Toxicology Letters 208 (2012) 1–11

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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

In vivo toxicological evaluation of Anisomycin

Tang Zhengle a , Xing Feiyue a,∗ , Chen Di a , Yu Yu b,c , Yu Chunyan a , Di Jingfang a , Liu Jing d,∗∗
a
Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China
b
Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
c
Department of Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
d
Department of Stomatology, Jinan University, Guangzhou 510632, China

a r t i c l e i n f o a b s t r a c t

Article history: Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. Recent studies have shown
Received 22 August 2011 that Anisomycin as a novel immunosuppressive agent is superior to Cyclosporine A (J. Immunother. 31,
Received in revised form 2 October 2011 858–870, 2008). In order to make toxicological evaluation of Anisomycin, acute and four-week continu-
Accepted 3 October 2011
ously intravenous toxicity studies were performed in mice. IC50 value tested on peripheral lymphocytes
Available online 8 October 2011
was 25.44 ng/ml. The calculated LD50 for Anisomycin was 119.64 mg/kg. The mice were intravenously
injected through mouse tail vein with a total dose of 5, 15, 30 and 60 mg/kg/mice of Anisomycin every
Keywords:
other day for 4 weeks. Just in the high-dose mice, death of three mice happened and body weight of the
Anisomycin
Cytotoxicity
mice was significantly decreased. Statistically significant changes in organ index included increases in
Acute and subchronic toxicity ratios of the spleen, liver, lung and brain to the body weight, and decrease in ratio of the thymus to the
Genetic toxicity body weight. Changes in clinical biochemistry parameters included increases in the aspartate aminotrans-
Mice ferase (AST) and alanine aminotransferase (ALT) activities, and decreases in the glucose (GLU) activity.
The distinct inflammation appeared in the lung, liver and kidney, and the number and size of megakary-
ocytes in the spleen were significantly increased. Anisomycin did not induce formation of the peripheral
blood micronucleus, but increased the number of micronucleated polychromatic erythrocytes in bone
marrow and sperm aberrations. However, the above aberrant changes occurred only in the mice treated
with the high-dose Anisomycin. These results indicate that although Anisomycin has no significant side
effects at effectively therapeutic doses, its over-dosage may lead to toxicity, particularly pulmo-, nephro-
and hepato-toxicity.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction to Cyclosporine A with relatively slight toxic signs, which is the

Anisomycin (2-(p-methoxybenzyl)-3,4-pyrrolidinediol-3-
acetate), as an antiprotozoal agent with little antibacterial or
antifungal activity, is a pyrrolidine antibiotic (Fig. 1) first iso-
lated from Streptomyces griseolus (Sobin and Tanner, 1954). It is
described as a potent, structurally specific and reversible protein
synthesis inhibitor in eukaryotic organisms (Grollman, 1967), it
binds to 60S ribosomal subunit and blocks peptide bond formation
and DNA synthesis. Anisomycin may also activate c-jun N-terminal
kinase/stress-activated protein kinase (Hori et al., 2008) and p38
mitogen activated protein kinase, being related to cell apoptosis
(Croons et al., 2009; Hong et al., 2007), thus it is frequently
used as an activation agent in mitogen-activated protein kinase
signaling pathways. Recently, our group accidentally found that
Anisomycin dramatically inhibited biological behaviors of T cells
and transplantation rejection, and its effect might be superior
first time to reveal the role of Anisomycin in T cell behavior
and function, and application, indicating a potentially applied
possibility to treat some autoimmune diseases and to inhibit
transplantation rejection (Xing et al., 2008). It is well known that
the immunosuppressive agents applied at present to clinic not
only damage hematopoietic and immune system, liver, kidney,
gastrointestinal function, but also result in neural and endocrine
dysfunction. Hence, ideal immunosuppressive agents should be
specific and effective with less adverse effect (Ballow and Nelson,
1997; deMattos et al., 1996). There is, up to now, no detailed
information about toxicity of Anisomycin to mammals, especially
in vivo toxicity. Therefore, it is essential to make a prospective risk
analysis of Anisomycin to evaluate its application perspective. The
objective of the present study is to evaluate in vitro cytotoxicity
of it toward lymphocytes, in vivo toxicity of a single dose and
consecutively intravenous exposure.
2. Materials and methods

2.1. Materials
∗ Corresponding author. Fax: +86 20 85220723.
∗∗ Corresponding author. Anisomycin (molecular weight: 265.30; CAS No.: 22862-76-6; purity >97%
E-mail addresses: tfyxing@jnu.edu.cn (F. Xing), tjliu@jnu.edu.cn (J. Liu). (TLC)) was purchased from Sigma–Aldrich and stored in a dark and dry place at

