendations[END_REF]. This is exemplified by the data given in Table I. The average level of agaritine in A. bisporus obtained directly from the growers was 723 ± 276 mg/kg fresh weight, whereas it was 475 ± 221 mg/kg fresh weight in mushrooms purchased on the open market. We have minimized the potential influence of storage on the agaritine content in the present study by analysing samples within three days after being collected.

Already in 1983, Speroni and co-workers suggested that wild-growing Agaricus mushrooms contain higher amounts of agaritine than the corresponding cultivated mushrooms. These investigators had noted that a cultivated strain of A. bisporus with brown colour phenotype, PSU-351, which was only recently isolated from nature, contained higher agaritine levels than other cultivated A. bisporus strains. It was 13 hypothesised that this strain had not yet adapted to the less stressing environmental conditions during cultivation and not yet lost or reduced its inherited ability to produce compounds inhibiting growth of certain competing fungi. A similar observation is made in the present study. In the Czech Republic, it is strains of A. bitorquis and 'A. hortensis' that are cultivated. Samples of these cultivated strains have been reported to contain comparable levels of agaritine, varying between 165 and 457 mg/kg fresh weight, with an average content of 272 mg/kg fresh weight [START_REF] Schulzová | Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom[END_REF]. As shown in Table II, A. bitorquis and A. bisporus collected from nature contain significantly higher amounts of agaritine, 1 473 and 746 mg/kg fresh weight, respectively.

Although too few samples were analysed for some Agaricus species to prove their normal agaritine content, enough samples were analysed to conclude on the agaritine content for other species. As shown in Table II and III, no less than 24 of the studied species contained agaritine levels above 1000 mg/kg fresh weight. This amount should be compared with the levels commonly found in A. bisporus, 200-500 mg/kg fresh weight. The highest amounts were observed in A. elvensis (mean 4445 mg/kg), A. purpurellus (4100 mg/kg), A. augustus (3995 mg/kg), A. fissuratus (3526 mg/kg) and A. sagatus (3479 mg/kg). As there is an ongoing discussion whether it is advisable or not to consume fresh cultivated mushroom [START_REF] Toth | Hydrazines and Cancer. A Guidebook on the Carcinogenic Activities of Hydrazines, Related Chemicals, and Hydrazine-Containing Natural Products[END_REF][START_REF] Andersson | Phenylhydrazines in the cultivated mushroom (Agaricus bisporus) -occurrence, biological properties, risk assessment and recommendations[END_REF], it seems highly questionable to advise consumption of wild-growing Agaricus species with even higher agaritine contents than the cultivated species. Some of the species with a high agaritine content, however, have a firm reputation as being good or even excellent culinary delicacies, e.g. A. augustus, A. fissuratus, A. essettei and A. substramineus. On the other hand, as indicated in Table II, some of them are The present study aimed at identifying tasteful, non-toxic Agaricus species with a low agaritine content. Such species would be potential sources for strain development for the mushroom cultivation industry, could it be shown they are devoid of other constituents with potentially harmful effects. Only nine of the analysed Agaricus species had an agaritine content bellow 100 mg/kg fresh weight. Of these at least five should be classified as non-edible as they may give rise to gastrointestinal symptoms due to formation of phenol. Based on the agaritine data presented in the present report, the most promising species for strain development seem to be A. caroli, A. maskae, A. silvaticus var. pallidus, and possibly A. squamulifer. Additional studies are required to establish whether these species fulfil the criteria necessary to be good substitutes for A. bisporus as cultivated mushrooms.

We have not addressed the question whether the presence of agaritine within the genus Agaricus may have chemotaxonomic significance. However, we have noted that the various sections of the genus Agaricus seem to be characterized by different abilities to produce agaritine (Table IV). [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF] have made a similar observation.

