6.2 II.6.2 Mushroom toxins by Kunio Gonmori and Naofumi Yoshioka

Introduction

As many as 5,000–6,000 mushroom species are growing in the world. Among them, only about 1,000 species are named; the majority of them are unnamed. Th e number of species of edible mushrooms in Japan is about 300; that of toxic mushrooms is said to be about 30. Various types of toxic mushrooms exist; some show high toxicity, while others show hallucinogenic actions. Morphological and chemical analyses for mushrooms are occasionally required in forensic sci- ence practice. In this chapter, the characteristics of the representative toxic mushrooms and some chemical methods for their toxins are presented.

Current situation of mushroom poisonings in Japan

According to “National Record of Food Poisoning Incidents” [1], the number of mushroom poisoning incidents taking place in Japan in 1974–1997 was 1,068; it was 431 in 1988–1997 (10 years) with 1,842 poisoned people, including 20 fatal victimsa. Among the 431 incidents, the numbers of incidents according to causative mushrooms are: Rhodophyllus rhodopolius plus Rhodophyllus sinuatus, 133; Lampteromyces japonicus, 127; Tricholoma ustale, 42; Amanita virosa plus Amanita verna, 16; Amanita pantherina, 15; Clitocybe acromelalga, 15; Psilocybe argentipes (a species of magic mushrooms), 12; other mushrooms, 36; not specifi ed, 35 (> Figure 2.1)b. Toxic mushrooms can be classifi ed into 6 groups according to their actions as follows. • Th ose which destroy cells, injure the liver and kidney and thus may cause death (latent period, 6–10 h; Amanita virosa, Amanita verna and Amanita phalloides). • Th ose which act on the autonomic nervous system and provoke symptoms, such as sweat- ing, lacrimation, vomiting and diarrhea (latent period, 20 min–2 h; Clitocybe gibba, Inocybe species and others). • Th ose which inhibit the metabolism of acetaldehyde in blood (disulfi ram-like eff ect), caus- ing a fl ushing phenomenon and palpitation upon drinking alcohol concomitantly (latent period, 20 min–2 h; Clitocybe clavipes, Coprinus atramentarius and others). • Th ose which act on the central nervous system and provoke abnormal excitement and hallucinations (latent period, 20 min–2 h; Amanita pantherina, Psilocybe argentipes and others). • Th ose which irritate the gastrointestinal tract and provoke symptoms, such as abdominal pain, vomiting and diarrhea (latent period, 30 min–3 h; Rhodophyllus rhodopolius, Lamptero- myces japonicus and others). • Others which cause swelling or necrosis of tips of extremities or sharp pain due to distur- bances of the peripheral nerves (Clitocybe acromelalga and others).

© Springer-Verlag Berlin Heidelberg 2005 470 Mushroom toxins

⊡ Figure 2.1

Incidence ratio of mushroom poisonings according to species in Japan. It is calculated from the data of “National Record of Food Poisoning Incidents”. The number of the mushroom poisoning incidents was 431; the poisoned subjects involved were 1,842 people.

> Table 2.1 shows the outline of the mushroom poisoning analyses, which the authors had undertaken in recent 9 years. As shown in this table, the number of the poisoning cases, in which Amanita virosa had been (suspected to be) causative, was as many as 10. Amanita virosa is highly toxic and sometimes causes fatalities. Th e highest incidence of the Amanita virosa in our laboratories is interpreted to mean that such fatal poisoning cases are selectively brought to our Department for analysis. Two cases were suspected of poisoning by Rhodophyllus rhodopolius (> Table 2.1).

Representative mushrooms causing poisoning cases

Rhodophyllus rhodopolius ( > Figure 2.2)

