Researching mycotoxicosis, biological characteristics and toxicity of some common poisonous mushrooms in cao bang province

MINISTRY OF EDUCATION

MINISTRY OF NATIONAL DEFENSE

AND TRAINING

VIETNAM MILITARY MEDICAL ACADEMY

NGUYEN TIEN DZUNG

RESEARCHING MYCOTOXICOSIS, BIOLOGICAL
CHARACTERISTICS AND TOXICITY OF COMMON
POISONOUS MUSHROOMS IN CAO BANG
PROVINCE
Major: Pharmacology - Toxicology
Code : 62 72 01 20

SUMMARY OF MEDICAL PhD. THESIS

HANOI - 2015


This Work is completed in:
VIETNAM MILITARY MEDICAL ACADEMY

Scientific Supervisors:
•

Assc. Prof., PhD. HOANG CONG MINH

•

Assc. Prof., PhD. PHAM DUE

Opponent 1:

Prof., PhD. NGUYEN THI DU

Opponent 2:

Prof., PhD. TRINH TAM KIET

Opponent 3: Assc. Prof., PhD. NGUYEN TRONG THONG

This thesis shall be defended at the Academy-level Examiner
Board, meeting at Vietnam Military Medical Academy
At: ....... on date.....month...2016. 1

This thesis may be found out in:
•

National Library

•

Central Medical Library

•

Library of Vietnam Military Medical Academy

1

PREAMBLE
• Necessity of the thesis
The poisonous mushroom includes many kinds of species, each species
have different morphological characteristics, toxicity and effects on the
body. Mycotoxicosis is often due to people’s impossible distinguishing
between the poisonous mushrooms and non- poisonous mushrooms.
In the world, the mycotoxicosis occupied 2,1% in total poisonings. In
America, in 11 recent years, there has occurred 85.556 mycotoxicosises. In
Vietnam, the mycotoxicosis constantly occurs in forested provinces such as
Ha Giang, Cao Bang, Bac Kan, ... In March 2014, Poison Control Center at
Bach Mai Hospital has treated 15 patients with mycotoxicosis from Thai
Nguyen, Tuyen Quang, Cao Bang province. In which, 10 patients were died
(66.7%). Up to now, the research works on poisonous mushroom in
Vietnam are very few. Until before 2008, posters on mycotoxicosis
prevention of the Ministry of Health as well as of the province mainly had
relies on images of poisonous mushrooms that grow in the United State,
Europe… In which, some mushrooms only grow in temperate climate
zones, thus the propaganda effectiveness is not good.
Cao Bang is a mountainous province in the North, with diversified and
plentiful forest ecosystem. According to the Center for Preventive
Medicine, Department of Food Safety, Provincial Health Department, in the
period from 2003-2009, there had 29 cases of mycotoxicosis leading to 81
poisonous patients with 17 fatal cases. Especially, the mycotoxicosis
occurred in 8 mortal people in a family. Most poisonings have not been
identified the species of poisonous mushroom yet.
From the above issues, we have studied the subject: “Researching the
mycotoxicosis, biological characteristics and toxicity of some common

poisonous mushrooms in Cao Bang Province”
2. Objectives:
2.1. Assessing the mycotoxicosis in Cao Bang Province from 2003 to 2009
and results of the post-intervention mycotoxicosis from 2010 to June 2014.
2.2. Identifying the morphological characteristics and allocation of
some common poisonous mushrooms in Cao Bang Province.
2.3. Identifying the toxicity and changes in biochemical, hematological,
cardiovascular and histopathological criteria under the extract effect of 4
common poisonous mushrooms on animals.
3. New contributions for the thesis
- The first time a research is conducted to assess the mycotoxicosis in
Cao Bang Province from 2003 to 2009 and results of the post-intervention
mycotoxicosis from 2010 to June 2014.


2
- Identifying and describing the morphological characteristics and
allocation of 13 species of common poisonous mushrooms in Cao Bang
Province.
- Identifying the toxicity and changes in biochemical, hematological,
cardiovascular and histopathological criteria under the extract effect of 4
common poisonous mushrooms on animals. In which, the Chlorophyllum
molybdites cause most of poisonings, Amanita virosa can cause mortality,
Russula emetica has not been researched and the Coprinus atramentarius is
found out to grow in Cao Bang and not in any other locality.
4. Thesis Layout
This thesis is included 156 pages, including the following parts:
Introduction (2 pages), Overview (42 pages), Researching Object and
Methods (22 pages), Results (44 pages), Discussion (42 pages), Conclusion
(3 pages), Recommendations (1 page). This thesis contains 35 tables, 3

diagrams, 46 images, 144 references of which 19 Vietnamese references,
125 English references, 66 references since 2010 up to now.
CHAPTER 1- OVERVIEW
1.1. Definition
Poisonous mushrooms are the ones that contain toxin to poison the
human body and animals when ingested. Previously, these mushrooms were
sorted into the flora but today they are separated into the fungi. In the world
now there have nearly 140,000 species of mushrooms to be identified, of
which about 2,000 edible mushrooms, 700 species with active ingredients to
be used in disease treatment and many poisonous mushrooms. According to
Trinh Tam Kiet (1996), Vietnam has 826 large mushroom species to be
recorded, including 512 species newly discovered on the territory of
Vietnam. Some poisonous mushrooms are listed herein.
1.2 Classification
* Classification of poisonous mushrooms under the toxin contained in
the mushroom:
Poisonous mushrooms include many species with different
morphological characteristics, toxin composition and characteristics acting
on the body, so there are many classifications of poisonous mushrooms. The
American scientists (DW Fischer, Bessette AE-1992, Cope RB-2007) have
classified the poisonous mushrooms according to toxin contained in the
mushrooms. According to this classification, the poisonous mushrooms are
divided into 8 kinds: Amatoxin (cyclopolypeptid), gyromitrin
(monomethylhydrazin), orellanin, muscarin, ibotenic acid and muscimol,
coprin, psilocybin and psilocin, as the toxin causing gastrointestinal
disorder.


