Human & Experimental Toxicology (2001) 20, 84-89
O 2001 Amold AJI rights reserved 0960-3271/01
www.arnoldpublishers.com/journals
Trichothecene toxicity on human
megakaryocyte progenitors (CFU-MK)
R Froquet, Y Sibiril and D Parent-Massin
Laboratoire de Microbiologie et Securit6 Alimentaire, Ecole Superieure de Microbiologie et Securit6 Alimentaire de
Brest, ISAMOR, Technop6le Brest-Iroise, 29280 Plouzane, France
Trichothecenes are mycotoxins produced by various spe-
cies of fungi, which can occur on various agricultural
products. Among these compounds, T - 2 toxin, HT - 2 toxin,
diacetoxyscirpenol (DAS) and deoxynivalenol (DON) are
the most naturally encountered and the most potent
trichothecenes. Consumption of trichothecene contami-
nated foods by farm animals and humans leads to
mycotoxicosis. Trichothecenes are known to induce hae-
matological disorders such as neutropenia, aplastic anemia
and thrombocytopenia in humans and animals.
Four trichothecenes, T-2 toxin, HT-2 toxin, DAS and
DON have been tested on human platelet progenitors (CFU -
MK) using a culture model of CFU-MK optimized for
Introduction
In order to determine the origin of the haematological
troubles observed in trichothecene intoxication, clo-
nogenic assays have been used by Parent-Massin et
a],1,2 Lautraite et a]3'6 and Rio et al.7 These authors
showed that human white blood cell progenitors
(CFU-GM) and red blood cell progenitors (BFU-E)
are the targets of trichothecene's cytotoxic effects. In
the present work, the sensitivity of platelet progeni-
tors (CFU-MiK) to trichothecenes is evaluated to
determine whether the thrombocytopenia and coagu-
lation troubles observed upon trichothecene con-
sumption could be related to a decrease in platelet
progenitor proliferation.
Trichothecenes are mycotoxins produced by
various species of fungi such as Fusarium, Stachy-
botrys, Trichothecium, Verticimasporium, Cephalospo-
rium and Cylindrocarpon.8-10 These fungi develop
under specific conditions of temperature and humid-
ity.9 Trichothecenes are known as major contami-
nants of cereals and cereal-containing foods.
*Correspondence: D Parent-Massin, Laboratoire de Microbiologie
et de Securit6 Alimentaire, Ecole Sup6rieure de Microbiologie et
S6curit6 Alimentaire de Brest, ISAMOR, TechnopBle Brest-Iroise,
29280 Plouzan6, France
Received 12 January 2000; revised 22 December 2000; accepted 7
January 2001
toxicological studies. Trichothecenes cause, at low concen-
trations, cytotoxic effects in megakaryocyte progenitors,
which could induce thrombocytopenia. Sensitivity of
human CFU-MK is compared to respective sensitivities of
human red blood cell progenitors (BFU -E) and white blood
cell progenitors (CF-U-GM) that were described in pre-
vious works. (2001) 20, 84-89.
Keywords: food toxicology; trichothecenes; haematotoxicity; plate-
let progenitors; mycotoxins
Trichothecene mycotoxins comprise a large group
of closely related compounds designated as sesqui-
terpenoids. Among these compounds, T-2 and HT-2
toxins, diacetoxyscirpenol (DAS), deoxynivalenol
(DON or vomitoxin) and nivalenol (NIV) are the
most commonly detected.
Consumption of trichothecene - contaminated foods
leads to severe mycotoxicosis. The three most known
human fusariotoxicoses induced by trichothecenes are
alimentary toxic aleukia (ATA, so named because the
number of white blood cells decreased markedly in
affected patients), stachybotryotoxicosis and akakabi
byo disease. In 1942-1947, over 10% of the population
of Orenburg (west Siberia) were fatally affected by
over-wintered millet and other kinds of cereals.