The question whether agaritine may be found in mushrooms outside the genus Agaricus has been addressed also by other investigators. [START_REF] Levenberg | Isolation and structure of agaritine, a γ-glutamyl-substituted arylhydrazine derivative from Agaricaceae[END_REF] was unable to detect agaritine in any of the 30 non-Agaricus species analysed (detection limit for the analytical technique not mentioned), and Stijve and co-workers in any of 39 wild-growing and 30 [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF]Stijve and Pittett 2000). Two reports on popular cultivated mushrooms are in conflict with these data. [START_REF] Hashida | Quantities of agaritine in mushrooms (Agaricus bisporus) and the carcinogenicity of mushroom methanol extracts on the mouse bladder epithelium[END_REF] found very low levels of agaritine in shiitake (Lentinus edodes), 0.82 mg/kg fresh weight, and [START_REF] Burini | Determination of agaritine in cultivated mushrooms using high performance liquid chromatography with fluorometric detection[END_REF] very high levels (average level 680 mg/kg fresh weight; range 478-905 mg/kg) in the Oyster mushroom (Pleurotus ostreatus). With such low quantities of agaritine in fresh shiitake, it came as no surprise that [START_REF] Andersson | Agaritine content in processed foods containing the cultivated mushroom (Agaricus bisporus) on the Nordic and the Czech market[END_REF] were unable to detect agaritine in a Swedish sample of canned shiitake, and that [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF] were unable to detect agaritine in fresh shiitake using an analytical technique having a limit of detection around 20-25 mg/kg fresh weight. Further studies are needed to determine whether fresh shiitake mushrooms contain low levels of agaritine. The report that agaritine occurs in the Oyster mushroom, is, however, most likely an artefact due to the use of an unspecific analytical method. The only other explanation, that the cultivated strains of Pleurotus ostreatus may vary substantially in their ability to produce agaritine seems less likely. Neither Swiss investigators [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF]Stijve and Pittett 2000), nor we in the present study could identify agaritine in this species, although the limit of detection was as low as 0.2 mg/kg fresh weight in the present study. Furthermore, there is no indication that agaritine production can be suppressed to negligible levels by the method of cultivation.

However, the data in the present study clearly show that agaritine is present in some species of the genera Leucoagaricus and Macrolepiota, which both are saprotrophic mushrooms closely related to the genus Agaricus (Singer 1986). The compound was detected in Leucoagaricus bresadolae, L. cretaceus, L. naucinus, and Macrolepiota konradii (detection limit 0.1 mg/kg), but not in one other species of Leucoagaricus and IV). [START_REF] Stijve | Agaritine, a p-hydroxymethylphenylhydrazine derivative in cultivated mushrooms (Agaricus bisporus), and in some of its wild-growing relatives[END_REF]

  present study reports on the quantity of agaritine detected in 53 different

  again. As he unfortunately died during the present study, his colleagues Borovička and Burel took over the confirmation step of a few samples.

  From this 100 µl were transferred into vials and evaporated to dryness by a stream of oxygen-free nitrogen. The vials were stored sealed in the freezer. Stability tests

  Total run time was 25 min, post time was 5 min. The flow rate was 1 ml/min, the column temperature being 35 o C. All analyses were run in duplicate. For identification of agaritine the retention time, DAD spectra and peak purity

  Agaricus were collected in various regions of the Czech Republic during different periods of the growing seasons 1998-2001, and identified by an experienced mycologist. Fifty-three different species were found.

  of the 53 analysed Agaricus species contained high levels of agaritine, more than 1000 mg/kg fresh weight (Table

  those of A. maleolens. In the years 1998 and 1999 A. maleolens contained significantly lower levels of agaritine (51±90 and 25±54 mg/kg fresh weight, respectively), than in year 2000 (165±50 mg/kg fresh weight), t-test, p=0.023. During the first two years the agaritine content was below the detection limit of the analytical method in the majority of the samples, whereas none of the samples collected in year 2000 contained below 100 mg/kg fresh weight. It is our interpretation that this significant difference might have disappeared had larger number of mushroom samples been analysed.

  freshly harvested wild mushrooms and, therefore, did not give specific information on the quantity of agaritine in the various species.The agaritine contents given for the various Agaricus species in the present study should fairly accurately describe the levels in freshly collected mushroom samples, at least when the number of samples studied for a species have been large enough. Several factors may influence the agaritine content of a mushroom. Studies performed with the cultivated

  -accumulating cadmium and other heavy metals from the soil[START_REF] Řanda | Trace elements in higher fungi (mushrooms) determined by activation analysis[END_REF]

  of Macrolepiota (Table

Fig. 3 .

 3 Fig. 1 The molecular structure of agaritine (N 2 -[γ-L-glutamyl]-4hydroxymethylphenylhydrazine).

Table II

 II

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	shows the

  inserted about here.]

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table I .

 I found no agaritine in Leucoagaricus pudicus, Macrolepiota procera and Macrolepiota rachodes. Agaritine content (mg/kg fresh weight) of methanolic extracts of fresh Agaricus bisporus as determined by HPLC. If samples were not freeze-dried (which does not influence the agaritine level) shortly after being obtained, they were analysed within 24 hours.