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1.3 Researches on poisonous mushrooms in the world

1.3.1 Researches on poisonous mushrooms with amatoxin
The poisonous mushrooms with amatoxin cause 90 - 95% fatal cases
due to mycotoxicosis in the world, so there have had many researches on
these fungi. Amatoxin is the common name of toxin contained in the
poisonous mushrooms in the genus Amanita, Galerina and Lepiota.
Amatoxin is contained in entire fruiting body of the mushroom (cap, gill,
stalk) and hyphae (mushroom roots).
Amatoxin includes 8 kinds: α-amanitin, β-amanitin, γ-amanitin, εamanitin, amanullin, amanullinic acid, proamanullin, amanin and 7 kinds
phallotoxin: phalloidin, phalloid, prophalloin, phallisin, phallacin,
phallacidin, phallisacin. Virotoxin found in these mushrooms.
1.3.2 Researches on poisonous mushrooms with muscarin
The group of mushrooms with muscarin often found out in the
mushrooms of genus Inocybe, Clitocybe and Omphalotus. The genus of
Inocybe: Inocybe patouillardi; Inocybe fastigiata (Inocybe rimosa),. .. The
genus of Clitocybe: Clitocybe dealbata, Clitocybe cerussata,… The genus
of Omphalotus: Omphalotus olearius; Omphalotus illudens…
All mushrooms in the genus of Inocybe have toxin. Previously, it was
thought that the Amanita muscaria caused poisoning symptoms muscarin.
However, making quantitative analysis of active substances in the
mushroom Amanita muscaria, the muscarin content in the Amanita
muscaria is very low (about 0,0003% fresh weight) so it can not enough to
cause poisoning despite eating the large quantities. The mushrooms in genus
of Inocybe and Clitocybe contain high quantity muscarin.
1.3.3. Researches on poisonous mushrooms with coprin
The group of poisonous mushrooms with coprin mostly in genus
Coprinus. Some fungi can cause poisoning as: Coprinus atramentarius,
small Coprinus atramentarius grew into clump (Coprinus disseminatus),
Coprinus micaceus, Coprinus fuscescens, Coprinus insignis.... Besides, the
mushroom Clitocybe clavipes in genus of Clitocybe may cause poisoning
similar to the mushroom with coprin although coprin has not been found out

in this mushroom.
1.3.4. Researches on Chlorophyllum molybdites, the toxin causing
gastrointestinal disorder.
Chlorophyllum molybdites, as the fungi cause most cases of poisoning
in many countries in the world and some provinces in Vietnam. Until 2004
Kobayashi Y. and CS (Japan) conducted to extract and refine from this
mushroom a kind of lectin as N-Glycolylneuraminic acid. In 2009 – 2010,
Gong Q.F. and CS separated 4 compounds from the hyphae (root) of this


4
mushroom by 5,6,(22E,24R)-5α,6α-epoxyergosta-8, 22-diene-3β,7α-diol,
(22E,24R)-ergosta-7,22-dien-3β-ol. Yamada M. and CS (2012), has
extracted a kind of toxic protein and named molybdophyllysin. Yoshikawa
K. (2001), has extracted 2 derivatives steroid as (22E,24R)-3α-ureidoergosta-4, 6, 8 (14), 22-tetraene and (22E,24R) -5α, 8α-epidioxyergosta6,9,22-triene-3β-ol-3-O-β-D-glucopyra-noside.
1.4 . Researches on poisonous mushroom in Vietnam
1.4.1. Researches on characteristics, allocation, toxicity of poisonous
mushrooms
The large studies on mushroom in Vietnam are primarily of biologists
and pharmacists on mushroom identification, determining the allocation in
different ecological zones and studies on farming breeding of fungi as the
food and pharmaceuticals. The “List of large mushrooms in Vietnam”
(1996) of Trinh Tam Kiet made a list of names and allocations of 826 kinds
of large mushrooms, in which there had names of about 20 kinds of
poisonous mushrooms. Tran Cong Khanh and Pham Hai (2004)
morphologically describe the common poisonous mushrooms. From 2007 –
2008, Hoang Công Minh and co-workers conducted to investigate and
identify the mushrooms causing poisoning in Ha Giang province. In this
study, 9 kinds of poisonous mushrooms have been found. In which, there
have 2 kinds of poisonous mushrooms. In Ha Giang province, the

mushroom with amatoxin causing dealth has been detected as the Amanita
verna. Hoang Cong Minh (2009) has studied the effect of the fungus extract
on rabbits and found that they have had AST, ALT, billirubin, ure, creatinin
increased highly, erythrocytes and hemoglobin decreased, coagulation
disorder after poisoning.
CHAPTER 2
RESEARCHING OBJECT AND METHODS
2.1 OBJECTS
* 93 patients who were suffered mycotoxicosis in the localities in Cao
Bang Province involved in this research. In which, 81 patients in the period
from 2003 to 2009 as there has no any communication interventions and 12
patients from 2010 to June 2014 after having communication interventions.
* Some samples of poisonous mushrooms growing in some
representative areas in Cao Bang Province.


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* Experimental animals:
- White mice in Swiss origination: 1280 mice, healthy, average weight
by 20 ± 2 gam (excluding the white mice used for survey of poisoning
dose). The white mice used to identify toxicity (Average lethal dose - LD50)
and making histopathological study for four species of poisonous
mushrooms.
- Rabbit: 60 rabbits, healthy, weight 2,0 ± 0,2 kg (excluding the rabbits
used to identify the minimum lethal dose (LDmin). Rabbits used to study the
biochemical and hematologic criteria of 4 kinds of mushroom.
- White rat in Wistar origination: 60 rats, healthy, weight 200 ± 20 gam
(excluding the white rats used to identify the minimum lethal dose (LDmin).
The white rat used to research the pulse, blood pressure for 4 kinds of
mushroom.

