Toxicology 146 (2000) 171 – 176

www.elsevier.com/locate/toxicol

In vitro inhibitory effects of antioxidants on cytotoxicity of

T-2 toxin
F. Shokri a,c,*, M. Heidari a,b, S. Gharagozloo a, M. Ghazi-Khansari b
a
Department of Immunology, School of Public Health, Tehran Uni6ersity of Medical Sciences, Tehran 14155, Iran
b
Department of Pharmacology, School of Medicine, Tehran Uni6ersity of Medical Sciences, Tehran 14155, Iran
c
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

Received 29 November 1999; received in revised form 7 February 2000; accepted 7 February 2000

Abstract

T-2 toxin is a secondary fungal metabolite produced by various species of Fusarium. It is capable of killing cells by
causing extensive damage to the cellular membrane. In this study, cytotoxicity of T-2 toxin in combination with
different antioxidant materials, including vitamin C (vit. C), vitamin E (vit. E) and selenium (sel) was investigated in
vitro using the neutral red cytotoxicity assay. Eleven primary and transformed cell lines established from different
tissues were used in pre-test experiments to identify the most sensitive and resistant lines by measuring the half lethal
concentration (LC50) of the toxin. Three cell lines including human gingival fibroblast (HGF), the most sensitive
(LC50 =0.25 ng/ml), human colorectal adenocarcinoma (SW742), the most resistant (LC50 = 5.5 ng/ml) and human
hepatoma (HepG2), with median susceptibility (LC50 =2 ng/ml) were selected to investigate the inhibitory effects of
the antioxidant agents, on cytotoxicity of T-2 toxin. Our results demonstrated that co-incubation of cell lines with
different concentrations of T-2 toxin and antioxidants decreased significantly, but did not totally inhibit, the
cytotoxicity of T-2 toxin (PB0.001). These findings suggest that in addition to lipid peroxidation, which is inhibited
by antioxidants, other unidentified mechanism(s) seem to be involved in cytotoxicity of T-2 toxin. © 2000 Elsevier
Science Ireland Ltd. All rights reserved.

Keywords: Cytotoxicity; T-2 toxin; Cell culture; Antioxidant; In vitro

1. Introduction ene) belongs to a large group of mycotoxins,

T-2 toxin (3-a-hydroxy-4-b, 15-diacetoxy-8-
a (3-methyl butyryloxy)-12,13-epoxytrichothec-9-
Abbre6iations: FCS, fetal calf serum; LC50, the 50% lethal
concentration; NCBI, National Cell Bank of Iran; OD, optical
density; sel, selenium; S.E.M., standard error of mean; vit. C,
vitamin C; vit. E, vitamin E.
* Corresponding author. Fax: + 98-21-6462267.
trichothecenes, which are widely encountered as
natural contaminants (Maurice, 1983; Frick et al.,
1989; Abramson et al., 1997). Upon exposure, T-2
toxin causes severe human and animal diseases
such as alimentary toxic aleukia (ATA) and
stachybotryotoxicosis, affecting the mucosa and
the immune system (Hayes, 1980). The im-
munotoxic effects of T-2 toxin mostly influence

0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 1 7 2 - 4
172 F. Shokri et al. / Toxicology 146 (2000) 171–176