Th is mushroom shows the highest incidence of poisoning in Japan, because a very similar edible species Rhodophyllus crassipes is available and grows at similar locations. Th e poisoning symptoms are vomiting, diarrhea and abdominal pain appearing 30 min–3 h aft er ingestion. Th e stem of Rhodophyllus rhodopolius is easily crushed by pressure with the fi nger, but that of the edible Rhodophyllus crassipes is not. Th e toxic compound being contained in the mush- room is reported to be muscarine or choline. Representative mushrooms causing poisoning cases 471 the mushroom mushroom mushroom mushroom detected from the mushroom detected from mushroom Outcome Outcome Specimen and detectability of the toxin number 111 dead dead1 and the mushroom the liver detected from or kidney the liver blood, not detected from dead1 blood not detected from alive2 alive blood or the mushroom not detected from 1 or contents alive blood stomach not detected from alive blood or the mushroom not detected from blood urine or the not detected from 7 1 dead blood of one patient detected from 511 alive alive blood or urine not detected from alive blood or urine not detected from blood not detected from 3 alive urine or the blood, not detected from 2 alive urine or the blood, not detected from 10 alive – blood not detected from the mushroom not detected from 21 alive but dead blood or urine, not detected from urine and the blood, detected from ?) ? ? ? ? ?) mushroom- Rhodophyllus Rhodophyllus rhodopolius Amanita virosa Amanita virosa not available) (mushroom Amanita virosa not available) (mushroom Amanita virosa available) (mushroom Amanita virosa available) (mushroom blazei Agaricus available) (mushroom Amanita virosa available) (mushroom Amanita virosa Amanita virosa Amanita virosa Amanita neoovoidea rhodopolius wheat-flour noodles containing not clear ( Lampteromyces japonicus Lampteromyces available) (mushroom Amanita neoovoidea Amanita neoovoidea available) (only mushroom not clear ( Amanita virosa available) (mushroom medium) with a culture and Dept. Nephrol and Dept. Nephrol and Dept. Nephrol. and Dept. Nephrol. and Dept. Nephrol. Med. and Dept. Pediat. Table 2.1 Table No. Year 1 Requesting institution 1993 Med. Dept. Legal H Univ. mushroom Causative patient The 2 1996 3 Dept. Anaesth. Hosp. T Kyodo 1996 4 Dept. Intern. Med Y Univ. 1997 5 Units Emerg. D Univ. 1997 6 Units Emerg. F Univ. 1998 7 Med. Dept. Intern. Hosp. A Pref. 1998 8 Units Emerg. Hosp. O Pref. 1998 Units Emerg. Univ. J Med. 9 1998 Units Emerg. Univ. J Med. 10 199811 199912 Units Emerg. Hosp. I Pref. 1999 Dept. Urol. Hosp. O Munic. Units Emerg. Univ. J Med. 13 1999 Units Emerg. Univ. J Med. 14 1999 Dept. Intern. Gen. Hosp. A Munic. 15 200016 Health Center Y Publ. 2000 Unit Emerg. Univ. K Med. 17 2001 H. Q. A Police (cultivated mushroom a magic ⊡ undertaken Akita Department School poisoning analyses of Medicine University by Medicine, Outline of mushroom of Legal 472 Mushroom toxins

⊡ Figure 2.2

Rhodophyllus rhodopolius.

⊡ Figure 2.3

Amanita virosa. Representative mushrooms causing poisoning cases 473

Amanita virosa (> Figure 2.3)

It is a very beautiful white mushroom growing in mountain areas; it is thus being called “de- stroying”. Only with one mushroom of Amanita virosa, 2 or 3 adult subjects can be killed. Th e Amanita genus mushrooms should be watched most carefully also in the forensic toxicological point of view. Th e main toxin of this genus is considered to be amanitin (> Figure 2.4) or phalloidin (> Figure 2.5). Th e amanitin is subdivided into α-, β- and γ-amanitins. In Japan, Amanita virosa and Amanita verna glow generally, while in Europe and America, Amanita phalloides is responsible for poisoning. Th ere is a report insisting that phalloidin does not exert toxic eff ect upon oral intake [2]. When chemical analysis was performed for 45 patients of

⊡ Figure 2.4

Structure of amanitin.

⊡ Figure 2.5

Structure of phalloidin. 474 Mushroom toxins

Amanita verna poisoning in France, amanitin could be detected from plasma in only 11 of 43 patients, from urine in 23 of 35 patients, from the contents of the stomach and duodenum in 4 of 12 patients and from feces in 10 of 12 patients [3]. Th e blood concentrations of amanitin are highly dependent on the intervals aft er ingestion; the concentrations in urine and the con- tents of the stomach and duodenum are much higher than those in blood, and these specimens are more suitable for analysis of amanitin [3].

Lampteromyces japonicus

Th is is one of the most common toxic mushrooms with the highest incidence of poisoning, like Rhodophyllus rhodopolius, in Japan. It is usually mistaken for the edible Lentinula edodes, Pleurotus ostreatus, Panellus serotinus or others. Th e shape of Lampteromyces Japonicus is semicircular or kidney-like; the size is as large as 10–25 cm. When it matures, the color of its cap part becomes purplish brown or dark brown. Th e stem is as short as 1.5–2.5 cm and located at a side part of the cap; there is a crater like protrusion in the reverse side of the cap just around the stem. When this part of the cap including the stem is cut, dark coloration can be observed there for the matured mushroom (> Figure 2.6), and the folds and hyphae lumi- nesce in a light yellow color in the dark; these are very useful for its discrimination. However, it should be cautioned that the above dark coloration is absent or obscure in the immature mushrooms. According to the growing circumstances, the Lampteromyces japonicus may show a round cap like Lentinula edodes, and thus is confusing (> Figure 2.7). Since the Lamptero- myces mushrooms can grow in colonies on the dead beech or maple trees, a great number of the mushrooms may be harvested at a single location. Th e harvester distributes them to neighbors and relatives, resulting in simultaneous occurrence of many poisoned patients. Its toxin is lampterol ( illudin S), which causes vomiting and diarrea. Th e fatality by the toxin is very rare.