2.2. METHODS
2.2.1. Survey methods for the mycotoxicosis
Stage 1 from 2003 to 2009, making survey for the poisonings of
poisonous mushroom under method of cross survey, retrospective records,
data, interviewing people suffered mycotoxicosis and people in information
collection sheet in the family people suffered mycotoxicosis in the localities
in Cao Bang Province. Stage 2 from 2010 to June 2014, surveying the
mycotoxicosis under statistic report of forest mycotoxicosis, poisonous
plants of Food Hygiene and Safety Department, Cao Bang Department of
Health (after communication inventions).
2.2.2 Surveying methods of poisonous mushrooms
Making survey of poisonous mushrooms in Surveying Form in the
field, where people picked the mushroom as the food and being poisoned. In
the localities without mycotoxicosis, we came to the areas where grow
many kinds of mushrooms as directed by the government, officer of medical
station and people in the commune.
2.2.3 Identifying methods of kinds of mushroom
Kinds of mushroom are identified under method of Trinh Tam Kiet,
Kuo M., identified basing on the morphological characteristics, spores, and
chemical reactions when compared with the standard mushroom sample.
The characteristics to be described as: Cap, gill, ……
2.2.4 Methods to research toxicity, extract effect of 4 kinds of poisonous
mushroom on animals
2.2.4.1 Methods to extract poisonous mushroom sample
♦ Extracting methods for dried mushroom:


6
• Dried mushroom, putting into weighting, crushed into powder and
put into the bottle. Depending on the kind of fungi to put methanol, water

soaking for 24 hours. Extracting and taking the whole solvent. Continue to
put methanol, water into the leaching tank and extract with 2 more with the
same way as to totally extract the active ingredient in the dry mushroom
samples.
• Gather all the solvent into a bottle, aeration to blow the solvent
evaporation to collect the residue. Residue in the bottle is the total amount
of active ingredients of the fungi, weighting the residue and calculating the
equivalent initial weight of the mushroom.
• Preparation of residue with distilled water to form extraction
solution. Before the animals used orally or by injection into abdomen, the
extract is boiled in a test tube, leave it cold and to ensure aseptic.
♦ Extracting method for fresh mushroom:
• Sample of fresh mushroom is stored in the alcohol 700 (weighting the
mushroom before soaking in the alcohol). Sampling the mushroom, alcohol
from the soaking bottle to the ceramic bowl, crushing into the suspension.
Distilling and taking the suspension into the separated bottle. Residue left in
the bottle is spread with a certain amount of distilled water and placed in a
ceramic bowl. Continue to put water into the ceramic bowl with mushroom
and mashed with water, filtered as above on 2nd, 3rd time to totally extract
the active ingredients.
• Gathering the whole extract, filtered through filter paper to obtain the
extract containing active substances of poisonous mushrooms. Aeration for
evaporation of alcohol and steam to obtain the active ingredient of the
extract residue. Weighing the residue and calculating the equivalent initial
weight of the mushroom.
• Residue of extracts is formulated to research on animals. Ensure
sterile extracts by boiled and leave it cold before use orally or injecting into
the animals.
2.2.4.2 Methods of researching acute toxicity of 4 poisonous mushrooms
on the white mice.

♦ Method causing poisoning on the white mice:
* Method causing acute poisoning through the digestion: Use a
dedicated tool to pump extracts of 4 kinds of poisonous mushroom to be
researched: Amanita virosa, Chlorophyllum molybdites, Russula emetica
and Coprinus atramentarius into the stomach of the white mice.
* Method causing acute poisoning by injection into the abdomen just to
be studied for Amanita virosa because the toxicity of these kinds of


7
mushroom are amatoxin poorly absorbed from the digestion of the white
mice, thus out of the digestion, the toxicity by injection into the abdominal
of the white mice will be further researched:
♦ Method identifying the average lethal dose (LD50):
LD50 white mice is identified in method of Karber G.
2.2.4.3 Method of conducting biochemical and hematological criteria
The biochemical and hematological criteria are studied at the time
before and after being poisoned in the morning at the day 1, 5 and 10 after
causing poisoning.
Get the vein blood in the rabbit ear, 2 ml for each into the test tube to
identify the biochemical and hematological criteria before causing
poisoning. Causing poisoning rabbit at the dose by 2/3 lethal dose (LDmin)
in each kind of poisonous mushroom (surveyin the dose before experiment).
At this dose, the rabbit is poisoned but not be in death to follow and take
blood for experiment at the time of after being poisoned. Specifically for
each kind of poisonous mushroom causing poisoning on experimental
rabbits:
+ Dried Amanita virosa: 0,618 g/kg body weight by abdominal
injection.
+ Dried Chlorophyllum molybdites: 5,734 g/kg body weight used

orally.
+ Dried Russula emetica: 6,628 g/kg body weight used orally.
+ Dried Coprinus atramentarius: 4,504 g/kg body weight used orally
with 5 ml alcohol 400/kg body weight.
+ Specifically for Coprinus atramentarius, making group for rabbits
drinking alcohol 5 ml 400/kg body weight.
- Centrifugally the blood to take serum. Putting the serum tube into the
biochemistry analyzer (CHEMIX-180), automatic hematology analyzer (XE
2100) (Japan) at biochemical and hematological laboratory of Research
Centre for Military Medicine and Pharmacy of Vietnam Military Medical
Academy, to define the testing criteria.
2.2.4.4 Researching cardiovascular criteria on the white rat of 4 kinds of
poisonous mushroom
♦ Methodology for cardiovascular criteria:
Pulse, blood pressure of the rate measured at the time of before and
after being poisoned at the time of 1 hour, 6 hours and 24 hours.


8
Pulse, blood pressure rat tail determined on dedicated devices
automatically measuring the pulse, blood pressure of the rat tail of Ugo
Basile (Italy) of the subject Military Toxicology and Radioactive Studies of
Vietnam Military Medical Academy.
2.2.4.5 Researching Methodology of histopathology as liver, kidneys,
spleen
Amanita virosa, Chlorophyllum molybdites, Russula emetica and
Coprinus atramentarius, researched the histopathology on general situation
of and microbials in the collation group and group causing poisoning on the
white mice with a dose by 1 average lethal dose (LD50), specifically:
+ Dried Amanita virosa: 0,322 g/kg body weight by abdominal injection.