T-cell mediated functions and delayed type hyper- Lafarge, 1983; International program on chemical
sensitivity in experimental animals (Miller, 1986; safety, 1990). Moreover, lipid peroxidation is sug-
Sharma, 1993). This seems to be partly controlled gested to be an important mechanism involved in
through reduced MHC class II expression and cytotoxicity of T-2 toxin (Iwahashi, 1982). At
antigen presentation which are directly induced by high concentration in vitro, T-2 toxin inhibits
T-2 toxin (Blaylock et al., 1993). Furthermore, mitochondrial respiration (Pace, 1988), alters cell
T-2 toxin has also been reported to induce selec- membrane function and structure (Gynogyossy-
tive destruction of lymphoid progenitors (Holla- Issa et al., 1986; Rizzo et al., 1994) and inactivates
day et al., 1993; Smith et al., 1994), and inhibition certain thiol-containing enzymes (Ueno, 1984;
of IL-2 and IL-5 production by T-cells (Marin et Krachenco, 1989). Despite increasing in vivo and
al., 1996). In addition to lymphocyte precursors, in vitro investigations, modulation of T-2 toxin
other hematopoietic progenitors, such as granulo- cytotoxicity has not been studied extensively. In
cyte, monocyte and erythrocyte colony forming the present study, the inhibitory effects of some
cells are also considered to be targets of tri- well-known antioxidant materials on cytotoxicity
chothecenes (Parent et al., 1994; Rio et al., 1997). of T-2 toxin have been assessed in a cell culture
Destruction of hematopoietic cells by T-2 toxin is system.
likely to be associated with apoptosis (Ihara et al.,
1997; Shinozuka et al., 1997). Massive destruction 2. Materials and methods
of hematopoietic cells results in leukopenia,
anaemia and bone marrow aplasia which are the 2.1. Cell lines
most important hematopathological symptoms in-
duced by these toxins. T-2 toxin is a potent in- Eleven well-characterised human and animal
hibitor of both protein and DNA synthesis cell lines were employed in this study. Major
particularly in eukaryotic cells (Rosenstein and characteristics of these lines are listed in Table 1.
HGF and LCL were established in our laboratory
Table 1 (F. SH.) and the remaining cell lines were ob-
Human and mouse cell lines employed in this studya tained from the National Cell Bank of Iran
(NCBI, Pasteur Institute of Iran, Tehran) and
Cell line Description Code number
European Collection of Animal Cell Culture
A375 Human malignant NCBI C136 (ECACC, Salisbury, Scotland).
melanoma
A549 Human lung carcinoma NCBI C137 2.2. Cell culture
SP2/0 Mouse myeloma NCBI C129
HepG2 Human hepatoma NCBI C158 The cells were cultured into a sterile plastic
LCLPI Human EBV transformed NCBI C159 flask (Nunc, Denmark). The flask was filled with a
B cells
EL-4 Mouse leukaemia NCBI C314
thin layer of complete culture medium [RPMI-
K562 Human chronic myeloge- NCBI C122 1640 (GIBCO, USA), 10% fetal calf serum (FCS)
nous leukaemia (CML) (GIBCO), supplemented with penicillin (100 U/
RAJI Human Burkitt’s NCBI C127, ml) and streptomycin (100 mg/ml) (Sigma, UK)]
lymphoma ECACC and incubated in a CO2 incubator at 5% CO2,
85011429
SW742 Human colorectal adeno- NCBI C146 37°C and saturated humidity. Following 48–72 h
carcinoma incubation, when cells reached 70–90% conflu-
3T3 Mouse embryo fibroblast NCBI C162, ency, the cells were detached by 1–2 min trypsin
ECACC (GIBCO) treatment (0.25% w/v in isotonic PBS)
93061524
and cell viability was assessed by the trypan blue
HGF Human gingival fibroblast NCBI C356
(Sigma) dye exclusion method. The cells were then
a
NCBI and ECACC code numbers denote accession codes passed to a 96-well tissue culture microplate
from NCBI and ECACC, respectively. (Nunc) for cytotoxicity assessment.
 F. Shokri et al. / Toxicology 146 (2000) 171–176 173