0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2011.10.001
2 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11
Fig. 1. Chemical structure of Anisomycin.
2–8 ◦ C. It was dissolved in phosphate-buffered saline (PBS), and the concentration
of the stock solution was 20 mg/ml and frozen at −20 ◦ C for subsequent analytical
determination.
2.2. Animals
Balb/c mice of both sexes (4–5 weeks old) were procured from the Center of
Laboratory Animals of Guangdong Province (Guangzhou, China), and were quar-
antined for about 10 d before the initiation of exposure. The mice were housed in
a room at a temperature of 22 ± 3 ◦ C, with 40–70% relative humidity. After quar-
antine, mice of each sex were randomly assigned into different groups. The weight
variation was within ±5% of the mean weight within each sex. All mice were housed
five per group for each sex in stainless steel cages. All husbandry conditions were
performed according to standard guidelines. All animal handling and experimental
procedures were approved by the Animal Care and Use Committee of Guangdong
Medical Animal Center.
2.3. Cytotoxicity
Cytotoxicity was assessed using lymphocytes. Mice were sacrificed and the
lymphocytes were isolated by filtering with 200-m nylon mesh screen. The cells
were washed twice with PBS, and resuspended in RPMI-1640 complete medium
with 10% FBS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2 mM l-glutamine and
50 ␮M 2-mercaptoethanol. The lymphocytes at a density of 2 × 109 cells/ml were
planted into a 96-well plate (200 ␮l/well), and treated with Anisomycin (5.0, 25.0,
50.0, 100.0, 200.0, 400.0 and 800.0 ng/ml) or medium without Anisomycin as a con-
trol. Evaluation of the lymphocyte proliferation for each group was performed in
triplicate by 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)
(Sigma–Aldrich) assay. Plates were incubated at 37 ◦ C in humidified atmosphere
of 5% CO2 for 24 h. At the end of stipulated time following Anisomycin treatment,
5 mg/ml of MTT was added to each well and the cells were incubated for 4 h. Then,
50 ␮l of DMSO (dimethyl sulphoxide) was added to each well, and plates were
shaken for 10 min at 37 ◦ C. The absorbance was recorded at 540 nm wavelength on
a microplate reader (BIO-RAD). Then the cell growth inhibition rate was calculated
as follows (Suman and Kaiser, 2006):
Mean OD of individual test group
Inhibition rate (%) = × 100
Mean OD of control group
OD = optical density.
2.4. Acute toxicity
Anisomycin, as an immunosuppressive agent, was applied to organ transplant
anti-rejection reaction and autoimmune diseases. A very effective way of adminis-
tration of drugs is through an intravenous route. An acute study for calculating LD50
was performed according to the modified Karber’s method (Wang et al., 2010). In
the acute toxicity study, mice were intravenously injected through mouse tail vein
with a total dose of 84, 99, 116, 136 or 160 mg/kg/mice of Anisomycin (0.2 ml per
mouse), respectively. The dosage levels of Anisomycin were determined before the
formal experiments through pretesting, respectively. Mice were randomly divided
into five groups with five of mice for each group. Before administered, mice were
fasted for 4 h. Mice were observed for 14 d for signs of toxicity, including dyspnea,
peripheral vasodilation, less movement and decreased food intake. The mouse body
weight was recorded before and at end of the test or time of mouse death. At the
end of the test, the survival animals were weighed and sacrificed for observation of
their systemic organs. The formula to calculate the LD50 values and 95% confidence
intervals (x) were shown as follows (Wang et al., 2010):
 