+ Dried Chlorophyllum molybdites: 3,718 g/kg body weight used orally.
+ Dried Russula emetica: 4,838 g/kg body weight used orally.
+ Dried Coprinus atramentarius: 2,976 g/kg body weight used orally
and with 5 ml alcohol 400/kg body weight.
+ Specifically for Coprinus atramentarius, making group for rabbits
drinking alcohol 5 ml 400/kg body weight.
The steps as follow:
• Killing rats and dissecting to take liver, kidneys, spleen and putting
into the bottle containing a fixed solution. Casting paraffin block, slicing
with thickness 5-6 microns on a Microtome. Dyeing slices under
hematoxylin – eosine staining method. Conducting morphological
observation of specimens on microscope. Color photography for illustration.
The experimental part to cause poisoning, dissecting to take liver,
kidneys, spleen and putting into the fixed solution conducted in the Military
Toxicology and Radioactive Studies, Vietnam Military Medical Academy.
Technique to case paraffin block, slicing, staining specimen, observing
the lesion morphology in the microscope, reading the results and imaging
were conducted at the Department of Disease Anatomy and Forensic
Medicine, Military Hospital 103.
2.3 Data processing
The data is processed with statistical method, using software as Excel,
Epical 2000, EpiInfo 3.5.4.
2.4 Ethical issues in research
Assuring of ethics in research.


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CHAPTER 3: RESEARCHING RESULTS
3.1 The mycotoxicosis in Cao Bang Province

No. of Poisonings
No. of Sufferers
No. of Mortality

Diagram 3.1: Allocation of number of poisoning, sufferers and mortality
due to eating poisonous mushrooms
Table 3.1: Allocation of number of poisonings, people being poisoned and
mortality due to eating poisonous mushrooms in the districts in Cao Bang
Province.
No.
1
2
3
4
5
6
7
8
9
10
11

District and
town

Poisonings

Thach An
Bao Lac
Tra Linh

Nguyen Binh
Hoa An
Ha Lang
Ha Quang
Phuc Hoa
Trung Khanh
Bao Lam
Thong Nong
Total

6
4
2
3
5
2
2
2
1
1
1
29

Number of
people
being
poisoned
18
17
10

7
6
6
6
5
4
1
1
81

Rate
(%)

Number of
people in
death

22,22
20,99
12,35
8,64
7,41
7,41
7,41
6,17
4,94
1,23
1,23
100

1
5
8
0
1
0
0
0
2
0
0
17

3.2 INVESTIGATION RESULTS OF POISONOUS MUSHROOMS IN
CAO BANG PROVINCE
3.2.1 List and allocation of poisonous mushrooms in Cao Bang Province


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The research has detected 13 kinds of poisonous mushroom. The poisonous
mushrooms growing in many communes and districts are distributed in Cao
Bang Province. Some mushrooms are detected to be available in all districts
such as Chlorophyllum molybdites. Some mushrooms are detected to be
grown in some investigated localities such as Inocybe fastigiata and
Amanita virosa.
Table 3.10: List and allocation of poisonous mushrooms in localities.
1

Vietnamese name
(other name)

Amanita verna

2

Amanita virosa

3

Inocybe
fastigiata

4

Chlorophyllum
molybdites

5

Russula emetica

Russula emetica (Schaeff)
Fr., Family: Russulaceae

6

Russula foetens

Russula foetens (Pers.) Fr.,
Family: Russulaceae

7

Scleroderma
citrinum

Scleroderma citrinum Pers.
or Scleroderma aurantium

No.

Scientific name; Family
Amanita verna (Bull.:Fr)
Roques. Family:
Amanitaceae

Amanita virosa Lam. Ex
Secr., Family:
Amanitaceae
Inocybe fastigiata
(Schaeff.:Fr) Quel or
Inocybe rimosa (Bull.:Fr)
Kumm. Family:
Cortinariaceae
Chlorophyllum molybdites.
Family: Lepiotaceae

Allocation
Quang Vinh Commune, Tra
Linh District. Chi Vien
Commune, Trung Khanh

District
Phan Thanh Commune, Bao
Lac District.
Hong Nam Commune, Hoa
An District.
Le Lai, Minh Khai
Communes, Thach An District
Area 7, Bao Lac Town

All districts are investigated
and the most in Hoa An,
Thach An, and Ha Lang
Districts
Chi Vien Commune, Trung
Khanh District
Cach Linh Commune, Phuc
Hoa District
An Lac, Thi Hoa, Ha Lang
Communes
Co Ba Commune, Bao Lac
District
Ngoc Khe Commune, Chi
Vien Commune, Trung Khanh
District.
Dao Ngan Commune, Ha
Quang District
Yen Tho Commune, Bao Lam
District
Chi Vien Commune, Trung
Khanh District

11
Pers. Family:
Sclerodermataceae

8

Agaricus
xanthodermus

Agaricus xanthodermus
Genev. Family:
Agaricaceae

9

Panaeolus
papilionaceus

10

Panaeolus
retirugis

11

Panaeolus
campanulatus

Panaeolus papilionaceus
(Bull. Ex Fr.) Quel.,
Family: Coprinus
atramentarius
(Coprinaceae)
Panaeolus retirugis (Fr.)
Quél. Family: Coprinus
atramentarius
(Coprinaceae)
Panaeolus campanulatus
(Bull.:) Quél, or:
Panaeolus sphinctrinus
(Fr.) Quél., Family:
Coprinus atramentarius
(Coprinaceae)

12

Coprinus
atramentarius

Coprinus atramentarius
(Bull.: Fr) Fr. Family:
Coprinus atramentarius
(Coprinaceae)

13

Coprinus
atramentarius

Coprinus disseminatus
(Pers. ex Fr.) S. F. Gray
Family: Coprinus
atramentarius
(Coprinaceae)

Le Lai Commune, Thach An
District
Son Lo, Co Ba, Bao Lac
Communes
Minh Tam, Hung Dao
Communes, Nguyen Binh
District
Ngoc Khe Commune, Trung
Khanh District Thi Hoa
Commune, Ha Lang District
Co Ba Commune, Bao Lac
District
Cach Linh, Trieu Au
Communes, Phuc Hoa
District.
Bao Toan, Son Lo – Bao Lac
Commune
Dao Ngan Commune, Ha
Quang District.
An Lac, Thi Hoa, Ha Lang
Communes.
Chi Vien Commune, Trung
Khanh District

Le Lai - Thach An
Communes. Quang Lam - Bao
Lam Communes. Thi Hoa Ha Lang Communes.
Minh Tam – Nguyen Binh
Communes.
Minh Khai – Thach An
Communes.
Minh Tam, Hung Dao
Communes, Nguyen Binh
District
Dao Ngan Commune, Ha
Quang District.
Truong Ha Commune, Ha
Quang District.
Yen Tho Commune, Bao Lam
District.
An Lac, Thi Hoa, Communes,
Ha Lang District.