Thrombocytes decreased to below 5000 per cubic
millimeter. The death of patients could occur during
this stage because of infections or haemorrhages. Some
cases of human stachybotriotoxicosis have been
observed and were induced by either inhalation of
aerosols during the handling of contaminated hay and/
or direct skin contact with hay.1' They are character-
ized by haematological disorders such as thrombocy-
topenia, coagulation troubles, leukopenia and
agranulocytosis. Akakabi byo disease was identified
in Asia after the ingestion of rice, wheat or oat induced
nausea, vomiting, diarrhea, feed refusal, congestion

and haemorrhages.12,3 These pathologies present
common symptoms. Among these, the most important
is the occurrence of haematological disorders respon-
sible for leukopenia, agranulocytosis, aplastic anae-
mia, thrombopenia and coagulation troubles.14,15
Haematological troubles can be due to either a toxic
effect on circulating blood cells or a decrease or a block
in blood cell renewal. Haematopoiesis is the general
mechanism by which haematopoietic progenitors can
give rise to mature blood cells of all lineages. It occurs
in the bone marrow. Megakaryocytopoiesis is the
complex process producing platelets from megakar-
yocyte progenitors (CFU-MK); the process includes
proliferation and differentiation of the progenitors,
thus allowing platelet release in the blood vessels.
Blood platelets are small cell fragments derived from
megakaryocytes. The first step in megakaryocytopoi-
esis is the prolifera-tion of megakaryocyte progenitors,
the second is the differentiation (synthesis of platelet
glycoproteins, polyploidization) of megakaryocyte
precursors. Megakaryocytopoiesis polyploidization
(or en-domitosis) is a process by which nuclear
division occurs without cytoplasmic separation. Only
mature polyploid megakaryocytes are able to produce
platelets by breaking long cytoplasmic extensions in
the blood vessel.
Myelotoxicity of xenobiotics, commonly measured
in standard animal toxicological studies, does not
allow the identification of target haematopoietic
progenitors in the bone marrow. To eliminate this
drawback, in vitro clonogenic assays (CFU-GM, BFU-
E) have been used. Pesticides, drugs, antineoplastic
agents, heavy metals and natural contaminants have
been evaluated in these in vitro assays.1 -7,16-20 Culture
conditions of CFU-MK usable in toxicology have been
proposed recently by Casati et al2l and Froquet et al.22
In this work, T- 2 toxin, HT- 2 toxin, DAS and DON,
known to be very toxic for human CFU-GM and BFU-
E, have been tested on human CFU-MK using the
protocol defined by Froquet et al.22
Material and methods
Chemicals
Trichothecenes were purchased from Sigma (St. Louis,
MO). T-2 toxin (4/3, 15-diacetoxy-3a-hydroxy-8a-
[3 -methylbutyryloxy]-12,13 epoxytrichothec -9- ene)
- phosphatase reaction step, which is followed by a
was produced from Fusarium sp. and chromatographi-
cally purified. DAS (4,3, 15- diacetoxy- 3a- hydroxy-
12,13 epoxy-trichothec-9-ene) was produced by
F. sambucinum. The origins of HT-2 toxin (15-acet-
oxy-3a, 4/3-dihydroxy-8a -[3 me thylbutyryloxyl - Megakaryocyte lineage cells were stained pink
12,13-epoxytrichothec-9-ene) and DON (3a, 7a,
15 -trihydroxy- 12,13 epoxytrichothec 9-en-8 -one)
- -
were not specified by Sigma.
In vitro trichothecene haematotoxicity
R Froquet et al
85
Culture of human CFU-MK
Human umbilical cord blood samples were col-
lected in heparinized tubes from placentas obtained
after normal deliveries. Because the cord blood
samples were collected from placentas destined to
be discarded after delivery, the informed consent of
the patients was not necessary. Light density cells
were separated by gradient density centrifugation
(d=1.077 g ml-1). Cells were seeded at a concen-
tration of 3.3x104 cells ml-' in a semisolid
medium containing collagen, Kit Megacult3'-C
(StemCell Technologies). Cultures were realized in
double-chamber slides. Each chamber contained 0.7
ml of the culture medium with cells. Human
recombinant thrombopoietin (TPO) (50 ng ml-')
and human recombinant IL-6 and IL-3 (10 ng
ml-1) provided cytokine stimulation. Cultures were
incubated at 370C at 100% humidity with 5% CO2
for 12 days.
Exposure to trichothecenes
The exposure to chemicals was performed as pre-
viously described.22 Trichothecenes were dissolved in
acetone and mixed with the culture medium before
plating. Based on previous studies,3-7 four concentra-
tions were tested for each mycotoxin: 10-10,
5x10 10, 10o- and 10-8 M for T-2 toxin
(MW= 466.5), 0-10, 5x10- 10, 2.5x10-9 and 10 -7
M for HT-2 toxin (MW=424.5), 5x10-10100 -,
5.3x10-9 and 10 -7 M for DAS (MW=366.4) and
10, 3.3x10-8, 7.5x10-8and 2.5x10-7 M for DON
(MW= 296.3).