log LD50 = xm − d p − 0.5
  
p(1 − p)
−1
X = log log LD50 ± 1.96 × d
n
xm : dosage logarithm for the group of maximal dosage; d: the difference of dosage
logarithm between two of  nearest groups; p: death rate of mice in each group comes
out in decimal fraction; p: sum of the death rate of mice in each group; n: the
number of the mice in each group.
2.5. Four-week intravenous toxicity
For 4-week continuous intravenous administration, mice were intravenously
injected every other day for 4 weeks through mouse tail vein with a total dose of
5, 15 and 60 mg/kg/mice of Anisomycin in PBS, a control group receiving PBS only,
and the dosed volume was 0.2 ml for each mouse. There were ten female mice per
group. The mice were observed twice a day for signs of toxicity and mortality. Body
weights and detailed clinical observations were evaluated weekly. Mice were fasted
for 4 h prior to the terminal sacrifice. Blood samples were obtained by retro-orbital
sinus puncture for biochemistry analysis and peripheral blood micronucleus test.
The liver, kidneys, brain, lungs, heart, spleen, thymus and uterus of the treated mice
were harvested after dissection and weighed. Organ-to-final body weight ratios
were calculated. The heart, liver, spleen, lung and kidney were collected from the
mice for standard histological evaluation.
2.5.1. Biochemical assay
Three mice for each group were bled and the serum was separated for clini-
cal biochemical assay. Clinical biochemical parameters evaluated included glucose
(GLU), blood urea nitrogen (BUN), creatinine (CREA), alanine aminotransferase
(ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and albumin
(ALB). The serum clinical biochemical parameters were determined on an Automatic
Biochemistry Analyzer (7600-020 type, Hitachi).
2.5.2. Histopathology
The organs were collected, containing liver, kidneys, brain, lungs, heart, spleen,
thymus and uterus. All the organs were carefully examined macroscopically and
gross lesions were recorded firstly. Then the organs were weighted after exami-
nation and the organ to body weight ratios were calculated. After being weighted,
the organs were fixed in 4% paraformaldehyde and processed for paraffin embed-
ding by standard methods. The paraffin-embedded sections of 5 ␮m thickness were
cut, stained with hematoxylin and eosin, and examined under a light microscopy.
Finally, the histological alterations were quantified using Image-Pro Plus (Mediacy),
following its manipulation instruction.
2.5.3. Peripheral blood micronucleus
The blood sample of each mouse was taken for peripheral blood smear speci-
mens. The specimens were air-dried, fixed in methanol, stained with Giemsa stain
and double-blindly evaluated for microscopic observation. Five thousand blood cells
per mouse were scored for the presence of micronuclei.
2.6. Sperm aberration
Twenty male mice were randomly divided into control and three dose groups
(5, 15, and 60 mg/kg). Each group included five mice. The mice were intravenously
injected with Anisomycin every other day for 10 d through mouse tail vein, but the
mice in the control group received PBS only. All the mice were sacrificed 30 d after
feeding. Epididymides were separated and put into 5 ml PBS, cut into pieces, filtered
and prepared for smears. The specimens were air-dried, fixed in methanol, and then
stained with Giemsa. One thousand sperms per mouse were recorded and the sperm
aberration rate was calculated.
2.7. Polychromatic erythrocyte micronucleus
Twenty female mice were randomly divided into control and three dose groups
(5, 15, and 60 mg/kg). Each group included five mice, and the mice were intra-
venously injected with Anisomycin twice at 24 h interval. The mice were euthanized
30 h after the treatment, and femurs and tibiae in the mice were removed and puri-
fied from the surrounding muscle tissue and connective tissue. Then, both ends of
intact bones were cut with scissors and the marrow was flushed with PBS in a syringe
with a 0.45-mm diameter needle. Clusters within the marrow suspension were dis-
integrated by vigorously pipetting. The suspension was centrifuged for 5 min at
1200 rpm and resuspended. The smears were made and fixed with methanol. The
smears were stained with Giemsa for 10 min, and used for microscopic evaluation.
Three thousand polychromatic erythrocytes per mouse were scored for the pres-
ence of micronuclei. The polychromatic erythrocytes and micronucleus cells were
calculated.
2.8. Statistical analysis
SPSS 13.0 program was used for the statistical evaluation. Variance homogeneity
was examined using Levene’s test. If Levene’s test indicated no significant deviation
from variance homogeneity, the data were analyzed with one-way analysis of vari-
ance (ANOVA). Otherwise, the Kruskal–Wallis non-parametric ANOVA was used to
analyze the data. The level of significance was considered to be P < 0.05. Values were
expressed as means ± S.D.
 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11 3

Fig. 2. Inhibition rate of Anisomycin on the lymphocyte proliferation. The lympho-
cytes at a density of 2 × 109 cells/ml were planted into a 96-well plate (200 ␮l/well),
and treated for 24 h with Anisomycin (5.0, 25.0, 50.0, 100.0, 200.0, 400.0 and
800.0 ng/ml) or medium without Anisomycin as a control. Evaluation of the lym-
phocyte proliferation for each group was performed in triplicate by MTT assay. Drug
concentration that would give 50% inhibition (IC50 ) of the lymphocyte proliferation
was determined from the dose–response curve.
Fig. 3. Mean body weight changes of the mice injected intravenously with the dif-
ferent concentrations of Anisomycin. The mice were intravenously injected every
other day for 4 weeks through the mouse tail vein with a total dose of 5, 15 and
60 mg/kg/mice of Anisomycin, and a control group received PBS only. There were
ten mice for each group. Their body weights were evaluated weekly. Means and stan-
dard deviations are presented. *P < 0.05, **P < 0.01, compared with the corresponding
control group.
3.3. Clinical signs and mortality

In the 60 mg/kg group, two mice were found dead on day 14

3. Results
3.1. Cytotoxicity
Sensitivity of original generation of the lymphocytes to Ani-
somycin was defined on the basis of IC50 value obtained from MTT
assay at the concentration range from 5.0 to 800.0 ng/ml. As shown
in Fig. 2, the inhibitory rate of the lymphocyte proliferation was
increased with enhanced concentration of Anisomycin, appearing
in a dose-related relationship. The percentage of the lymphocyte
viability was calculated and the dose–response curve was fitted to
a Hill equation using a non-linear least square method and the IC50
value was 25.44 ng/ml for mouse lymphocytes.
3.2. Acute toxicity
and one mouse dead on day 21, and post mortem examination of
the mice that died during the study did not reveal any recognized
cause of death. The mice in this group exhibited the following clin-
ical findings: thin appearance, fur-upright and less movement. No
clinical signs or death were found in other dose groups and all the
mice appeared to be in good health at the time of sacrifice.
3.4. Body weight change
Compared with the control group, the mouse body weight was
significantly decreased by 60 mg/kg of Anisomycin in a dose-related
manner, slight and transient decrease of the mouse body weight
was observed in the 15 mg/kg group, but there was no significant
difference of the mouse body weight in 5 mg/kg group (Fig. 3). The
result suggests that the observed decrease is related to the treat-
ment of the high dose of Anisomycin.

