Mechanism
of Cytotoxicity of Ribavirin in the Rat Bone Marrow and Testis
URBAN J. A. D’SOUZA* AND NARAYANA
K.**
Departments of Physiology* and Anatomy,**
Centre for Basic Sciences,
Kasturba Medical College,
Bejai, Mangalore – 575 004
(Received on February 15, 2002)
Abstract:
Mechanisms of cytotoxicity of an antiviral drug, ribavirin was
studied in the rat home marrow and testis. Ribavirin at the dose
levels of 20, 100 and 200 mg/kg was treated (i.p.) either as single
(for bone marrow) or 5 (for testis) treatments. Bone marrow smears
were obtained at 24, 48 and 72 h following the exposure and stained
with the May-Gruenwald-Giemsa combination. Smears were screened
for the incidence of dead cells, and at 24 h, a total of 2000
erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) (P/N).
Step 19 spermatids/stage VII tubule, dividing cells (meiotic figures)/stage
XIV tubule and the incidence of tubules with dead cells were counted
in periodic acid – Schiff’s reaction and haematoxylin (PAS-H)
stained testicular sections on days 14, 35, 70 and 105. Significant
decrease in the step 19 spermatids and meiotic figures, and increase
in the incidence of tubules with dead cells (P<0.05-0.01) were
observed mainly on days 14 and 35. The cell death was observed
in the bone marrow mainly at the two higher dose levels and significant
decrease (P<0.001) in P/N ratio was observed. This present
study concludes that the cytotoxicity of ribavirin in these two
target cell-lines is due to the induction of cell death and prevention
of the cell division.
Key words: ribavirin cell death bone
marrow testis apoptosis
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Although
ribavirin is one of the approved antiviral drugs indicated for
various viral infections, its toxicity has limited its therapeutic
use (1). But, it is a more appropriate drug to treat the respiratory
syncytial virus (RSV) infection and hepatitis, either alone or
in combination with interferon alfa (2, 3). Ribavirin induces
dyspnoea, chest soreness, worsening of respiratory status and
reversible progressive anaemia in humans (4). On the other hand,
in experimental animal, it acts as a teratogen (5) and imparts
cytotoxicity to developing limb bunds and affects the nucleic
acid synthesis (6). In monkeys, it produces marrow erythroid hypoplasia,
megakaryocytic hyperplasia, phagocytosis of erythroid elements,
vacuolation of erythroid precursors (7) and dose dependent suppression
of the late erythroid precursors in the bone marrow (8). Ribavirin
also decreases the platelet and leucocyte counts in cats (9),
and the polychromatic erythrocytes (PCEs) in mice (10). Further,
ribavirin is known too decrease the sperm count in the rat epididymal
suspension with some relation to the dose levels and time intervals
(11). Even though the cytotoxic effects of ribavirin are how increasingly
clear, the mechanisms by which this drug exerts its cytotoxicity
are not known. The present study investigates the mechanisms of
cytotoxicity of ribavirin in the rat bone marrow and testis.
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METHODS
In
this present study, albino Wistar rats (11-13 weeks old; 130-160
g body weight) were used. They were maintained under standard
laboratory conditions with the free access to food and water.
Cell death in the bone marrow
Rats
(3 males+ 2 females/group) were treated with 0.1 ml water (control),
20, 100 and 200 mg/kg of ribavirin as single i.p. treatment (Table
I). Dose selection was based on previous studies (10, 11), which
were equivalent to 1/265, 1/53 and 1/26.5 of LD50 respectively.
Animals were anaesthetized (pentobarbital sodium, 45 mg/kg, i.p.)
at 24, 48 and 72 h post exposure and the bone marrow was aspirated
from the femora into 2 ml of 5% bovine albumin in phosphate buffered
saline (pH 7.2). The suspension was then centrifuged at 1000 r.p.m.
for 5 minutes and the smears were prepared from the pellet on
clean glass slides. The smears were stained as previously described
(12) with the May-Gruenwald-Giemsa combination. Briefly, the smears
were fixed in methanol for five minutes, immersed in May-Gruenwald
solution for 5 minutes, then in buffered May-Gruenwald (pH 6.8)
for 5 minutes and the slides were washed with buffer, dried and
mounted. Slides were coded and evaluated for the cell death.
Only
those cells which showed fragmentation were identified as dead
cells. The cells with the pyknotic nuclei were not included the
incidence of cell death was noted as ‘0’ for no cell death (smear
without any dead cells) ‘+’ for few dead cells (5—7 cells) and
‘++’ for moderate number of dead cells (more than 7 cells)/ 2000
PCEs observed in the smear. In same smears, only at 24 h, a total
of 2000 PCEs and consequently found normochromatic erythrocytes
(NCEs) were counted to obtain the ratio of PCEs to NCEs (P/N).
Cell death in the testis
To
evaluate the mechanism of cytotoxicity of ribavirin in the testis,
the animals (n = 5/group) were treated with ribavirin for 5 days
at the dose levels of 20, 100 and 200 mg/kg intraperitoneally.