Cultures with acetone alone and cultures without
solvent and without xenobiotics were also used as
controls.
Assays of each concentration were carried out in
duplicate and each experiment was performed at least
three times.
Staining procedures
Following fixation in an acetone/methanol solution
for 20 min, the slides were air dried. Megakaryocyte
lineage cells were identified with immunocytochem-
ical staining. A primary monoclonal antibody directed
against CD 41, a component of the glycoproteins lIb/
IlIa present on the surface of megakaryocytes, was
used. A secondary staining system utilizes an alkaline
nuclear counterstain such as Evan's blue.
Criteria of toxicity
CFU-MK development was scored after staining.
whereas the nuclei were stained blue. Among the
megakaryocyte lineage cells, two main cellular types
could be observed: small circular cells with little blue
 In vitro trichothecene haematotoxicity
R Froquet et al
86
nuclei and large cells with large nuclei and invagi-
nated cytoplasms. Platelets were also more numerous
in the proximity of large cells.
A colony was defined as an arrangement of at least
three cells. The inhibition due to trichothecene
exposure was expressed as a percentage of the control 5S +.CFu-GM
growth, 100% corresponding to the growth in control
dishes. Three types of colonies were differentiated + CFUUW
I
according to their cell number and have been defined
as large colonies (more than 50 cells), medium
colonies (from 20 to 49 cells) and small colonies
(from 3 to 19 cells).
Statistical analysis OM 10-10 5S10 M 10- M 2.2.10-M M
15-"M
Different groups of mean values were compared to T-2 TOXIN CONCERMTION
control values according to the least significant Figure 1 T -2 toxin myelotoxic effects on human CFU -MK, CFU -

GM3 and BFU-E7

differences (LSD) test of the multifactor ANOVA
analysis using Statgraphic Plus for Windows (version
1.4) Effect of HT-2 toxin
All cells were destroyed in the presence of 10-7 M
HT-2 toxin. A slight decrease in the total colony
Results number was observed for 2.5x10-9 M. Taking into
account the distribution between the three types of
Results are presented in Table 1 and Figures 1-4. colonies, a shift from large colonies (51%) to
medium (136%) and small (180%) colonies was
Effect of T-2 toxin observed. In the presence of the lowest concentra-
Not a single cell was detected in the presence of 10-8 tions (5 x 10 - 10 and 10 -10 M), the growth percentage
M T-2 toxin. Only 52% of total colonies were was not affected.
observed in the presence of 10-9 M T-2 toxin. Large
colonies were more severely affected (22%) than Effect of DAS
medium and small colonies (57% and 103%, DAS was cytotoxic at 10-7 M. In the presence of
respectively). The growth percentage was more than 5.3 x 10 - 9 DAS, the total colony growth was decreased
70% for the two lowest concentrations (5 x 10-10 and to 31% compared to control, and the large colony
1010 M). A no-effect concentration could not be growth (5%) was more affected than the medium
determined. (45%). However, small colony growth percentage
Table 1 Proliferation of human CFUI-MK in the presence of T- 2 toxin, HT- 2 toxin, DAS and DON trichothecene mycotoxins. Growth is
expressed as percentage of colonies, 100% corresponding to the number of colonies in control dishes
Trichothecene Mycotoxin Growth percentage Total colonies
mycotoxins concentration (M) meon number
Small colony Medium colony Large colony Total colonies
T-2 toxin 10- 8 0 0 0 0 0
10 - 9 103 57** 22*** 52 10*** 34
5x10-10 89 76 63* 77±24* 51
10 - 10 83 65* 64* 72 14* 47
HT-2 toxin 10 -7 0 0 0 0 0
2.5x10- 180* 136* 51*** 82 11* 87
5xlO-1° 119 126 90 100 9 106
10 - 10 106 128 91 98 6 104
DAS 10 - 7 0 0 0 0 0
5.3x10-9 143 41*** 5*** 31 1*** 22
l0 - 183* 119 75 98 29 65
5x10-10 127 98 78 88 5 61
DON 2.5x10- 7 194* 82 22*** 55 20* 50
7.5x10 8 147 85 77 87 15 75
3.3x10 152 100 102 107 20 87
10 -8 115 94 102 100 19 82

***,* ** Differences statistically significant at P< 0.05, P< 0.01, P< 0.001, respectively when compared with control cultures under similar
experimental conditions.