2.3. Cytotoxicity assay calculated as follows:

Percent inhibition
The neutral red cytotoxicity assay was used to
determine the cytotoxic effects of T-2 toxin. The = Percent cytotoxicity of toxicant− Percent
assay was performed basically as reported by cytotoxicity of (toxicant+ antioxidant).
Babich and Borenfreund (1991a) with some mod-
All experiments were repeated at least four to
ifications (Gheshlaghi Azar et al., 2000, in press).
six times.
Briefly, 25 000 cells were seeded in each well of a
96-well tissue culture plate in 200 ml of complete
2.4. Statistical analysis
culture medium and incubated for 24 h, in a
humidified CO2 incubator at 37°C. Culture super-
LC50 was determined by probit analysis using
natant from each well was then replaced with 200
the Pharm. PCS (Springer-Verlag, New York)
ml of fresh culture medium containing various
statistical package. Statistical differences were
concentrations of T-2 toxin in the presence or
performed by analysis of variance (ANOVA) fol-
absence of antioxidants. To determine the LC50
lowed by Tukey–Kamber multiple comparison
concentration of T-2 toxin, serial concentrations
test. Differences were regarded as significant at
from 0.02–160 ng/ml were prepared in complete
PB 0.05.
culture medium from a stock solution of 5 mg/ml
of T-2 toxin pre-dissolved in ethanol. Stock solu-
tions of other materials, including vit. C, vit. E
3. Results
(Merck, Germany), sel (Sigma) and neutral red
(Merck) were dissolved in distilled water. The
3.1. Cytotoxic effects of T-2 toxin on 6arious cell
supernatant was subsequently removed and the
lines
wells were filled with 200 ml of neutral red dye (40
mg/ml) freshly prepared in RPMI-1640 from a
Comparative toxicity of T-2 toxin on various
stock solution of 5 mg/ml in distilled water. After
cell lines is shown in Fig. 1. The results depicted
3 h incubation at 37°C, viable cells were evalu-
in Fig. 1 represent only the concentration of the
ated. The cells were washed and fixed with CaCl2
toxin giving 50% cytotoxicity (LC50). Among the
(1%) in HCHO (0.5%) and finally lysed with
11 cell lines studied, HGF was found to be the
CH3COOH (1%) in C2H5OH (50%) to extract the
most sensitive (LC50 = 0.25 ng/ml), SW742, the
dye from viable cells. The optical density (OD) of
most resistant (LC50 = 5.5 ng/ml), and HepG2
extracted dye was then measured with a visible
displayed median sensitivity (LC50 = 2 ng/ml).
spectrophotometer at 540 nm. Cells treated with
These cell lines were selected for further
distilled water representing 100% cytotoxicity
experiments.
were used as a positive control to subtract back-
ground dye absorption from all test OD values.
3.2. Inhibitory effects of antioxidants on T-2
Cells incubated with culture medium alone, repre-
toxin cytotoxicity
senting 100% viability were included as a negative
control in all experiments to estimate percent of
The selected cell lines were cultured in combina-
cytotoxicity of each sample based on the follow-
tion with LC50 and LC90 doses of T-2 toxin in
ing calculation:
presence or absence of vit. C (0.01 mM), vit. E
(0.01 mM) and sel (0.001 mM). The selected
Percent cytotoxicity=
molarities of antioxidants were determined in pre-
test experiments using different concentrations
mean absorbance of toxicant
1− ×100 from 0.0001 to 40 mM (data not presented).
mean absorbance of negative control As shown in Table 2 all the antioxidants in-
Inhibition of cytotoxicity by antioxidants was duced significant inhibition on cytotoxicity of T-2
174 F. Shokri et al. / Toxicology 146 (2000) 171–176

toxin in all three cell lines. The results obtained as

OD values from spectrophotometrical analysis
were always in agreement with microscopic obser-
vations. The morphologic changes observed in
HepG2 cells are illustrated in Fig. 2.