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3.3 TOXICITY RESEARCHING RESULTS OF SOME POISONOUS
MUSHROOMS ON EXPERIMENTAL ANIMALS
3.3.1 Researching results on Amanita virosa
* Acute toxicity of Amanita virosa:
Table 3.11: LD50 of Amanita virosa for the white mice
Toxicity indicator
LD50 by abdominal injection for dried mushroom
LD50 by abdominal injection for fresh mushroom
LD50 via ingestion for dried mushroom

LD50 via ingestion for fresh mushroom

Weight of mushroom
(g/kg body weight)
0,322
3,270
3,896
28,632

* Researching results on extract effect of Amanita virosa on some
biochemical tests on rabbit:
Table 3.12: Change in some biochemical tests on the rabbit ( X ±SD; n = 10)
After being poisoned (day)
Before being
Criteria
poisoned
1
5
10
AST
50,4 7,8
226,8 25,4 1093,9 112,6 435,4 51,7
(U/l)
p <0,001
p <0,001
p <0,001
ALT
62,3 9,5
432,7 51,2
798,5 90,3

371,9 45,6
(U/l)
p <0,001
p <0,001
p <0,001
GGT
18,2 3,4
75,4 8,0
113,6 12,3
45,2 5,4
(U/l)
p <0,001
p <0,001
p <0,001
Bilirubin TP
4,77 0,65
9,22 0,97
47,38 5,36
22,45 2,38
(mol/l)
p <0,001
p <0,001
p <0,001
Glucose
6,81 0,55
7,94 0,82
4,13 0,49
5,42 0,59
(mmol/l)
p <0,01

p <0,001
p <0,001
Ure
5,73 0,67
7,56 0,81
14,71 1,63
9,24 0,82
(mmol/l)
p <0,001
p <0,001
p <0,001
Creatinin
66,8 6,91
95,9 9,67
172,3 18,77 99,7 11,59
(mol/l)
p <0,001
p <0,001
p <0,001
* Researching results of effect of extracts Amanita virosa on
hematological criteria on rabbit:


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Table 3.13: Change in quantity of erythrocytes, platelets, leukocytes,
hemoglobin content ( X ± SD; n = 10)
Criteria
Before being
After being poisoned (day)
poisoned

1
5
10
Erythrocytes
4,62 0,41
4,97 0,56
3,41 0,52
3,89 0,42
(T/L)
p>0,05
p<0,001
p<0,01
Hemoglobin
97,3 9,2
111,0 12,3
76,7 8,2
88,5 9,0
(g/l)
p<0,05
p<0,001
p<0,05
Platelet
284,6 31,5
295,2 34,8
194,5 26,7 234,6 31,9
(G/L)
p>0,05
p<0,001
p<0,01
Number of

8,65 0,81
13,04 1,42 11,34 0,98
9,45 0,93
leukocytes (G/L)
p<0,001
p<0,001
p>0,05
* Image of histopathology on liver, kidneys, spleen of the white mice
being poisoned with Amanita virosa in day 10 after being poisoned.
+ Image of histopathology (general situation of and microbials) liver of
the white mice:

Image 3.31: Image of general
situation of liver of the normal
white mice.

Image 3.32: Image of general situation
of the liver of the white mice being
poisoned with Amanita virosa

The liver cell kernel is broken and clotted

Hemorrhage
area

Necrosis of
liver cells

Image 3.35: Image of liver of the white mice being poisoned with Amanita
virosa. HE, 400x

14
+ Image of histopathology (general situation of and microbials) of the
white mice

Image 3.36: Image of general
situation of of the normal white
mice.

Image 3.38: Image of covering
microbials of the normal white
mice. HE, 400x.

Image 3.37: Image of general
situation of of the white mice being
poisoned with Amanita virosa.
Glomus tumor

Image 3.39: Image of atheist microbials
of the white mice being poisoned with
Amanita virosa. HE, 400x

+ Image of histopathology (general situation of and microbials) spleen
of the white mice being poisoned with Amanita virosa

Image 3.43: Image of normal
general situation of spleen of the
white mice.

Image 3.44: Image of general
situation of spleen of the white mice
poisoning Amanita virosa


15
White
pulp

Normal
white pulp
area

Congestion
of red pulp
area

Normal red
pulp

Image 3.45: Image of spleen microbials
of the normal white mice. HE, 200x.

Image 3.46: Image of spleen
microbials of the white mice
being poisoned with Amanita
virosa. HE, 200x.

3.3.2 Researching results of Chlorophyllum molybdites
* Researching results of acute toxicity of Chlorophyllum molybdites

Table 3.16: LD50 for the white mice of Chlorophyllum molybdites
Weight of mushroom
Toxicity indicator
(g/kg body weight)
LD50 via ingestion for dried mushroom
3,718
LD50 via ingestion for fresh mushroom
35,253
3.3.3 Researching results of Russula emetica
* Researching results of acute toxicity of Russula emetica
Table 3.21: LD50 for the white mice of Russula emetica
Mushroom weight
Toxin criteria
g/kg body weight
LD50 via ingestion for dried mushroom
4,838
LD50 via ingestion for fresh mushroom
41,326
3.3.4 Researching results of Coprinus atramentarius
* Researching results of acute toxicity of Coprinus atramentarius
Table 3.26: LD50 for the white mice of Coprinus atramentarius
Weight of mushroom
Toxicity indicator
(g/kg body weight)
LD50 via ingestion dried mushroom
2,976
LD50 via ingestion fresh mushroom
34,248