Hf-2 TOWN CONCrNRATION
Figure 22 HT -2 toxin myelotoxic effects on human CFU -MK,, CU
HT-t7
GM4 and BFU-ET
GM
increased (143%%). Lower concentrations down to
10o- M had no effect on human megakaryocyte
progenitor proliferation, with the exception of an
increase in small colony growth.
Effect of DON
No cytotoxic level was detected for DON. Total colony
growth was decreased to 55% compared to control in
the presence of 2.5 x 10 -7 M. This decrease was
mainly observed with large colony growth that was
more affected (26%). However, small colony growth
percentage increased to 139%. The three lowest
concentrations (10- 8, 3.3x10 -8 and 7.5x10 8 M)
did not show significant effects on human megakar-
yocyte progenitors proliferation.
Platelets were observed in all treated cultures
including those with cytotoxic concentrations of
mycotoxins, but they have not been scored.
OM 5.tO-1OM 10-SM 510-SM 53.10-SM
DA0S CONCENTRATION
Figure 3 DAS myelotoxic effects on human CFUJ-MK, CFU-GM6
and BFBU-E
In vitro trichothecene haematotoxicity
R Froquet et al
87
z u
- CFR-GM
4 BFtJE
+FL
Om 10-8 M 25.10-SM 3.3.10- M M751M 10-t7 M 2.510-7M
CFUJ- "oNcaATON
Figure 4 DON myelotoxic effects on human CFU-MK, CFU-GM5
and BFU-E7
FUGM
Discussion
Each colony detected in CFU-MK cultures corre-
sponds to one megakaryocyte progenitor. The size of
the colony, i.e., the number of cells in the colony,
reflects the number of mitoses entertained by the
initial progenitor. T-2 toxin was cytotoxic for human
megakaryocytes progenitors in the presence of 10-8
M. For 10 -10 and 5x10- 10 M, 75% of CFU-MK gave
rise to colonies. The cytotoxic concentration for HT - 2
toxin was equal to 10- 7 M. In the presence of
2.5x10-9 M, a decrease in large colony number
compensated by an increase in small colony number
was observed. These results seem to reflect a
cytostatic effect of HT-2 toxin on CFU-MK prolifera-
tion. Level without effect of HT-2 toxin was obtained
for 5x10-10 M. In the presence of 10-7 M of DAS,
human CFU-MK progenitors were destroyed. The
decrease of large colonies compensated by the
increase of small colonies showed a cytostatic effect
of DAS in the presence of 5.3 x10 -9 M. DAS had no
effect when its concentration was equal to 5 x 10- 10
M. DON had no cytotoxic effects at tested concentra-
tions. In the presence of the highest concentration, a
cytostatic effect was observed.
In comparison, T-2 toxin appears to be the most
potent toxin among the four tested trichothecenes
and DON the least. DAS and HT - 2 toxin have a
similar profile of myelotoxicity. T- 2 toxin, HT- 2
toxin and DAS present an antimitotic activity
revealed by a cytostatic effect. Megakaryocytic pro-
genitors exposed to the lowest concentrations of HT-
2 toxin, DAS and DON, seem to recover their capacity
to undergo mitosis.
10-TM These results show that human megakaryocyte
progenitors are very sensitive in vitro to trichothe-
cenes. In consequence, platelet production could be
 In vitro trichothecene haematotoxicity
R Froquet et al
88
Table 2 Toxic effects in vitro of trichothecenes on human hematopoietic progenitors: CFU - GM (white blood cells progenitors), BFU - E (red
blood cells progenitors) and CFU- MK (platelet progenitors). IC50 is defined as a statistical value corresponding to a concentration inducing
50% of growth inhibition, 100% corresponding to the number of colonies in control dishes
T-2 toxin (M)
CFU-MK Cytotoxic level lo-
No effect level <10 _ 10
BFU-E7 Cytotoxic level lo-1
No effect level 2.2x10-
CFU-GM3-6 Cytotoxic level 10 lo108
No effect level < 10lo 10 < lo_10
IC50 2.6xlO-9 1.8xlO-9
affected by destruction of CFU-MK due to tri-
chothecenes. In the presence of the lowest concen-
tration tested, the slight of large colonies (>50
cells) to small colonies could induce a decrease in
progeny from platelet production. The number of
megakaryocytes produced by one CFU-MK could
decrease and in consequence, platelet number
could be less important. Taking into account these
aspects, trichothecenes could induce two types of
toxic effects: a cytotoxic effect on megakaryocyte
progenitors (CFU-MK), and a cytostatic effect
causing a decrease in megakaryocyte production
and consequently in platelet.