⊡ Figure 2.6

How to discriminate Lampteromyces japonicus. Representative mushrooms causing poisoning cases 475

⊡ Figure 2.7

Lampteromyces japonicus mushrooms having circular umbrellas, which tend to be mistaken for edible Lentinula edodes mushrooms.

Magic mushrooms (> Figure 2.8)

Th e magic or hallucinogenic mushroom is a popular name for ones which exhibit hallucina- tion (visual and auditory), mental derangement and muscle fl accidness. In central and south America, such mushrooms were being used in religious ceremonies since ancient times. Th e hallucinogenic eff ects vary according to diff erent individuals; they are similar to those ob- tained with LSD, though they are much weaker than those of LSD. Th ey were illegally sold, in the forms of cultivation kits, dried pieces or tablets, on the streets and via the Internet before 2002. Various species of the Psilocybe genus are being used as magic mushrooms. Most magic

⊡ Figure 2.8

Cultivation of “magic mushrooms” (Psilocybe cubensis). 476 Mushroom toxins

⊡ Figure 2.9

Structures of psilocybin and psilocin.

mushrooms being circulated in Japan are Psilocybe cubensis and/or P. subcubensis and Cope- landia genus. Th e responsible toxins are psilocybin and psilocin. Th e psilocybin is metabolized into psilocin in human bodies (> Figure 2.9). From January 1997 to June 1999, 24 inquiries about magic mushrooms were received by the offi ce of Japan Poison Information Center [4]; the numbers of inquiries were 1 in 1997, 10 in 1998 and 13 in 1998 (6 months). An article entitled “Dangerous proliferation of hallucino- genic mushrooms” appeared in the Asahi morning newspaper on July 18, 1999. It described a case, in which a person had had a delusion of being capable of fl ying in the air, had jumped from a window of the 2nd fl oor and had been severely injured, and also a case, in which a uni- versity student had been mentally deranged on the campus; the article raised the alarm on such dangers. In January, 2001, there was a case, in which a youngster ate a grown magic mushroom, which had been purchased in the form of a cultivation kit via the Internet, and provoked hal- lucinatory symptoms to result in his death due to cold inside a roadside gutter in the nude. Accidents and incidents by ingestion of magic mushrooms are increasing recently; such abuse should be controlled strictly. In the United States and Japan, the possession, cultivation and intake of magic mushrooms have been completely prohibited recently.

Chemical analyses

For identifi cation of a mushroom, in addition to the morphological method using the observa- tions of its appearance and the form of its spores, chemical methods for analysis of toxins of mushrooms are also important. In this section, some examples of such chemical methods are described; especially, those for toxins of Amanita and Psilocybe mushrooms are presented.

Analysis of toxins of Amanita mushrooms

Th e toxins of Amanita mushrooms are usually analyzed by HPLC. As toxins, α-amanitin, β-amanitin, γ-amanitin and phalloidin are known. Th eir authentic standards can be purchased from Sigma (St. Louis, MO, USA). Chemical analysis 477 i. HPLC conditions (> Figures 2.10 and 2.11) Column: Inertsil OD-3 (150 × 4 mm i.d., particle size 5 µm, GL Sciences, Tokyo, Japan); mobile phase: 0.01 M ammonium acetate-acetic acid buff er solution (pH 5.0)/acetonitrile (84:16); its fl ow rate: 1.0 mL/min; detector: diode array detector (DAD); detector wavelengths: 302 nm for amanitin and 292 nm for phalloidin. ii. Extraction from a mushroom Aft er a mushroom is minced into small pieces with a knife or scissors, they are extracted with 3 mL of methanol/ water/0.01 M HCl (5:4:1) by shaking the mixture at 4 °C for 24 h.

⊡ Figure 2.10

HPLC chromatograms for amanitins and phalloidin. A 0.25-µg aliquot each of the compounds was injected into HPLC.

