T-2 toxin, a trichothecene mycotoxin, is considered to be one of the most toxic compounds that are produced by molds, particularly the Fusarium species. Fusarium species have been recognized as a great agricultural problem. They occur worldwide on a variety of plant hosts and cereal grains. The aim of this study was to investigate T-2 toxin-induced liver injury using in situ perfused rat liver. The in situ perfused rat liver (IPRL) was chosen because it permits studies of liver function in a system that resembles normal physiology. Elevation of aminotransferase activities have shown to be a good indicator of hepatocellular damage. In addition, glutathione levels have also shown to be an indicator of liver damage through lipid peroxidation. Male Sprague-Dawley rats (6-8 weeks) weighing 250-300 g were used in this study. They were randomly divided into 5 groups of 3-4 rats per cage. In group 1, liver was perfused by Krebs-Henseleit buffer alone (Control). Groups 2-5 received different concentration of T-2 toxin (4, 9, 21, 43 rmol/L) in Krebs-Henseleit buffer and biochemical changes in the liver were examined within 2 h. There was a significant increase in both ALT and AST activity in all dose levels compared with the control group (p<0.01 and p<0.05). T-2 toxin treatment enhanced lipid peroxidation in the liver, as indicated by the increased MDA content in liver homogenates. The MDA level was maximal 2 h after the T-2 toxin challenge (p<0.01 and p<0.05). The results also show that T-2 toxin causes an increase in lipid peroxidation while causing a decrease in glutathione (GSH) content in bile secretion (p<0.01). This result suggests that both lipid peroxidation and glutathione (GSH) depletion play a role in T-2 toxin liver induced damages.
T2-Toxin Hepatotoxicity in the in situ Rat Liver Model
Iranian Journal of Pharmaceutical
Research (2004) 4: 225-230 Received: June 2004 Accepted: October 2004
Copyright ? 2004 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
T2-Toxin Hepatotoxicity in the
in situ Rat Liver
Model
Bahram Daraeia, Mahmoud Ghazi-Khansari*b,
and Hamid Reza Rasekha
aDepartment
of Pharmacology and Toxicology, School of Pharmacy, Shaheed
Beheshti University of Medical Sciences, Tehran, Iran. bDepartment of
Pharmacology, Medicine School, Tehran University of Medical
Sciences, Tehran, Iran.
T-2 toxin, a trichothecene mycotoxin, is
considered to be one of the most toxic compounds that are
produced by molds, particularly the Fusarium species. Fusarium
species have been recognized as a great agricultural problem.
They occur worldwide on a variety of plant hosts and cereal
grains. The aim of this study was to investigate T-2
toxin-induced liver injury using in situ perfused rat liver.
The in situ perfused rat liver (IPRL) was chosen because it
permits studies of liver function in a system that resembles
normal physiology. Elevation of aminotransferase activities
have shown to be a good indicator of hepatocellular damage. In
addition, glutathione levels have also shown to be an indicator
of liver damage through lipid peroxidation. Male Sprague-Dawley
rats (6-8 weeks) weighing 250-300 g were used in this study.
They were randomly divided into 5 groups of 3-4 rats per cage.
In group 1, liver was perfused by Krebs-Henseleit buffer alone
(Control). Groups 2-5 received different concentration of
T-2 toxin (4, 9, 21, 43 rmol/L) in Krebs-Henseleit buffer and biochemical
changes in the liver were examined within 2 h. There was a
significant increase in both ALT and AST activity in all dose
levels compared with the control group (p<0.01 and
p<0.05). T-2 toxin treatment enhanced lipid peroxidation in
the liver, as indicated by the increased MDA content in liver
homogenates. The MDA level was maximal 2 h after the T-2 toxin
challenge (p<0.01 and p<0.05). The results also show that
T-2 toxin causes an increase in lipid peroxidation while
causing a decrease in glutathione (GSH) content in bile
secretion (p<0.01). This result suggests that both lipid
peroxidation and glutathione (GSH) depletion play a role in T-2
toxin liver induced damages.