16
CHAPTER 4: DISCUSSION
4.1 MYCOTOXICOSIS IN CAO BANG PROVINCE
* Statistics of poisonings of poisonous mushroom:
The statistical results of mycotoxicosis in Cao Bang Province at table
3. The resultd showed that from 2003 to 2009, when there were not any
communication interventions, Cao Bang province had 29 poisonings which
caused by poisonous mushroom with 81 sufferers, 17 deaths (20,99%),
averagely 11,57 sufferers/year, much higher compared with the stage from
2010 to June 2014 as deploying the communication interventions: 06
poisonings with poisonous mushroom, 12 sufferers, 01 deaths (8,3%),
averagely 2,4 sufferers/year.
• Propaganda on prevention of mycotoxicosis for ethnic minorities in
communes is still ineffective. The media like the leaflets, posters on
mushrooms..., not the kinds of mushrooms grow locally, it’s easy to confuse
with the kinds of edible mushrooms. From 2010 to June 2014 as the
communication interventions were synchronously deployed such as:
Pictures, leaflets with image of mushrooms growing in Cao Bang Province,
the medical staffs were trained on diagnosis, treatment of mycotoxicosis,
contributing to decrease the mycotoxicosis, sufferers and the rate of
mortality due to poisonous mushrooms in Cao Bang Province.
* Epidemiology:
The patients in research are available over the districts of Cao Bang
Province. Researching results in table 3.2 showed that, the mycotoxicosis
occurs in 11/12 districts in Cao Bang Province. In March 2014, there had 2
patients in Quang Uyen District suffered the mycotoxicosis, 1 patient in
death on day 5st of poisoing. Thus, the poisonings of poisonous mushroom
have occurred almost districts and towns in Cao Bang Province.
4.2 INVESTIGATION RESULTS OF POISONOUS MUSHROOMS IN
CAO BANG PROVINCE

* Investigation results of the poisonous mushrooms:
Cao Bang is a border province with many forests and kinds of
mushrooms. According to the domestic and foreign authors, the same
poisonous mushroom but growing in different climates and soils, then the
shape, size, color may also be different. Through investigation, it is found
out 13 kinds of poisonous mushroom, in which there are 2 kinds of
mushroom with amatoxin as Amanita verna, Amanita virosa, 1 kind of
mushroom with muscarin as Inocybe fastigiata or Inocybe rimosa and 5


17
kinds of mushroom causing digestive disorder as Chlorophyllum
molybdites, Russula emetica, Russula foetens, Scleroderma citrinum,
Agaricus xanthodermus, 3 kinds of mushroom with psilocybin and psilocin
cause psychosis as Panaeolus papilionaceus, Panaeolus retirugis,
Panaeolus campanulatus, 2 kinds of mushroom with coprin (only causing
poisoning if accompanied by alcohol) as Coprinus atramentarius and small
Coprinus atramentarius grow in clump (Coprinus disseminatus).
Allocation of 13 kinds of poisonous mushroom is very different. Many
kinds of mushroom are detected in most of investigated districts such as
Chlorophyllum molybdites. However, there also had many kinds of
mushroom found out in some localities such as: Inocybe fastigiata found out
to grow in Bao Lac. Amanita virosa found in Thach An District.
4.3 RESEARCHING RESULTS ON EFFECT OF SOME
POISONOUS MUSHROOMS ON EXPERIMENTAL ANIMALS
4.3.1 Toxicity and effect of Amanita virosa on experimental animals
* Toxicity of Amanita virosa:
Researching results on toxicity of Amanita virosa picked in Cao Bang
Province (table 3.11) showed that LD50 of Amanita virosa for the white
mice by abdominal injection by 0,322 g/kg weight for dried mushroom and

3,270 g/kg weight for fresh mushroom. Through ingestion by 3,896 g/kg
weight for dried mushroom and 28,632 g/kg for fresh mushroom. From
above result, it is showed that Amanita virosa has high toxicity for the white
mice by abdominal injection and low toxicity via ingestion. According to
the foreign authors, toxicity of Amanita virosa is amatoxin and these are
cyclopolypeptid (including: alpha-amanitin, beta-amanitin, gammaamanitin, epsilon-amanitin, amanullin, amanullinic acid, proamanullin).
Toxicity of Amanita verna for animals is different due to the different
toxicity absorption in gastrointestine and different characteristics of
individual. The toxicity absorption of mushrooms cyclopolypeptid in the
gastrointestine for the white mice and rats happened very slowly. It is
possible that gastrointestine in the rat has no catalyst (carrier) attached with
molecule cyclopolypetid or concentration of these substances is very low
then the absorption of toxicity occurred slowly. In the foreign countries,
researches on toxicity of amatoxin on the white mice or rats are oftern
carried out via abdominal injection. Different from the white mice and rats,
the other animals such as dogs, cats, guinea pigs rapidly absorbed the
molecule cyclopolypeptid, thus toxicity of Amanita verna via gastrointestine


18
for these kinds of mushroom is very high. According to the foreign authors,
the lethal dose of amatoxin for people via ingestion about 0,1 mg/kg body
weight. For dogs, cats, guinea pigs the LD50 about 0,3 - 0,5 mg/kg body
weight. Concentration amatoxin in Amanita virosa is in range of about 5 15 mg/ 40 gam fresh mushroom. So, it is possible to cause death if only
eating a plant of Amanita verna.
Researching results of toxicity of Amanita virosa showed that these
kinds of mushroom have toxicity in either dried or fresh form. Amatoxin in
the mushroom is the stably-thermal toxicity (toxicity is not lost in case of
boiling for several hours) and storage in dry form via 10 years, the toxicity
keeps unchanged.