⊡ Figure 2.11

Tridimensional HPLC-DAD chromatograms for amanitins and phalloidin. 1: α-amanitin; 2: β-amanitin; 3: phalloidin. The amount of the compounds injected into HPLC was 0.25 µg each in an injected volume. 478 Mushroom toxins

Aft er centrifugation, the supernatant solution is condensed under a stream of nitrogen and injected into HPLC for analysis.

iii. Extraction from a body fluid i. A 5-mL volume of serum is mixed with 10 mL acetonitrile, shaken for 10 min and centri- fuged at 1,000 g for 10 min. ii. Th e supernatant solution is mixed with 30 mL dichloromethane, shaken for 20 min and centrifuged at 1,000 g for 5 min. iii. Th e supernatant solution is condensed under a stream of nitrogen and injected into HPLC for analysis.

Analysis of toxins of magic mushrooms (Psilocybe species)

For analysis of hallucinogenic toxins, such as psilocybin and psilocin, GC, GC/MS, LC and LC/MS are being used. Th e authentic standards of psilocybin and psilocin are not commer- cially available in Japan; the solution vials of psilocin can be imported aft er an appropriate procedure from Sigma, USA.

i. HPLC For HPLC, a spectrophotometric detector or an electrochemical detector (ECD)c can be used. If LC/MS or LC/MS/MS is available, analysis with much higher sensitivity and reliability can be realized. Here, an HPLC method with a relatively cheap and highly sensitive ECD detector is described [5]. Column: Inertsil ODS-3 (150 × 4 mm i.d., particle size 5 µm, GL Sciences); mobile phase: pH 3.8 buff er solution (300 mL of 0.1 M citric acid solution + 160 mL of 0.1 M sodium di- hydrogenphosphate solution)/ethanol (9:1); its fl ow rate: 1.0 mL/min; detector: ECD (+1.0 V).

ii. GC or GC/MS Aft er ingestion of psilocybin, it is easily metabolized into psilocin in human bodies. In a recent report [6], psilocin is said to exist in the glucuronide-conjugated form in human samples; they have insisted that enzymatic hydrolysis with glucuronidase is required before analysis. Psilo- cybin is dephosphorylated into psilocin in an injection chamber of GC at high temperature; TMS derivatization is required for GC or GC/MS analysis. Th e readers can refer to the refer- ence [6] on the details of the method. Scan range: m/z 50–550; retention index: 2,099; psilocin-di-TMS: m/z 290, 291 and 348.

iii. Extraction from a mushroom [5] i. A 300-mg aliquot of a mushroom is mixed with 30 mL methanol and homogenized. ii. Aft er shaking for 24 h, the homogenate is passed through a paper fi lter. iii. Th e clear solution is evaporated to dryness under a stream of nitrogen; the residue is dis- solved in 3.0 mL methanol and a 10-µL aliquot of it is injected into HPLC.

iv. Extraction from a dried mushroom [7] i. A 100-mg aliquot of a dried mushroom is mixed with 9 mL methanol and extracted by sonication for 120 min. Toxic concentrations 479 ii. Th e volume of the mixture is adjusted to 10 mL and centrifuged at 1,000 g for 15 min. An aliquot of the supernatant solution is injected into HPLC. v. Extraction from cerebrospinal fluid (CSF) [5] i. A 5-mL volume of CSF is mixed with 0.35 mL of 70 % perchloric acid solution, and centri- fuged at 1,000 g for 30 min. ii. Aft er decanting the supernatant solution, its pH is adjusted to 12 by adding 45 % KOH solution with cooling, followed by centrifugation at 1,000 g for 5 min. iii. Th e supernatant solution is mixed with 1 g NaCl, extracted with 6 mL dichloromethane by shaking for 15 min and centrifuged. Th e organic phase is transferred to another tube, and 6 mL dichloromethane is again added to the aqueous phase; the same extraction procedure is conducted. Th e resulting organic phases are combined. iv. Th e combined extract is dehydrated with anhydrous Na2SO4 and centrifuged at 1,000 g for 5 min. v. Th e organic extract is evaporated to dryness under a stream of nitrogen, and the residue is dissolved in 200 µL methanol. An aliquot of the solution is injected into HPLC. vi. Extraction from blood or urine [7] i. A 1-mL volume of blood or urine is mixed with 10 µL of β-glucuronidase (E. coli origin, Sigma) and incubated at 45 °C in a water bath with shaking for 1 h. ii. Th e mixture is diluted with 5 mL of 0.1 M potassium phosphate-NaOH buff er solution (pH 8) and poured into a Bond Elut Certify LRC 300 mg column (Varian, Harbor City, CA, USA). Th e column had been activated by passing 2 mL methanol and 2 mL of 0.1 M potassium phosphate-NaOH buff er solution (pH 8) in advance. iii. Th e above sample solution is poured into the column at a fl ow rate of 1–2 mL/min. Th ere- aft er, nitrogen gas is passed through the column to dry it. iv. Th e column is washed with 2 mL water, 2 mL of 0.2 M acetic acid-sodium acetate buff er solution (pH 4) and 2 mL of 30 % methanol aqueous solution. v. Aft er passing nitrogen gas through the column to dry it up, 2 mL of methanol/concen- trated ammonia solution (98:2) and 1 mL of the same solution are passed for elution of the target compound. vi. Aft er both solutions are combined, they are evaporated to dryness under a stream of ni- trogen with warming at 40 °C. vii. Th e residue is mixed with 50 µL of N-methyl-N-trimethyl- silyltrifl uoroacetamide (MSTFA), capped airtightly and heated at 80 °C for 15 min. viii. Aft er cooling to room temperature, an aliquot of the solution is injected into GC/MS.