T-2 toxin, a trichothecene
mycotoxin, is considered as one of
the most toxic compounds that are produced by molds,
particularly the Fusarium species (1). Structurally, they are
sesquiterpenes and have an epoxy ring at C-12, 13 (1).
Contamination of cereals such as barley, wheat, rice and maize
with Fusarium mycotoxins has been reported worldwide (2-7).
Because of their widespread natural occurrence and diverse
toxic effects, the presence of these mycotoxins in food and
feed is considered potentially hazardous to humans and animals
(4-7). Different mechanisms of action have been proposed for
T-2 toxin. They react with the thiol groups of sulfhydryl
enzymes and as a result are potent protein and DNA synthesis
inhibitors (8, 9, 10, and 25). Further, morphological and
functional changes in biological membranes have been observed
in the heart (11), red blood cells (12) and liver (13).
Previous studies have shown that there is a similarity between
the hemolysis of rat erythrocytes caused by T-2 toxin and that
caused by the free radicals (8). Furthermore, T-2 toxin
administration leads to a pronounced increase in the
thiobarbituric acid reactive compounds in liver homogenate of
T-2 toxin-treated rats. This was interpreted as an indicator of
the presence of lipid peroxidation substances such as
2-alkenals, 4-hydroxy-alkenals and malondialdehyde (MDA), which
were reported to be present in the liver after acute exposure
to T-2 toxin (8, 25). Hoehler et al. used a specific species of
yeast (Kluveromyces marixianus) to study oxidative damage induced by T-2 toxin
(26). They demonstrated that MDA content increased when the
concentration of T-2 toxin was raised. These studies further
suggest that T-2 toxin stimulates lipid peroxidation by
promoting free radical production. Despite reports on the
induction of lipid peroxidation by T-2 toxin, others reported
no effect on lipid peroxidation property of trichothecenes (1).
Overall, the evidence suggests that one of the modes of action
of T-2 toxin is to enhance the peroxidation of lipids, with
free radicals involved in the process. Studies on liver tissue
were undertaken since this organ has been shown in previous
studies (8, 9, 13, and 27) to be a target for T-2 toxin.
Experimental
Materials
T-2 toxin and all other chemicals were of
analytical grade obtained from Sigma-Aldrich (St Louis, MO,
USA).
Methods
Animal
Male Sprague-Dawley rats (6-8 weeks)
weighing 250-300 g were obtained from Razi Vaccine Institute,
Tehran, Iran. They were housed in standard stainless-steel
cages at a 12 h cycle of light and dark. Room temperature was
kept at 22°C and humidity maintained at 50%. Rats were
allowed to become acclimatized to standard laboratory condition
for at least 7 days and standard food and water was provided ad libitum. Food
was withdrawn 12 h before starting the experiment.
Administration of T-2 toxin
One mg crystalline T-2 toxin was dissolved
in 1 ml ethanol (96%) and stored at 4°C until use. The
stock solution was appropriately diluted to the concentration
needed (25, 28).
Experimental design
Animals were divided into 5 groups. Each
group contained 3-4 male rats. In group 1, liver was perfused
by Krebs-Henseleit buffer alone (Control). Groups 2-5 contained
different concentrations of T2 toxin, with the same buffer.
Before performing the experiment, perfusate was circulated for
30 min to permit stabilization. Then, the perfusion was
recirculated with Krebs-Henseleit buffer.