All the tested mice were found dead within 1 h after the intra- 3.5. Biochemical change
venous administration of 160 mg/kg of Anisomycin, eight mice
dead at 136 mg/kg, four mice dead at 116 mg/kg and one mouse From serum clinical biochemical analysis, the significant differ-
dead at 99 mg/kg (Table 1). Their depression and huddle up, less ence was observed between the highly dosed group (60 mg/kg)
movement, piloerection and dyspnea were observed. At necropsy, and the control group. The significant increase in ALT level was
peripheral vasodilation on the skin appeared and hemorrhagic exhibited. Consistent with the alteration of ALT, AST level was
spots on the surface of lungs of all the dead mice were noted. The also augmented. But the significant decrease in GLU level occurred.
survival mice had a transient reduction in their body weight 2–3 d These abnormalities were found only in the 60 mg/kg group. Other
after the administration of Anisomycin, and then recovered rapidly. parameters, including ALP, ALB, BUN and CREA, were not affected
The LD50 value of Anisomycin was 119.64 mg/kg with an approxi- by the treatment of the used doses of Anisomycin (Table 2). As to
mate 95% confidence interval ranging from 110.94 to 129.27 mg/kg. GLU, the alteration of it may result from hepatocyte damage and
decrease of food intake. The data indicate that the high dose of
Anisomycin may result in the hepatocyte damage of the mice.
Table 1
Experimental outcome of the acute test. 3.6. Organ index
Doses of Number of Number of
Anisomycin animals animal Relative organ weight data are summarized in Table 3. The dose-
(mg/kg) dead/dosed related increase in spleen-to-body weight ratio was observed in
84 10 0
both 15 mg/kg and 60 mg/kg groups, but the dose-related decrease
99 10 1 in thymus-to-body weight ratio was done in all the treated groups.
116 10 4 The increases in liver, lung and brain to body weight ratios were
136 10 8 also noted only in the 60 mg/kg group. In the ratio changes, the
160 10 10
alteration of spleen and thymus was much bigger. However, the
4 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11

Table 2
Serum biochemical values in female mice given Anisomycin for four weeks.

Parameter Dose of Anisomycin (mg/kg)

0 5 15 60

ALT (U/l) 30.33 ± 6.43 32.33 ± 8.62 33.00 ± 5.20 41.33 ± 0.58*
AST (U/l) 89.67 ± 18.61 80.00 ± 25.63 94.33 ± 29.11 185.33 ± 23.35**
ALP (U/l) 118.33 ± 43.10 146.67 ± 27.21 138.33 ± 29.77 106.00 ± 10.82
ALB (g/l) 28.43 ± 3.81 30.03 ± 4.11 31.60 ± 1.22 26.40 ± 3.78
GLU (mmol/l) 5.54 ± 0.12 6.89 ± 2.23 4.60 ± 0.67 3.06 ± 0.77*
BUN (mmol/l) 11.35 ± 2.92 11.77 ± 2.25 11.45 ± 1.10 12.74 ± 0.42
CREA (␮mol/l) 11.33 ± 1.53 10.33 ± 0.57 12.67 ± 1.53 12.00 ± 1.00

ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ALB, albumin; GLU, glucose; BUN, blood urea nitrogen; CREA, creatinine.
Means and standard deviations are presented.
*
Significantly different from control group at P < 0.05.
**
Significantly different from control group at P < 0.01.

Table 3
Mean organ-to-body weight ratiosa in the female mice dosed with Anisomycin.

Organ Dose of Anisomycin (mg/kg)

0 5 15 60

Spleen 6.08 ± 0.65 6.55 ± 0.54 7.38 ± 0.79** 15.94 ± 0.73**

Heart 6.26 ± 0.76 6.12 ± 0.79 6.15 ± 0.52 6.36 ± 0.57
Liver 44.06 ± 2.48 45.38 ± 4.64 45.43 ± 3.03 49.74 ± 3.97**
Lungs 7.64 ± 0.52 7.49 ± 0.83 7.86 ± 1.15 12.73 ± 2.50**
Kidneys 12.67 ± 0.82 12.43 ± 0.59 12.24 ± 0.88 13.00 ± 0.90
Brain 20.82 ± 1.00 20.54 ± 0.63 21.31 ± 1.41 22.34 ± 1.75*
Uterus 2.40 ± 0.21 2.45 ± 0.25 2.19 ± 0.21 2.28 ± 0.43
Thymus 2.51 ± 0.40 2.09 ± 0.24* 1.82 ± 0.56** 0.42 ± 0.14**