Following the last exposure, on days 14, 35, 70 and 105, the tests
were removed by laparotomy under anaesthesia. The tunica vaginalis
was removed from the testis, the two testicular poles were punctured
by 24G needle and immediately fixed in Bouin’s fluid for 18 h
(13). Tissues were then processed for paraffin embedding and 5
thick sections were cut (Richert-Jung microtome, Germany) and
stained with periodic acid-Schiff’s reaction and haematoxylin
(14, 15).
One
hundred seminiferous tubular cross sections per animal were screened
to identify the cell death. Darkly stained cells were distinguished
from dark-type spermatogonia and the tubules in which dead cells
observed were counted. In each animal, 10 stage VII tubules were
selected and the step 19 spermatids were counted and expressed
as per tubule incidence. This is a most sensitive parameter to
evaluate the cell death in the testis (16). Ten stage XIV tubular
cross sections per animal were selected and the meiotic figures
were counted and expressed as per tubule incidence.
For
each group, mean and SE were calculated and the data were analysed
by the Mann-Whitney ‘U’ test.
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RESULTS
Analysis
of bone marrow smears from ribavirin treated rats revealed the
cell death in the marrow. These cells were burst and found as
numerous bodies, which darkly stained with the May-Gruenwald Giemsa
combination (Fig. I). Very few dead cells were seen in 20 mg/kg
and 100 mg/kg dose levels, but at 200 mg/kg, moderate number of
dead cells were observed (Table I). The P/N ratio was decreased
(P<0.001) almost in a dose independent pattern (Fig. 2).
Fig.1
click for full view |
Fig.2
click for full view |
TABLE
I: Grading the of cell death in the bone marrow of control and
ribavirin treated rats*.
|
Drug
/ dose
|
24
h
|
48
h
|
72
h
|
|
Control
|
0
|
+
|
0
|
|
Ribavirin
20 mg/kg
|
+
|
+
|
+
|
|
Ribavirin
100 mg/kg
|
+
|
++
|
+
|
|
Ribavirin
200 mg/kg
|
++
|
++
|
++
|
On
the other hand, in the testis, the cell death was significant
and number of tubules with dead cells increased at two higher
dose levels except on day 105. The maximum effect was seen on
day 14 however, the effect was dose-dependent. These dead cells
were darkly stained and found in the seminiferous epithelium as
distinct bodies (Fig. 3). The incidence of step 19 spermatids/stage
VII tubule decreased at all the dose levels except on day 105.
The decrease was more or less dose-dependent on days 14 and 70,
but not on day 35 (Table II). This decrease was maximum on day
14. Frequency of dividing cells (meiotic figures) decreased but
without any significance at 20 mg/kg. The meiotic figures decreased
on days 14 and 35 in animals treated with 100 mg/kg. In 200 mg/kg
treated rats, the meiotic figures decreased significantly on all
sample times. This effect was dose-dependent (Table II). On day
70 only the highest dose induced the decrease in step 19 spermatids,
decreased the number of dividing cells and increased the incidence
of tubules with the cell death.
Fig.3
click for full view |
TABLE
II: Incidences of step 19 spermatids, meiotic figures and cell
death in the testis exposed to ribavirin.
|
Parameter
|
Drug/dose
|
14
|
35
d
|
70
d
|
105
d
|
|
|
Control
|
148.60±2.21
|
149.20±4.41
|
139.20±1.86
|
139.40±2.71
|
|
Step19spermatids/
|
20
mg/kg
|
134.60±2.06**
|
131.80±1.86**
|
130.40±1.91
|
139.40±2.32
|
|
Stage
VII tubule
|
100
mg/kg
|
87.80±1.69**aa
|
115.80±3.67**aa
|
130.20±4.21
|
137.60±2.80
|
|
|
200
mg/kg
|
85.60±2.99**bb
|
122.00±2.21**b
|
112.20±1.93*bbc
|
134.80±2.25
|
|
|
|
|
|
|
|
|
|
Control
|
24.60±0.93
|
2.80±1.28
|
27.00±1.22
|
28.20±1.66
|
|
Dividing
cells/
|
20
mg/kg
|
22.00±1.00
|
23.00±0.71
|
26.40±0.51
|
23.80±1.39
|
|
Stage
XIV tubule
|
100
mg/kg
|
15.80±0.80**aa
|
18.60±0.51**aa
|
25.00±1.01
|
27.20±1.36
|
|
|
200
mg/kg
|
11.60±0.51**bbcc
|
16.00±0.71**bbc
|
20.00±1.18*bbc
|
20.80±1.02**c
|
|
|
|
|
|
|
|
|
|
Control
|
0.40±0.24
|
0.60±0.40
|
0.61±0.24
|
0.82±0.37
|
|
%
Tubules with dead cells
|
20
mg/kg
|
1.00±0.32
|
1.20±0.37
|
0.59±0.40
|
1.20±0.37
|
|
|
100
mg/kg
|
4.20±0.58**aa
|
6.00±0.71**aa
|
1.80±0.37*
|
0.80±0.36
|
|
|
200
mg/kg
|
4.60±0.51**bb
|
9.60±0.40**bbcc
|
4.20±0.58**bbc
|
0.81±0.38
|
Mean±
SE
*P<0.05, **P<0.01, control vs
treated
a) aP<0.01, 20 mg/kg vs
100 mg/kg
b) P<0.05, bbP<0.01,
20 mg/kg vs 200 mg/kg
c) P<0.05, ccP<0.01,
100mg/kg vs 200 mg/kg
d) T 3 parameters in same animals, 5
animals/each treatment and sample time
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DISCUSSION
Ribavirin
inhibits the activity of inosine monophosphate dehydrogenase (IMPDH),
that catalyses the oxidation of inosine 5’ monophosphate to xanthosine
5’ monophosphate (1). It is one of the key enzymes of de novo
guanine nucleotide biosynthesis (17). As IMPDH inhibitors selectively
reduce the guanylate concentration, the incomplete guanosine triphosphate
level possibly down-regulates the G-protein function, a process
that hinders the cell growth or induces the apoptotic cell death
(18), such effects of ribavirin were not previously reported.
