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Original Article
Volume 46 - No.4:January 2002 index
Indian J Physiol Pharmacol 2002;46 (4);

Mechanism of Cytotoxicity of Ribavirin in the Rat Bone Marrow and Testis
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





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.



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.


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).


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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





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.


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TABLE II: Incidences of step 19 spermatids, meiotic figures and cell death in the testis exposed to ribavirin.





35 d

70 d

105 d








20 mg/kg





Stage VII tubule

100 mg/kg






200 mg/kg

















Dividing cells/

20 mg/kg





Stage XIV tubule

100 mg/kg






200 mg/kg

















% Tubules with dead cells

20 mg/kg






100 mg/kg






200 mg/kg





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


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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