Means and standard deviations are presented.

a
Organ weight/body weight × 1000.
*
Significantly different from control group at P < 0.05.
**
Significantly different from control group at P < 0.01.

alteration of kidneys and uterus was not apparent. It is specu- Table 4

Effect of Anisomycin on the aberration rate of sperms in mice.
lated that the increases in liver, lung and brain to body weight
ratios might be related to both occurrence of organ inflammation Group Number of animal Aberration rate (%)
and transient loss of mouse body weight by the treatment of Ani- (mean ± S.D./3000
cells)
somycin.
Control 5 1.69 ± 0.06
3.7. Pathological finding 5.0 mg/kg 5 1.78 ± 0.15
15.0 mg/kg 5 1.79 ± 0.14
60.0 mg/kg 5 2.15 ± 0.16**
At necropsy, hyperemia spots on the surface of lungs and spleen
Means and standard deviations are presented.
were observed in the 60 mg/kg group, and the slightly swelling **
Significantly different from control group at P < 0.01.
of liver was also done in the 60 mg/kg group. But there were no
changes in the control, 5 mg/kg and 15 mg/kg groups.
Histopathological study of spleen showed the profile of splenic
Compared with the control, a significant increase in the abnormal
corpuscles in the spleen became indistinct, the red pulp expanded,
rate of sperms was observed in the 60 mg/kg group (Table 4). The
and the number and volume of splenic megakaryocytes were sig-
types of sperm abnormalities are presented in Fig. 9.
nificantly increased in the 60 mg/kg group (Fig. 4). It was revealed
in the 60 mg/kg group that a bulk of cells and fluid leaked out into
alveolar wall interspaces and alveolar spaces in the lungs, resulting 3.9. Peripheral blood micronucleus rate
in the much thicker alveolar wall, alveolar space consolidation and
inflammatory cell infiltration (Fig. 5). In the liver, hepatic lobules The results of peripheral blood micronucleus assay are sum-
appeared to be indistinct, hepatic sinusoid interspaces disappeared marized in Table 5. There were no significant changes in the
for the swollen hepatocytes. Some of central venules markedly frequencies of micronucleus at any dose level.
expanded, with the infiltration of a large number of inflammatory
cells (Fig. 6). In kidney, the number of renal glomeruli in the renal
cortical area seemed to be reduced, glomerular capsule interspaces Table 5
disappeared, which might result from swollen glomerulus cells Peripheral blood micronucleus rate in mice treated with Anisomycin.
(Fig. 7). However, there were no changes in the control, 5 mg/kg and Dose (mg/kg) Number of animal Micronucleus rate (‰)
15 mg/kg groups. No abnormal alteration was found in the heart at (mean ± S.D./5000
all the used doses of Anisomycin (Fig. 8). cells)

0 10 0.80 ± 0.27
3.8. Sperm aberration rate 5 10 0.78 ± 0.20
15 10 0.84 ± 0.16
60 7 0.86 ± 0.10
The high dose of Anisomycin (60 mg/kg) induced various types
of aberration sperms, such as hookless or amorphous heads. Means and standard deviations are presented.
 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11 5

Fig. 4. Representative histopathological changes of the spleen in the Anisomycin-treated mice. (A and B) Morphology of the spleen in the PBS-treated group was observed
under a microscope at the amplification of 100-fold (A) and 250-fold (B). The long arrow shows the distinct splenic corpuscles and red pulp, and the head arrow the splenic
megakaryocytes. (C and D) The profile of splenic corpuscles in the spleen became indistinct, the red pulp expanded (long arrow), and the number and volume of the splenic
megakaryocytes (head arrow) were significantly increased in the 60 mg/kg group, as observed under a microscope at the amplification of 100-fold (C) and 250-fold (D). (E
and F) 15 of randomly visual fields for each group were quantified for number of the splenic megakaryocytes and area of the splenic megakaryocytes (magnification: 250×).
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3.10. Polychromatic erythrocyte micronucleus rate Table 6

Effect of Anisomycin on the polychromatic erythrocyte micronucleus formation in
mice.
There was no statistically significant increase in the micronu-
cleus rate in the 5 mg/kg and 15 mg/kg groups. But in the 60 mg/kg Group Number of animal Micronuclear rate (‰)
(mean ± S.D./3000
group, the polychromatic erythrocyte micronucleus was readily
cells)
formed (Fig. 10) and the statistically significant increase in the
micronucleus rate was found (Table 6). Control 5 1.67 ± 0.56
5.0 mg/kg 5 2.67 ± 0.58
15.0 mg/kg 5 3.67 ± 0.58
60.0 mg/kg 5 10.33 ± 1.5**
4. Discussion
Means and standard deviations are presented.
**
Significantly different from control group at P < 0.01.
The recent studies show that Anisomycin significantly inhibits
the biologic behaviors of T cells and transplantation rejection, pro-
viding important evidence for the development and application of
6 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11