Decrease in erythrocyte and leucocyte counts in cats (7) and PCEs
in mice by ribavirin (10) are well known, but the mechanism that
brings out these phenomena has not been investigated. In the present
study, ribavirin was found to elevate the incidence of dead cells
in the bone marrow smear and in the testis. This cell death was
similar to the apoptosis, and the damaged cells appeared as darkly
stained bodies in the marrow (Fig. 1). Number of such cells increased
when the dose was increased. Thus, this cell death is a direct
reason for decreased erythrocyte count in the marrow or sperm
count in the epididymis. However, whether the cell death induced
by ribavirin is apoptosis or not should be confirmed further.
But, simple staining solutions such as haematoxylin (19) and May-Gruenwald-Giemsa
combination (20) could confirm the apoptotic cell death accurately.
Therefore, the present data confirm the ribavirin induced cell
death in the marrow and testis is by apoptosis. The decreased
P/N ratio (Fig. 2) indicates the death of blast (nucleated) cells
in the marrow, which otherwise convert into PCEs. Hence, in the
bone marrow, the PCEs (early erythrocytes) decrease in number
and as a result the number of mature erythrocytes also decrease.
However, soon after the treatment, the existing PCEs during treatment
get convert into NCEs, and still later, they also may decrease
due to the inhibition of the entire erythropoeisis. This was the
reason for decreased erythrocyte and possibly for leucocyte counts
observed in the previous studies (8, 9) of animals treated with
ribavirin.
In
the seminiferous tubules, the cells stained darkly with haematoxylin
and the incidence of tubules with such cells increased as the
dose was increased. Probably, ribavirin may cause chromosomal
damage, that begins as DNA strand breaks and cross links, a mechanism,
that is known to induce the apoptotic cell death (21). The chromosomal
damage is known to occur in the bone marrow cells of mice exposed
to multiple treatments of ribavirin (10). Whether ribavirin damages
the chromosomal integrity in the testis is not known, even though
it induces the point mutation in the rat testis as evaluated indirectly
by sperm morphology assay (22). It is possible that the chromosomal
damage may occur in ribavirin exposed germ cells and such mutagenic
or pre-carcinogenic cells are known to be removed by the apoptosis
(23). Moreover, other IMPDH inhibitors such as tiazofurin, selenazafurin
and benzamide riboside, which inhibit DNA synthesis also induce
the apoptosis (18). Therefore, the cell death in the testis too
is apoptosis, like that in the bone marrow and the staining method
used in this study is sufficient to illustrate this phenomenon.
Following
the cell death, the process of spermatogenesis is also hindered
as indicated by decreased number of step 19 spermatids in stage
VII tubule. This happens only when there is cell death and therefore
it is a sensitive indicator of cytotoxicity (24). These late spermatids
were found decreased drastically on day 14, which is therefore
the reason for around 50% decrease in epididymal sperm count on
day 35 as previously reported (11). Decrease in step 19 spermatids
was due to the immediate effect of ribavirin on dividing cells
and promoting the cell death. Decreased meiotic figures in the
stage XIV tubules indicate therefore two important lines of toxicity
of ribavirin; first, the total number of cells available for cell
division was decreased due to the cell death; and second, prevention
of cells entering into the process of the cell division as some
non-dividing cells wee also observed in this stage tubule. We
conclude that, the cytotoxicity of ribavirin is imparted on both
rapidly cell-dividing target tissues where the cells are removed
by the cell death somewhat similar to the apoptosis and this cell
death is responsible for decrease in erythrocyte and sperm counts
in the rat.
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REFERNECES