	Reference	Number	Diameter of	Content
		of	cap (cm)	Average	Range
		samples
	F o r F o r F o r F o r F o r Agaritine content of 53 Agaricus species collected from nature F o r Samples directly from growers Kelly et al., 1962* 1 not given ~ 400 -Daniels et al., 1961* 1 not given ~ 220 -Levenberg, 1964* 1 not given ~ 3300*** -Liu et al., 1982 14 ** 3.0-4.5 880 330-1730 F o r
	P e e r P e e r P e e r P e e r P e e r P e e r Speroni and Beelman, 1982 1 Speroni et al., 1983 8 ** P Fischer et al., 1984 2 ** e Sharman et al., 1990 2 ** Andersson et al., 1994 1 e r	not given 2.5-5.0 5.0-6.0 not given not given	2 190*** -304±6.0; 438±2.5 ~ 180 340	-1700-5100*** --80-250 -
	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w 2 not given 11 2.0-5.5 R 1 not given e 5** not given 5 not given 2 not given v i e * gravimetric method; ** different strains: *** mg/kg dry weight (approximately 10% dry Samples from the market Ross et al., 1982b 440; 720 170-1170 Fischer et al., 1984 368±45 94-629 Hashida et al., 1990 228 -Stijve et al., 1986 160-650 Burini et al., et al., 1999 820 630-980 Andersson et al., 1999 212; 229 -matter) w
			O n l O n l O n l O n l O n l O n l O n l
				y y y y y y y
					1 2 6

Table III .

 III Agaricus species with low, intermediate or high levels of agaritine. Section, according to Nauta (2001) is given within brackets (Ag= Agaricus;

	Ar=Arvenses; B=Bitorques; M=Minores; Sa=Sanguinolenti;
	Sp=Spissicaules; X=Xanthodermi).
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	7

Table IV .

 IV Average agaritine content (mg/kg fresh weight) in some mushroom species of the genera Leucoagaricus and Macrolepiota collected in the Czech Republic.

	Species as determined by	Species as interpreted by	Content
	Hlavácek	Knudsen (1992) and	(mg/kg fresh weight)
		Noordeloos et al. (2001)	(n=sample size)
	Leucoagaricus bresadolae	Leucoagaricus americanus	368 (n = 1)
	(Schultz.) Bon	(Peck) Vellinga
	L. cinerascens Migl. & Coccia L. leucothites (Vitt.) Wasser	n.d. (n = 3)
		var. carneifolius (Gillet)
	Vellinga L. cretaceus (Bull.:Fr.) Moser L. cretaceus (Bull.:Fr.) Moser L. naucinus (Fr.) Sing. L. leucothites (Vitt.) Wasser Macrolepiota rachodes var. bohemica (Wichansky) Bellù same F o r	838 (n = 2) 68 (n = 2) n.d. (n = 2)
	& Lanzoni M. konradii (Huijsman ex P P.D. Orton) Marchand M. permixta (Barla) Pacioni e M. mastoidea (Fr.) Sing. same M. rachodes (Vitt.) Sing. same e r	42 (n = 1) n.d. (n = 2) n.d. (n = 2)
		R
		e
		v i e
		w
		O n l
			y

Table V .

 V Agaritine levels reported by other investigators in various Agaricus species collected from nature.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

The synthesis of agaritine was supported by a grant from the Nordic Council of Ministers, and the chemical analysis by grant MSM 6046137305 given by the Ministry of Education, Youth and Sports of the Czech Republic. We are very much indepted to Jan Borovička, Jiří Burel and Henning Knudsen for very valuable information on Agaricus taxonomy.

 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 Agaricus species collected from the nature in the Czech Republic.

Culinary usefulness

Agaricus species (preferred nomenclature according to [START_REF] Hlaváček | Systém rodu Agaricus L. -pečárka -I[END_REF]Hlaváček ( , 1984aHlaváček ( , 1984bHlaváček ( , 1984c) ) Agaricus species -preferred nomenclature according to [START_REF] Knudsen | Agaricaceae Fr[END_REF] [START_REF] Hlaváček | Poznámky ke studiu žampionů (Agaricus) III. Pečárky rostoucí trsnatĕ 2[END_REF](3) Hlaváček, 1987;(4) Pilat, 1951;(5) [START_REF] Berkeley | Notices of British Fungi 34[END_REF](6) Hlaváček, 2000;(7) Bon, 1983;(8) Møller, 1952;(9) Hlaváček, 2002;(10) Pilat, 1954;(11) Freeman, 1979;(12) Bon and Cappelli, 1983;(13) Wasser, 1977;(14) Courtecuisse, 1985