Fig. 5. Representative histopathological changes of the lungs in the Anisomycin-treated mice. (A and B) Morphology of the lungs in the PBS-treated group was observed
under a microscope at the amplification of 100-fold (A) and 250-fold (B). The long arrow shows the alveolar wall, the short arrow the terminal bronchiole and the head
arrow the less infiltrating cells in alveolar wall interspaces. (C and D) In the 60 mg/kg group, a bulk of cells leaked out into alveolar wall interspaces and alveolar spaces in
the lungs, resulting in the much thicker alveolar wall, alveolar space consolidation (long arrow), the blockage of the terminal bronchiole (short arrow) and the inflammatory
cell infiltration (head arrow), as observed under a microscope at the amplification of 100-fold (C) and 250-fold (D). (E and F) 15 of randomly visual fields for each group were
quantified for thickness of the alveolar wall and number of the terminal bronchiole occlusion (magnification: 250×).

Anisomycin as a novel immunosuppressant. But little study has
tested the safety/toxicity of Anisomycin, especially in vivo eval-
uation. Therefore, this study was conducted to test the safety of
Anisomycin mainly in four aspects, including cytotoxicity, genetic
toxicity, acute and subchronic intravenous toxicity.
It is well known that Cyclosporine (CsA) is an effective immuno-
suppressive agent and widely applied to treat organ transplant
rejection and autoimmune diseases. The IC50 value for CsA is
0.13 ␮M in mouse lymphocytes and 1.00 ␮M in human lympho-
cytes, respectively (Carfi et al., 2007). Our data showed that the IC50
value for Anisomycin was 0.09 ␮M (25.44 ng/ml) in mouse lympho-
cytes. On the basis of this toxicological datum we consider that the
inhibitory effect of Anisomycin on mouse lymphocytes is stronger
than CsA, which further supports the obtained evidence that the
in vitro or in vivo inhibition of Anisomycin on the behaviors of T
lymphocytes is superior to methylprednisolone or CsA, especially
on original generation of T lymphocytes (Xing et al., 2008). It should
be noted that IC50 for the same drug is much different in various
species. For example, the IC50 for benzopyrene is more than 160 ␮M
in mouse lymphocytes and 12.82 ␮M in human lymphocytes. And
the IC50 for CsA is 10 times higher in human than in mouse lym-
phocytes, while the IC50 for verapamil is similar between mouse
and human lymphocytes (Carfi et al., 2007).
In the acute toxicity test, our results indicated that the LD50
for Anisomycin was 119.64 mg/kg. The survival mice with slightly
poisoning signs had a transient reduction in body weight, which
was remarkable between the second day and the third day, and
then rapidly recovered. Contrasted with CsA, Anisomycin had less
 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11 7

Fig. 6. Representative histopathological changes of the liver in the Anisomycin-treated mice. (A and B) Morphology of the liver in the PBS-treated group was observed under
a microscope at the amplification of 250-fold (A) and 400-fold (B). The long arrow shows the central venules, the short arrow the hepatic sinusoid interspace and the head
arrow the less infiltrating cells. (C and D) In the 60 mg/kg group, the hepatic lobules appeared to be indistinct, the hepatic sinusoid interspaces disappeared (short arrow) for
the swollen hepatocytes. Some of the central venules markedly expanded (long arrow) with the infiltration of a large number of inflammatory cells (head arrow), as observed
under a microscope at the amplification of 250-fold (C) and 400-fold (D). (E and F) 15 of randomly visual fields for each group were quantified for number of the infiltrating
lymphocytes around central veins (magnification: 400×) and area of the central veins (magnification: 250×).

toxicity than CsA. Ryffe et al. reported that the LD50 of CsA given
intravenously in mice was 107 mg/kg (Ryffel et al., 1983). It should
be emphasized that there are big differences of the LD50 for differ-
ent administration routes. The LD50 of CsA given intravenously to
mice was 107 mg/kg, whereas the LD50 of CsA given orally to them
was 2329 mg/kg. Hence, LD50 of Anisomycin for various species
and different administration routes needs to be further investi-
gated. The mainly found primary signs that occurred in the high
dose Anisomycin-treated mice, such as mouse depression and hud-
dle up, less movement, fur-dim, piloerection, hyperventilation and
dyspnea, were as well observed in the acute toxicity study of CsA
(Ryffel et al., 1983) and toxic signs became apparent immediately
after drug administration. Differently, the above signs were much
more serious under the same given dose in CsA-treated mice than in
Anisomycin-treated mice (Xing et al., 2008), and other signs includ-
ing mouse hunched posture and strong muscular spasm existed
in the CsA-treated mice other than the Anisomycin-caused signs.
Nevertheless, the CsA-treated mice were looking very irritable.
Consistent with the observed clinical signs of the hyperventilation
and dyspnea, there were vasodilation and hemorrhagic spots on
the surface of lungs of all the dead mice at necropsy. It suggests
that the acute toxicity of Anisomycin might be at lest relevant to
circular system dysfunction. Similarly, CsA can also produce the
vascular toxicity through directly affecting vascular smooth mus-
cle cells to cause endothelial dysfunction, which may be relevant
to the development of arterial hypertension (Berkenboom et al.,
1996; Hennersdorf et al., 2002). Regarding this, it was explored
that in isolated rat arteries CsA affects endothelial function by
8 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11