Sensitivity of human CFU-MK to T-2 toxin, HT-2
toxin, DAS and DON can be compared to the
respective sensitivities of human BFU-E7 and CFU-
GM3-6 (Figures 1-4 and Table 2). The shape of the
dose-response curves is very similar for the HT-2
toxin, DAS and DON in the case of BFU-E and CFU-
MK. However, a decrease of growth percentage of
CFU-MK is detectable for 2.5 x 10- 7 M DON. Erythro-
blastic progenitors exposed to T-2 toxin seem less
sensitive than CFU-M1K. Megakaryocyte progenitors
are more sensitive to T-2 toxin than CFU-GM. The
cytotoxic concentration is 10- 8 M for CFUI-MK
compared to 10-7 M for CFU-GM. Concerning HT-2
toxin, CFU-GM are more sensitive than CFU-MK.
Sensitivity to DAS is very similar except for 5 x 10 -10
M. The shape of curves obtained from CFU-GM and
CFU-MK cultures in the presence of DON are very
different. Whereas DON exhibits cytotoxic effects for
2.5 x10 - 7 M on CFU-GM, CFU-MK proliferation is
inhibited at 50%. T- 2 toxin was the most potent toxic
trichothecene on haematopoietic progenitors and
DON was the least.
Sensitivity of other cell lineages to trichothecenes
had also been tested in vitro. Neoplastic cells were
sensitive to T-2 toxin. Cytotoxic concentrations were
3.5 x 10 M for B16 and K562 cells.23 HeLa cells were
destroyed at a concentration higher than 5.5 x10-7
M.23 In the presence of 10 - 8 T- 2 toxin, baby hamster
kidney cells (BHK 21) were totally destroyed.25 CTLL
and EL425 mouse T-cell line presented IC50 equal to
HT-2 toxin (M) DAS (M) DON (M)
10 10 - >2.5x 10
5x 10 -10 l-9 3.3x 10- 8
10 -1 10-7 >2.5x10 -7
< 10 10 5xl0 -i 7.5x10 -8
10-7 10-7
5 10-9 lo-8
6.2xlO-9 3.8x10-8
4.5x10 -9 and 4.1x10 -9 M, respectively. In the same
study, the IC50 for rat stimulated spleen T cells was
2.8x10-9 M. Data concerning the three other myco-
toxins were less numerous. BHK 21 were totally
destroyed by 2.4x10- 7 M HT-2 toxin and 2.7x10 8
MDAS.23Inthe presence of 25 ,ugml '(6.8x10-5M)
DAS, chicken macrophage viability decreased to
29.6%.26 The bone marrow is rich in fat tissue and
trichothecenes are rapidly accumulated in this type of
tissue. Thirty minutes after an intramuscular injec-
tion of 1.04 mg kg -1 T-2 toxin (DL 50) in guinea pig,
3H-labelled T- 2 toxin was found in the plasma and in
the fat tissue at concentrations of 9.2 x 10 - 8 and
117x10 -8 M, respectively.27
The strong myelotoxicity of trichothecenes shows
that the haematological troubles observed in tri-
chothecene intoxication have, in part, a central origin.
However, mycotoxins present in blood could have a
direct effect on the survival of peripheral blood cells
and could provoke neutropenia, thrombopenia and
coagulation troubles, observed in cases of severe
intoxication. The haemolytic effect of trichothecenes
has been demonstrated by incubating human or
mouse erythrocytes under in vitro conditions
(3 x10 -4 M T- 2 toxin) .28 The direct cytotoxic effect
of the toxins on white blood cells has also been
studied in vitro but only with human leukocytes.29 In
1984, the study by Gentry et a130 demonstrated that
trichothecenes interfere with bovine blood cells and
coagulation. Haematopoietic progenitor sensitivity to
trichothecenes has to be compared to human blood
cell sensitivity in order to determine the kinetic origin
and nadir of cytopenia induced by these mycotoxin
intoxications. In consequence, further studies have to
be done to explore the effect of trichothecenes on
human coagulation.
Acknowledgement
The Brittany Regional Council provided financial
support for this research.

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