* Effect of extracts of Amanita virosa on some biochemical criteria
Researching results of at table 3.12 showed that: AST, ALT, GGT and
bilirubin entirely in the serum of rabbit being poisoned with Amanita virosa
increase and higher than before mycotoxicosis. Compare activity of AST
and ALT before anf after poisoning in research show that, on the first day of
research, activity of AST serum increased 4-time higher and ALT increased
7-time higher. On 5st day after being poisoned, activity of AST serum
increased 21-time higher and ALT increased 13-time higher and activity
AST increased higher than ALT. This proved that the liver cells suffer
seriously damage, necrosis in the liver cells. When the liver cells are in
necrosis, the mitochondria has been destroyed and made AST from the
mitochondria escaping from the blood plus AST in the Cytoplasm leading to
the AST serum increased higher than ALT. Results of our researching are
also similar with the foreigner authors, that the patients being poisoned with
kinds of mushroom containing amatoxin like Amanita verna, Amanita
virosa, Amanita phaloides… injury of the liver became seriously with
expressions of destroying the liver cells, dysfunction of liver, depletion of
clotting factors especially coagulation factors generalized by the liver and
subsidiary coagulation factors vitamin K. Injury due to toxicity of
mushrooms may cause consequences of coagulopathy, bleeding, liver coma,
multi-organ failure. Previously, almost patients getting liver failure due to
poisonous mushrooms were in mortality.
- In term of effect of extracts of Amanita virosa on on some criteria to
assess kidney function, researching results at table 3.14 showed that ure
concentration and creatinine in the blood on the rabbit being poisoned with
acute Amanita virosa highly increased for statistical significance (p<0,001)
compared with the moment before poisoning at all researching times on 1st


19

day, 5 st day and 10 st day after being poisoned. Therefore, toxicity of
Amanita virosa causes injury to the kidney cells.
- In term of effect of extracts of Amanita virosa on glucose
concentration:
Researching results showed that glucose concentration in the blood
increased compared with the time before poisoning in day 1 and decreased
in day 5, day 10 after being poisoned. Glucose concentration decreased may
be due to the liver injury and lead to decreasing the generalization of
enzymatic catalysis of glycogen in the liver and glycogen resolution
process in the liver became disorder, the liver may be in necrosis and make
the glycogen concentration in the liver cells decreased, after being poisoned,
the animals stop eating then the feeding supplied from outside for the body
shall decrease and amatoxin cause injury gastrointestine and thereby
limiting the absorption of glucose. Our researching results are also similar
with other ones as Floersheim G.L. (1987) and Chan A.K. (2007) that the
glucose concentration in the blood decreased in case of poisoning
mushroom with amatoxin and in some cases glucose in the blood may
decrease very low.
* In term of effect of extracts of Amanita virosa on hematological
criteria
Researching results at table 3.13 showed that: Number of erythrocyte,
hemoglobin concentration and platelet in the rabbit being poisoned with
Amanita virosa decreased for statistical significance (p<0,05) compared
with the time before poisoning in 5 st day and 10 st day after being poisoned.
One of the characteristics of mycotoxicosis containing amatoxin was long
bleeding in organs. According to Floersheim G.L. (1987) the poisonings of
amatoxin at the serious level mostly had bleeding in the internal organs. The
cases strongly decreasing the clotting factors often have a poor prognosis.
* On histopathological image of liver, kidneys, spleen of the white
mice being poisoned with extracts of Amanita virosa:

- On image of general situation of the liver, kidneys, spleen of the mice
being poisoned with Amanita virosa (image 3.32; 3.37; 3.44) clearly
showed the organs to be edema, dark brown, limp and less elastic. With the
naked eye we may see the pattern of these organs changed when compared
with the collation group.
- On image of microbials:


20
+ For the liver: Image of microbials liver of the white mice being
poisoned with Amanita virosa (image 3.35) clearly showed the necrosis in
the liver cells with the damage and degeneral of nuclear, congestive and
bleeding
The liver cells are very sensitive with amatoxin and liver may be
considered as the key target organ for this kind of toxicity. As entering into
body, the toxicity of mushrooms will penetrate inside the liver cells and
disrupt information ARN synthesis and lead to disorder of protein-enzyme
synthesis, finally killing cells. In addition, amatoxin starts the factors
activating the death under cycle of the cell (apoptosis) and creating necrosis
hole, under the microscope it may clearly see that the cell kernels has been
shattered into pieces, the cells have not had kernel (kernel dissolved). Final
result is liver failure, coagulopathy, bleeding of organs. Thus, observation
of liver organization under the microscope will see bleeding mass scattered.
Researching results of histopathology are also suitable with biochemical
blood tests to evaluate liver function (activity enzym AST, ALT, billirubin
entirely increased very highly).
+ Researching results of histopathology with kidneys showed that
glomerular in the rats being poisoned with Amanita virosa (image 3.39) got
injury with image of congesting glomerular, widening the Bowman cavity
of glomerular, there had cell kernels in clot, kernels broken into pieces. At

the kidney medulla there had bleeding mass at the slit area and in the
tubular (image 3.41). In the tubular there had Renal cast (image 3.42).
Renal injury often accompanied with liver injury in mycotoxicosis with
amatoxin. The kidneys in injury due to direct impact of toxicity on
glomerular, tubulars from complications of liver failure. Amatoxin is the
toxicity is not metabolized in the body, this substance is excreted in the
intact form. Normal, there had 40% of amatoxin amount in the body is
excreted in the urine. According to Puschner B. and CS (2007), on the dogs
there had about 70 – 80% amanitin is excreted in urine, toxins in the urine
will cause renal tubular epithelium vulnerability peeling combined with
other components such as red blood cells, protein forming casts. This causes
acute tubular necrosis, renal tubular switch, whereas if normal blood
pressure, the urine flows into the bag and filter vanduoc Bowman. However,
because the kidney tubules are clogged up urine retention and microscopic
images we see how Bowman extended. Image also appropriate
histopathological clinical in patients poisoned with mushrooms have
amatoxin manifest oliguria or anuria..