Toxic concentrations

Although there are great variation in concentrations among references, there is a report [3] describing that the concentrations of α-amanitin and β-amanitin are 8–190 and 15.9–162 ng/mL in blood plasma, respectively. Amanitin usually disappears from blood about 36 h aft er in- gestion. Aft er oral ingestion of 10–20 mg (0.224 ± 0.02 mg/kg) of psilocin, its blood plasma con- centrations were reported to be 8.2 ± 2.8 ng/mL [7]. 480 Mushroom toxins

Conclusion

Th ere are some toxic mushrooms, the toxins of which are not clarifi ed; for such types of mush- rooms, chemical analysis is useless. Especially for Amanita neoovoidea, which has been found to be a toxic mushroom very recently, its toxin is only estimated to be a kind of peptides. Th e structure of the toxin remains to be clarifi ed. Upon analysis of mushroom toxins, the causative foods, such as miso soup and sukiyaki, even aft er being cooked, and/or the corresponding mushrooms should be obtained together with specimens of blood, urine and/or stomach contents. Raw mushrooms should not be frozen, because their forms are destroyed upon thawing; they should be stored in a refrigerator at 4 °C. Th ey should be transported as soon as possible to reach a laboratory for analysis. Th e morphological fi ndings of mushrooms themselves and their spores are very useful for interpre- tation of the results obtained by chemical analysis.

Notes

a) Th e numbers are based on only poisoning cases, which had been reported to a local public health center; the unreported cases are not included in the numbers. b) Upon totaling the numbers of each mushroom poisoning, the task may be undertaken by a nonexpert for mushrooms. Th erefore, it seems diffi cult to expect the exact classifi cation of mushrooms; the analogous species or genuses may be treated as a whole. For example, they are probably treated as Rhodophyllus rhodopolius plus Rhodophyllus sinuatus and Amanita virosa plus Amanita verna. In the Psilocybe argentipes mushrooms, other species of halluci- nogenic mushrooms may be included. c) HPLC-ECD is being widely used for sensitive analysis of catecholamines. Although it is not a common detector, it enables the detection of trace levels of compounds, which cannot be detected by the usual spectrophotometric detector. In addition, by using a CV stabilizer (CV 1000 Denken), clean chromatograms without noises or drift can be obtained.

References

1) Life Hygiene Bureau of the Ministry of Health, Labour and Welfare (ed) (1974–1997) National Record of Food Poisoning Incidents. Japanese Association of Food Hygiene, Tokyo (in Japanese) 2) Yamashita M, Furukawa H (1993) Mushroom poisonings. Kyoritsu-shuppan, Tokyo, p 110 (in Japanese) 3) Jaeger A, Jehl F, Flesch F et al. (1993) Kinetics of amatoxins in human poisoning. Therapeutic implications. J Toxicol Clin Toxicol 31:63–80 4) Madono K, Sakai Y, Hatano Y et al. (1999) Hallucinogenic “magic mushroom” poisoning: a report by JPIC. Jpn J Toxicol 12:443–447 (in Japanese) 5) Kysilka R, Wurst M, Pcakova V et al. (1985) High-performance liquid chromatographic determination of halluci- nogenic indoleamines with simultaneous UV photometric and voltammetric detection. J Chromatogr 320: 414–422 6) Sticht G, Kaferstein H (2000) Detection of psilocin in body fluids. Forensic Sci Int 113:403–407 7) Musshoff F, Madea B, Beike J (2000) Hallucinogenic mushrooms on the German market. Simple instructions for examination and identification. Forensic Sci Int 113:389–395