Liver perfusion
Livers from male Sprague-Dawley rats were
perfused at 37°C with Krebs-Henseleit solution. D-glucose
(0.1% w/v) was added to provide energy source. The perfusion
medium was gassed continuously with carbogen (95% O2, 5% CO2)
essentially as described by Wolkoff et al. (14). Briefly, an
incision was made along the length of the abdomen to expose the
liver under ketamine and xylazine (70 mg/kg and 15 mg/kg, i.p.,
respectively) anesthesia. Heparin, 500 units, may also be
injected i.p. to prevent blood clotting, although this is not
obligatory for successful perfusion. An incision was made along
the length of the abdomen to expose the liver. Sutures were
placed loosely around the common bile duct, which were
canulated with PE10 tubing, secured and bile was collected for
a period of 120 min. Sutures were then placed loosely around
the inferior vena cava, above and below the renal veins. The distal
suture around the vena cava was tightened and then an 18G polyethylene
catheter was inserted, placed above the renal vein and secured
with the proximal suture. The portal vein was immediately
cannulated. The diaphragm was incised and the inferior vena cava was
legated super-hepatically. Following attachment of the
perfusion tubing to the cannulae, the liver was perfused in
situ through the portal vein. Temperature, perfusion fluid
pressure, flow rate and perfusion fluid pH were closely
monitored during the perfusion (14) and the experiment was not
begun until they all had reached to constant and acceptable
values. Perfusion pressure was not raised above 10-15 cm of
water with a flow rate of approximately 3 ml/min/g liver weight
to provide an adequate oxygenation. The pH of perfusion fluid
was always set between 7.2 and 7.4 by adjusting the
Carbogen gas. As soon as perfusion was begun, the liver should
blanch to a uniform, light brown color. Blotches or
discoloration means that liver is not well-perfused. Serum
aminotransferase activities (ALT and AST), central vein
pressure (CVP), bile flow and pH, serve as indicators of liver
viability during perfusion, which were determined in samples of
perfusion medium every 15 min (Figure 1).
Determination of the activities of
aminotransferases
The activities of alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) in the perfusion
fluid and homogenate sample were assayed using the commercial
kit of Darman Kave (Tehran, Iran).
Determination of the total protein and
glutathione (GSH) contents
Glutathione contents in tissue homogenates
and bile secretion were determined as described by Kuo and Hook
(15). Briefly, the tissue was homogenized in 20% (w/v)
trichloroacetic acid and centrifuged at 6000 rpm for 20 min. To
determine glutathione (GSH) concentration in the tissue, an
aliquot of the deproteinized supernatant fraction was added to
2 ml of 0.3 M Na2HPO4 solution followed by the addition of 0.5
ml of 0.04 %, 5,5-dithiobis-[2-nitrobenzoic acid] dissolved in
10% sodium citrate. The absorbance at 412 nm was measured
immediately after mixing and the glutathione (GSH) values were
determined by extrapolation from standard curve. The amount of
total protein was determined in liver homogenates after 120 min
of perfusion, colorimetrically according to the Bradford
method, using the coomassi brilliant blue G-250 reagent (16).
Lipid peroxidation
Lipid peroxidation was determined in liver
tissue homogenates according to the thiobarbituric acid (TBA)
method (17).
Statistical analysis
Statistical analysis was performed using
the one way analysis of variance (ANOVA) followed by the
Student Newman Keuls post test. Level of significance was set
at p<0.05 and p<0.01.
Results and Discussion
T-2 toxin has the highest toxicity among
the 16 trichothecene mycotoxins studied (18). In this study we
showed that T-2 toxin causes an alteration in the biochemical
parameters in the in situ perfused rat liver. Evaluation of
viability of the perfused liver by different parameters in
control groups showed no significant change, which indicated
stability and consistency of the method. The data is in
agreement with those reported by Wolkoff et al. (Figure1) (14).
Perfusion of rat liver with the Krebs-Henseleit buffer
containing different concentrations of T-2 toxin (4, 9, 21, 43 rmol/L) showed a
significant increase in both AST and ALT activities in all
concentrations, compared to the control group at p<0.01 and
p<0.05 (Figure 2). However, this increase was more
significant at p<0.01 with doses of 4 and 9 rmol/L of T-2 toxin.
T-2 toxin doses of 4, 9, 21, 43 rmol/L caused a significant decrease in
glutathione (GSH) level in liver homogenates, compared to the
control group, with p<0.01 and p<0.05 (Figure 3). It was
also shown that the addition of toxin to perfusion buffer
resulted in a significant decrease (p<0.01) in GSH
concentration in biliary excretion at doses of 21 and 43 rmol/L, compared
with their respective control groups (Figure 4). T-2 toxin is
known to bind to SH-proteins (22). The epoxy group of T-2 toxin
competes with the substrates of glutathione-s-transferase (19).
This could result in a decrease in the content of GSH.
