6.2
6.2
© Springer-Verlag Berlin Heidelberg 2005
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.  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 victims
a
. 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 speci ed, 35
(
> Figure 2.1)
b
.
Toxic mushrooms can be classi ed into 6 groups according to their actions as follows.
•  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).
•  ose which act on the autonomic nervous system and provoke symptoms, such as sweat-

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.
⊡ Figure 2.1
471
⊡ Table 2.1
Outline of mushroom poisoning analyses undertaken by Department of Legal Medicine, Akita University School of Medicine
No. Year Requesting institution Causative mushroom The patient
number
Outcome Specimen and detectability of the toxin
1 1993 H Univ. Dept. Legal Med. Amanita virosa 1 dead detected from the liver and the mushroom
2 1996 T Kyodo Hosp. Dept. Anaesth. Amanita virosa?
(mushroom not available)
1 dead not detected from blood, the liver or kidney
3 1996 Y Univ. Dept. Intern. Med Amanita virosa?
(mushroom not available)
1 dead not detected from blood
4 1997 D Univ. Emerg. Units Amanita virosa?
(mushroom available)
1 alive not detected from blood or the mushroom
5 1997 F Univ. Emerg. Units Amanita virosa?
(mushroom available)
1 alive not detected from blood stomach contents or
the mushroom
6 1998 A Pref. Hosp. Dept. Intern. Med. Agaricus blazei
(mushroom available)
2 alive not detected from blood or the mushroom
7 1998 O Pref. Hosp. Emerg. Units Amanita virosa
(mushroom available)
1 alive not detected from blood urine or the

16 2000 K Med. Univ. Emerg. Unit
and Dept. Pediat.
Amanita virosa
(mushroom available)
2 alive not detected from blood or urine, but
detected from the mushroom
17 2001 A Police H. Q. a magic mushroom (cultivated
with a culture medium)
1 dead detected from blood, urine and the
mushroom
Representative mushrooms causing poisoning cases
472 Mushroom toxins
Rhodophyllus rhodopolius.
⊡ Figure 2.2
Amanita virosa.
⊡ Figure 2.3
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.  e
Amanita genus mushrooms should be watched most carefully also in the forensic toxicological
point of view.  e main toxin of this genus is considered to be amanitin (
> Figure 2.4) or
phalloidin (
> Figure 2.5).  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.  ere is a report insisting that phalloidin does not exert
toxic e ect upon oral intake [2]. When chemical analysis was performed for 45 patients of
Structure of amanitin.
⊡ Figure 2.4

How to discriminate Lampteromyces japonicus.
⊡ Figure 2.6
475
Magic mushrooms (> Figure 2.8)
 e magic or hallucinogenic mushroom is a popular name for ones which exhibit hallucina-
tion (visual and auditory), mental derangement and muscle  accidness. In central and south
America, such mushrooms were being used in religious ceremonies since ancient times.  e
hallucinogenic e ects vary according to di erent individuals; they are similar to those ob-
tained with LSD, though they are much weaker than those of LSD.  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
Lampteromyces japonicus mushrooms having circular umbrellas, which tend to be mistaken for
edible Lentinula edodes mushrooms.
⊡ Figure 2.7
Cultivation of “magic mushrooms” (Psilocybe cubensis).
⊡ Figure 2.8
Representative mushrooms causing poisoning cases
476 Mushroom toxins
mushrooms being circulated in Japan are Psilocybe cubensis and/or P. subcubensis and Cope-
landia genus.  e responsible toxins are psilocybin and psilocin.  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 o 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  ying in the air, had jumped
from a window of the 2nd  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,

in an injected volume.
⊡ Figure 2.11
Chemical analysis
478 Mushroom toxins
A 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.  e supernatant solution is mixed with 30 mL dichloromethane, shaken for 20 min and
centrifuged at 1,000 g for 5 min.
iii.  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.  e authentic standards of psilocybin and psilocin are not commer-
cially available in Japan; the solution vials of psilocin can be imported a 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 bu 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  ow rate: 1.0 mL/min; detector: ECD (+1.0 V).
ii. GC or GC/MS
A er ingestion of psilocybin, it is easily metabolized into psilocin in human bodies. In a recent

and centrifuged at 1,000 g for
5 min.
v.  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.  e mixture is diluted with 5 mL of 0.1 M potassium phosphate-NaOH bu er solution
(pH 8) and poured into a Bond Elut Certify LRC 300 mg column (Varian, Harbor City,
CA, USA).  e column had been activated by passing 2 mL methanol and 2 mL of 0.1 M
potassium phosphate-NaOH bu er solution (pH 8) in advance.
iii.  e above sample solution is poured into the column at a  ow rate of 1–2 mL/min.  ere-
a er, nitrogen gas is passed through the column to dry it.
iv.  e column is washed with 2 mL water, 2 mL of 0.2 M acetic acid-sodium acetate bu er
solution (pH 4) and 2 mL of 30 % methanol aqueous solution.
v. A 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. A er both solutions are combined, they are evaporated to dryness under a stream of ni-
trogen with warming at 40 °C.
vii.
 e residue is mixed with 50 µL of N-methyl-N-trimethyl- silyltri uoroacetamide (MSTFA),
capped airtightly and heated at 80 °C for 15 min.
viii. A 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 a er in-
gestion.
A er oral ingestion of 10–20 mg (0.224 ± 0.02 mg/kg) of psilocin, its blood plasma con-

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