Fig. 7. Representative histopathological changes of the kidneys in the Anisomycin-treated mice. (A and B) Morphology of the kidneys in the PBS-treated group was observed
under a microscope at the amplification of 100-fold (A) and 250-fold (B). The arrow shows the renal glomerulus and renal glomerular capsule interspace. (C and D) In the
60 mg/kg group, the number of glomerulus in the renal cortical area seemed to be reduced, glomerular capsule interspaces disappeared (arrow), which might result from
swollen glomerulus cells, as observed under a microscope at the amplification of 100-fold (C) and 250-fold (D). (E and F) 15 of randomly visual fields for each group were
quantified for number of the renal glomerulus and interspace of the glomerular capsules (magnification: 250×).

uncoupling the acetylcholine-mediated relaxation and interfer-
ing with an endothelium-mediated pathway that regulates 45Ca2+
uptake by a mechanism reversed by an l-arginine-dependent cGMP
generation (Gallego et al., 1994).
In the 4-week intravenous toxicity study, the body weight of
the mice was decreased, three of ten mice were dead, and some
of the above clinical signs were also found only in the high-dose
group of Anisomycin (60 mg/kg). Furthermore, a statistically sig-
nificant increase of ALT/AST and decrease of GLU were found only
in the high-dose group. ALT is considered as a liver specific enzyme
and its increase hints increased cell membrane permeability and
even hepatocyte necrosis. AST exists most in cardiocytes, then in
hepatic cells. Because in the high dose group there was not any
alteration of histopathology in the heart, but the obvious inflam-
mation reaction in the liver that suggests hepatocyte injury, their
increases combined with the histopathologic alteration of the liver
and heart indicate that the alteration of AST mainly results from the
liver, rather than the heart, and that in vivo intravenous adminis-
tration of the high dose of Anisomycin results in the liver injury. In
addition, ALP activity is considered as a marker of cholestasis. The
levels of ALB and GLU in serum reflect synthetic and metabolic func-
tion of liver. Our data demonstrated that the levels of ALP, ALB and
GLU were not changed markedly by the treatment of the high, mid
and low doses of Anisomycin, indicating that the extent of the liver
impairment caused by the high dose of Anisomycin is not enough to
give rise to decrease of the synthetic function and cholometabolism
aberrance in it. Compared to Anisomycin, the cultured rat hepato-
cytes exposed to CsA exhibited concentration-dependent signs of
apoptotic cell injury, including chromatin condensation and frag-
mentation, increased caspase-3 activity, and release of cytosolic
 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11 9

Fig. 8. Representative histopathological changes of the heart in the Anisomycin-treated mice. (A and B) Morphology of the heart in the PBS-treated group was observed
under a microscope at the amplification of 100-fold (A) and 250-fold (B). (C and D) No abnormal alteration was found in the heart at all the used doses of Anisomycin, as
observed under a microscope at the amplification of 100-fold (C) and 250-fold (D).

Fig. 9. Influence of in vivo administration of Anisomycin on sperm morphology in mice. Twenty male mice were randomly divided into control group (A) and 5 mg/kg group
(B), 15 mg/kg group (C) and 60 mg/kg group (D). Then they were intravenously injected with Anisomycin every other day for 10 d through the mouse tail vein. Compared with
the control, a significant increase in the abnormal sperms, such as hookless or amorphous heads, was observed in the 60 mg/kg group, indicating the high dose of Anisomycin
may induce the various types of aberration in sperm morphology.
10 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11

Fig. 10. Effect of in vivo administration of Anisomycin on polychromatic erythrocyte micronucleus. Twenty male mice were randomly divided into control group (A) and
5 mg/kg group (B), 15 mg/kg group (C) and 60 mg/kg group (D). And the mice were intravenously injected with Anisomycin twice at the 24 h interval. There was no statistically
significant increase in the micronucleus formation in both the 5 mg/kg and the 15 mg/kg groups. But in the 60 mg/kg group, statistically significant increase in the micronucleus
formation (arrow) was found.