Prevention of lipid peroxidation is primarily connected with
the glutathione (GSH) metabolism and also the ability of
hepatic glutathione peroxidase to remove H2O2 and organic
peroxides in the compartments where catalase is absent (23). It
is possible that T-2 toxin reduces the level of glutathione
(GSH), when it is required for the elimination of peroxide
radicals.
The TBA values increased in the perfused
liver, 2 h after administration of T-2 toxin, reaching values
of approximately six (21 rmol/L) and seven (9, 43 rmol/L) times higher than
control (Figure 5). This study showed that T-2 toxin causes an
increase in lipid peroxidation, while causing a decrease in
glutathione (GSH) in the in situ perfused rat liver.
Glutathione (GSH) and lipid peroxidation are both sensitive
indicators of oxidative stress. Some investigators have
suggested that T-2 toxin produces its toxic effects by inducing
membrane lipid peroxidation mediated by superoxide anion
radicals (19). Since lipid peroxidation is one of the causes of
cell membrane disruption and necrosis, T-2 toxin may induce
modifications in membrane permeability, releasing significant
quantities of aminotransferase enzymes into the perfusion
fluid. Moreover, glutathione cycle imbalance induced by T-2
toxin produced an increase in lipid peroxidation which affected
membrane integrity, in turn causing enzymes leakage. Increasing
both lipid peroxidation and aminotransferase enzymes release
could result either from a primary increase in membrane
vulnerability to oxidant damage, or a possible increase in free
radical production. Liver lipid peroxidation caused by certain
drugs (20) and ethanol (21) has been shown to be due to the
depletion of hepatic reduced glutathione (GSH). Moreover, T-2
toxin is a lipophilic substance and is readily absorbed into
the membrane, resulting in structural alteration within the
membranes that triggers the stimulation of membranous lipid
peroxidation. Stimulation of lipid peroxidation by T-2 toxin in
liver cells rich in membranes appears to be carried out through
the electron transport system and polyunsaturated fatty acids.
T-2 toxin stimulates lipid peroxidation by promoting free
radical production. The reactive oxygen molecules interact with
phosopholipids of cellular membranes, resulting in an increased
level of lipid peroxidation especially in the liver (25). In
this study, toxin treatment also enhanced lipid peroxidation in
the liver, as indicated by the increased MDA content in liver
homogenate. The MDA level was maximal 2 h after the T-2 toxin
challenge. The formation of lipid peroxides following T-2 toxin
treatment in mammals has been reported in previous studies
(24-26). Schuster et al., in contrast to the results obtained
by several different groups, concluded that lipid peroxidation
is not involved in T-2 toxicity (1). The reason for these
differences was not established. Nevertheless, the collective
results of several different research groups together with the
results discussed in this study, suggests that T-2 toxin
induces lipid peroxidation in the biological systems. T-2 toxin
and some other mycotoxins are believed to enhance the
production of oxygen radicals to such extent that the normal
body radical scavengers are overwhelmed, resulting in cell
injury (25).
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Daraei, B., Ghazi-Khansari, M., Rasekh, H. (2010). T2-Toxin Hepatotoxicity in the in situ Rat Liver Model. Iranian Journal of Pharmaceutical Research, Volume 3(Number 4), 225-230. doi: 10.22037/ijpr.2010.605
MLA
B Daraei; M Ghazi-Khansari; HR Rasekh. "T2-Toxin Hepatotoxicity in the in situ Rat Liver Model". Iranian Journal of Pharmaceutical Research, Volume 3, Number 4, 2010, 225-230. doi: 10.22037/ijpr.2010.605
HARVARD
Daraei, B., Ghazi-Khansari, M., Rasekh, H. (2010). 'T2-Toxin Hepatotoxicity in the in situ Rat Liver Model', Iranian Journal of Pharmaceutical Research, Volume 3(Number 4), pp. 225-230. doi: 10.22037/ijpr.2010.605
VANCOUVER
Daraei, B., Ghazi-Khansari, M., Rasekh, H. T2-Toxin Hepatotoxicity in the in situ Rat Liver Model. Iranian Journal of Pharmaceutical Research, 2010; Volume 3(Number 4): 225-230. doi: 10.22037/ijpr.2010.605
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