21
+ For spleen: On image of spleen microbials of the normal white mice
(image: 3.45) clearly see the white cord catch the dark blue, red medulla
area with the red and blue. The spleen in white mice in poison by
mycotoxicosis (image: 3:46) visible in the red marrow congestive areas and
clusters of dengue. Spleen congested as a result of the toxic effects on the
blood vessels in the spleen and as a result of liver failure, kidney failure.
4.3.2 Toxicity and effect of Chlorophyllum molybdites on experimental
animals
* In term of toxicity of Chlorophyllum molybdites:
Chlorophyllum molybdites is one of two mushrooms causing poisoning

the most in Cao Bang Province. Chlorophyllum molybdites grows in Cach
Linh Commune, Phuc Hoa District to select for research of toxicity.
Researching results of toxicity of Chlorophyllum molybdites at table
3.16 showed that: LD50 of Chlorophyllum molybdites for the white mice via
ingestion is 3,718 g/kg body weight for dried mushroom and 35,253 g/kg
body weight for fresh mushroom. Thus, Chlorophyllum molybdites has low
toxicity.
According to the foreign materials, the Chlorophyllum molybdites are
the fungus with quick toxin effect. The first symptoms after eating only
about 30 minutes to 2 hours with the expression of digestive disorders
(vomiting, abdominal pain, diarrhea) but usually not fatal. Some documents
have cited the clinical picture of mycotoxicosis Chlorophyllum molybdites.
All patients in this fungus poisoning are vomiting, abdominal pain, diarrhea,
dehydration and loss of electrolytes. Patients usually recover after 2-3 days
after complete rehydration.
4.3.3 Toxicity and effect of Russula emetica on experimental animals
* On toxicity of Russula emetica:
Russula emetica is a kind of mushroom growing in some districts in
Cao Bang Province. The outside characteristic of this mushroom are: Red
cap thus people often consider this mushroom to be very toxic, dangerous
and lethal. However, researching results at table 3.21 showed that: LD50 via
ingestion for the white mice of Russula emetica at the dry form is 4,838
g/kg weight and at the fresh form is 41,326 g/kg weight.
Researching results proved that the Russula emetica has low toxicity.
The foreign materials also confirmed that this mushroom just cause
gastrointestinal disturbance (nausea, vomiting, abdominal pain, diarrhea,
....), especially vomiting violently after eating mushrooms so this species


22

named Russula emetica. Presently,the toxicity of this mushroom has not
been clearly stated.
4.3.4 Toxicity and effect of Coprinus atramentarius on experimental
animals
* On toxicity of Coprinus atramentarius:
Coprinus atramentarius is a kind of conditional poisonous mushroom,
i.e. the mushroom only has toxic effects from eating mushrooms together
with alcohol or patients drink alcohol after eating mushrooms. Researching
results at table 3.26 showed that LD50 of Coprinus atramentarius (in dry
form) via ingestion for the white mice is: 2,976 g /kg weight and when
drinking with 5 ml alcohol in concentration by 400/kg weight
(corresponding with 0,1 ml alcohol in concentration 400/animal). Coprinus
atramentarius is the mushroom containing coprin. Toxicity of this
mushroom is explained under the following mechanism:
Coprin in the body is converted into 1-aminocyclopropanol. This
substance inhibits enzym aldehyd dehydrogenase (ALDH) to make
acetaldehyd not converted into acetic acid and acetic acid not go into the
Krebs cycle and not broken down into CO2 and water, leading to the
accumulation of acetaldehyd in the body and causing poisoning. Thus,
nature of the poisoning coprin is poisoning acetaldehyd, an intermediate
product in the metabolism of alcohol. Therefore, only the people drink and
eat Coprinus atramentarius containing coprin to be poisoned. Normally,
enzym ALDH inhinited within 5-7 days after eating mushrooms. During
this time in case of disease, every drink will appear the symptoms of
poisoning.
CONCLUSION
• Mycotoxicosis in Cao Bang Province in stages from 2003 to 2009 and
from 2010 to June 2014.
• In the stage without communication interventions (2003 – 2009), Cao
Bang province had 29 poisonings which caused by poisonous mushroom, 81

sufferers and 17 deaths (20,99%) have occurred. After deploying the
communication interventions (from 2010 to June 2014) the mycotoxicosis
decreased down to 6 cases with 12 sufferers and 01 death (2,4%).
• The mushroom causing poisoning the most is Chlorophyllum
molybdites: 18 cases, 39 sufferers, not mortal. The mushroom causing
mortality is Amanita verna (16 people) and Amanita virosa (01 person).


23
2. Morphological characteristics and allocation of common poisonous
mushrooms in Cao Bang Province: The research has found and described
morphological characterization of 13 kinds of poisonous mushroom of 11 in
12 districts in Cao Bang Province, in which the research has identified 4
kinds of poisonous mushroom commonly causing poisoning in Cao Bang
Province: The Amanita verna, Amanita virosa, Chlorophyllum molybdites,
Inocybe fastigiata.
3. Acute toxicity, changes in some biochemical, hematological,
cardiological and histopathological criteria of extracts of 4 kinds of
common poisonous mushroom on animals.
* Amanita virosa:
• LD50 for the white mice:
• Abdominal injection: 0,322 g/kg (dried mushroom), 3,270 g/kg (fresh
mushroom).
• Gastrointestine: 3,896 g/kg (dried mushroom), 28,632 g/kg (fresh
mushroom).
• AST, ALT, GGT, bilirubin, ure, serum creatinine on rabbits
increased highly in total following time. Glucose increased in day 1 and
decreased in day 5, day 10 after being poisoned (p<0,001). erythrocytes,
platelet, hemoglobin decreased in day 5 and day 10, number of leukocytes
increased in day 1, day 5 after being poisoned (p<0,05).

• Pulse increased, blood pressure decreased at 24 hours after being
poisoned (p<0,01).
• Image of histopathology of liver, kidneys, spleen of the white mice:
+ General situation of: Liver, kidneys, spleen with the dark brown, soft,
elastic decreased.
+ Microbials: Liver bleeing, necrosis in cells. Congestion in kidneys,
Bowman cavity widened, spleen congestion red in the marrow.
* Chlorophyllum molybdites:
• LD50 in white mice via ingestion: 3,718 g/kg (dried mushroom),
35,253 g/kg (fresh mushroom).
• ALT increased in day 1 after being poisoned (p<0,001).
• Erythrocytes, hemoglobin, leukocytes and the polymorphonuclear
leukocytes rate increased in day 1 (p<0,05).