4. Discussion

In the present study, the effect of three well

known antioxidant agents, including vit. C, E and
sel on cytotoxicity of T-2 toxin was investigated in
a cell culture model.
In pre-test experiments cytotoxicity of various
concentrations of T-2 toxin on 11 cell lines (in-
cluding normal diploid and transformed cells) was
studied to determine the LC50 and LC90 doses of
the toxin and to select the most sensitive and
resistant cell lines. There was a good linear rela-
tionship between the amount of neutral red ab-
Fig. 1. Comparative toxicity of T-2 toxin on various cell lines.
sorption and various doses of toxin in all cell lines
Vertical lines depicted on each bar represent S.E.M.
studied (data not shown). The pattern of linearity
Table 2 was dependent on sensitivity of the cell line used.
Effect of vit. C, E and sel on cytotoxicity of T-2 toxina Similar findings were obtained by other investiga-
tors with regard to cytotoxic effects of T-2 toxin
Experiment Percent cytotoxicity
in SW742 and HepG2 cell lines, using the same
HepG2 HGF 5W742 methodology (Babich and Borenfreund, 1991b),
suggesting reproducibility of this method. Vari-
Control 3.5 90.65 2.89 1.80 1.89 0.48 ability in cellular sensitivity to T-2 toxin has also
Vit. C 3.5 90.95 4.0 91.47 1.69 0.29
been observed in a variety of other cell lines
Vit. E 3.3 91.60 2.390.63 2.09 0.41
Sel 3.8 9 0.85 4.091.08 2.09 0.41 (Juranic et al., 1998).
T-2 toxin 44.0 92.14 43.591.70 42.09 1.23 Our results have demonstrated significant inhi-
(LC50) bition of T-2 toxin cytotoxicity by all the three
T-2 toxin 90.7 92.14 85.09 0.94 88.0 91.47 antioxidants used (Table 2). Interestingly, the in-
(LC90)
Vit. C+LC50 23.0 94.06** 28.39 2.32** 34.591.32**
hibitory effect was similarly induced at both LC50
Vit. C+LC90 72.0 93.85** 74.89 0.48** 78.090.85** and LC90 dose of the toxin and in both the least
Vit. E+LC50 20.5 92.66** 34.79 3.45** 32.092.75** and the most sensitive cell lines tested, indicating
Vit. E+LC90 68.3 94.13** 73.59 1.90** 77.09 0.85** consistency of the results and involvement of the
Sel+LC50 25.8 93.20** 30.29 3.23** 30.092.48**
same (or similar) mechanism(s) underlying in-
Sel+LC90 67.0 9 3.58* 79.09 0.90** 76.091.70**
hibitory effect of all the antioxidants used.
a
Data are the mean and S.E.M. of four triplicate determi- It has long been known that antioxidants dis-
nations. Significant differences compared to controls (T-2 play a protective role against pathological changes
toxin LC50, LC90) cultures lacking the corresponding antioxi- induced by free radicals (Bus, 1979; Frick et al.,
dants are shown as *, PB0.01; and **, PB0.001. Control
represents cells treated with culture medium alone. 1989). One of the suggested cytotoxicity mecha-
nisms of T-2 toxin is lipid peroxidation which
toxin (P B0.01, PB 0.001). This was similarly occurs in various organs and tissues such as
represented at both LC50 and LC90 doses of the spleen, bone marrow, thymus, kidney and particu-
 F. Shokri et al. / Toxicology 146 (2000) 171–176
Fig. 2. Photomicrographs ( × 20) of human hepatoma cell line (HepG2) cultured in presence or absence of T-2 toxin and vit. C. (A)
Culture medium alone (control); (B) vit. C; (C) T-2 toxin; (D) T-2 toxin + vit. C.
larly in liver (Rizzo et al., 1994). Since T-2 toxin is
reported to bind to the thiol end of certain thio-
lated enzymes (Ueno, 1984; Krachenco, 1989),
therefore this may cause alteration in cell mem-
brane structure, resulting in lipid peroxidation.
The oxidative damage caused by T-2 toxin may
be one of the underlying mechanisms for T-2
toxin-induced cell injury, DNA damage and
apoptosis (Atroshi et al., 1997). T-2 toxin-induced
DNA damage and apoptosis is thought to be
mediated by increased level of intracellular cal-
cium ion which transduces activation signals for
endonuclease and protease enzymes (Yoshino et
al., 1996).
The anti-oxidative effects of vit. E and sel
against T-2 toxin have frequently been reported
mainly in vivo, in experimental animals (Tutelyan
et al., 1990; Rizzo et al., 1994; Hoehler and
Marquardt, 1996; Atroshi et al., 1997; Yazdan-
panah et al., 1997). The effect of vit. C, however,
has been a matter of controversy (Rizzo et al.,
1994; Hoehler and Marquardt 1996). These obser-
vations have not been widely reported in vitro.
Our results confirm and extend previous find-
ings regarding the efficiency of vit. C, E and sel in
entrapping of free radicals and inhibition of lipid
peroxidation (Rizzo et al., 1994). Cytotoxicity of
T-2 toxin, however, has been partially counter-
acted by these antioxidants, suggesting involve-
175
ment of other unidentified mechanisms in this
process.
Acknowledgements
The authors wish to express their gratitude to
Dr Zahra Samadi Bahrami from the National Cell
Bank of Iran and Jalal Khoshnoodi from School
of Public Health, Tehran University of Medical
Science for their technical help. Thanks are also
due to Dr S. Mehdi Rezayat from the Department
of Pharmacology, Tehran University of Medical
Sciences for the generous supply of T-2 toxin.
This study was supported in part by a grant from
Tehran University of Medical Sciences.
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