lactate dehydrogenase, showing hepatocellular toxicity of it (Grub
et al., 2003).
BUN and CREA are usually used as parameters of renal func-
tion, in which serum CREA is more sensitive than BUN for detecting
nephropathy (Travlos et al., 1996). They were not markedly altered
in the treatment of the high, mid and low doses of Anisomycin.
However, in kidney the number of nephroglomerulus in the renal
cortical area seemed to be reduced and glomerular capsule inter-
spaces disappeared in the high dose group, suggesting that the
extent of the renal injury is not enough to cause the remarkable
decrease of filtration function of nephroglomerulus. Compared to
Anisomycin, CsA has a severe glomerular toxicity. An extensive ele-
vation in the activities of xanthine oxidase was noted in the renal
tissue of the CsA-administered rats, which might be correlated
with significant increase in the levels of plasma lipid peroxidation
with high protein carbonyl contents and 3-nitrotyrosine forma-
tion coupled with diminished protein thiols (Amudha et al., 2007).
Another study proves that the glomerular toxicity of CsA might be
mediated by the recruitment of vasoconstricting peptides (Castello
et al., 2005). But until now, there lacks any research of mecha-
nism involving Anisomycin-induced toxicity. Therefore, it remains
to be elucidated. In lungs, the high dose of Anisomycin results in
the much thicker alveolar wall, alveolar space consolidation and
inflammatory cell infiltration. This pathological alteration may be
related to the clinically observed mouse hyperventilation and dys-
pnea.
Histopathological study of spleen showed that the number and
volume of splenic megakaryocytes were significantly increased
in the treatment of the high dose Anisomycin. This seems to be
consistent with the observed enhancement of the spleen-to-body
weight ratio in the mice treated with the same dose of Anisomycin.
Splenic megakaryocytes might be associated with thrombopoiesis
in mice. What is significance of this change? Whether is the
Anisomycin-stimulated spleen enlargement implicated in direct or
indirect immune reaction in spleen? This deserves further study.
On the contrary, we noted that Anisomycin resulted in the atro-
phy of thymus. The thymus-to-body weight ratios in the high, mid
and low dose group of Anisomycin all were markedly decreased.
Considered from this point, Anisomycin might become a promised
immunosuppressant to be applied in autoimmune diseases and
transplantation rejection. In addition, Anisomycin exhibited no
influence on the heart at the tested doses. But CsA has myocar-
dial toxicity through inhibition of calcium ATPase and nitric oxide
synthase activities, and the inhibition can be attenuated in vitro by
fructose-1,6-diphosphate (Hutcheson et al., 1995).
Sperm aberration may be caused by naturally occurring errors
during the differentiation or an abnormal chromosome (Bruce et al.,
1974) and Y chromosomes have an important role in this process
(Krzanowska, 1976; Styrna et al., 1991). According to some related
reports, the exogenous factors induced alterations in sperm mor-
phology by point mutations (Chauhan et al., 2000; Narayana et al.,
2002). CsA-induced oxidative stress leads to the structural and
functional damages in the testicular tissue and sperm quality of
rats (Turk et al., 2007). In the present study, a slightly increased
percentage of abnormal sperm occurred in the mice treated by
the high-dose Anisomycin, showing that the high-dose Anisomycin
may affect spermatogenic tissues. In other aspect, the frequency
of the micronucleus formation in bone marrow cells had also a
significant increase in the high dose group of Anisomycin, but no
changes happened in peripheral blood cells at all the tested doses.
It is well known that micronucleus assay is an effective way for
the identification of genotoxic effects (Heddle, 1973). Micronuclei
may be formed from clastogenic and aneugenic effects. At anaphase
of mitosis, chromosome fragments or whole chromosomes lagged
behind are not incorporated into daughter nuclei, forming single or
multiple micronuclei and distributing in the cytoplasm. According
 Z. Tang et al. / Toxicology Letters 208 (2012) 1–11
to our results, higher concentration of Anisomycin has a proba-
ble genotoxic effect on mouse bone marrow, but it does not affect
peripheral blood cells. Maybe, administration time of Anisomycin
within one month might be too short to produce genotoxic effect on
peripheral blood cells. Additionally, even therapeutic CsA is capa-
ble of generating the pancreatic toxicity in rats (Schulz et al., 1991).
And neurotoxicity is a recognized complication with the use of CsA
in bone marrow and organ transplantation patients. Most common
symptoms are seizures and altered mental status which are usually
transient (Pace et al., 1995).
In summary, Anisomycin has no significant side effects at
effectively therapeutic doses, compared with CsA. Neverthe-
less, over-dosage leads to various toxicities, particularly pulmo-,
nephro- and hepato-toxicity as well as slight micronucleus for-
mation and sperm aberration. Therefore, Anisomycin, which will
be hopefully developed and applied as an efficacious immunosup-
pressant in clinic, needs further test for other toxicity, including
neurotoxic, myelotoxic, teratogenic, mutagenic and carcinogenic
effects, and elucidation of its toxic mechanism.
Conflict of interest statement
The authors declare that they have no conflicts of interest.
Acknowledgements
This project was supported by the National Natural Sci-
ence Foundation of China (No. 81172824, No. 30471635, No.
30971465), the Fundamental Research Funds for the Central Uni-
versity (21610608) and “211” project grant.
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