Investigation of the embryo-toxicity of the antiviral drug “Ribavirin” in Wistar rats during different gestation periods

ABSTRACT Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a water-soluble, guanosine nucleoside analog that mimics other purines, including inosine and adenosine. The current study was designed to assess the embryotoxic effect of ribavirin and investigate its teratogenic effect on the placental and fetal liver tissues. In this study, 18 pregnant Wistar rats (165–185 g) 11–13 weeks old were orally administrated ribavirin at a dosage of 82.3 mg/kg at intervals of the organogenesis period (5th −13th) gestation day and fetal developmental period (13th −19th) gestation day. At gestation day 20, the pregnant rats were postmortem, and their fetuses were examined via morphological, histopathological investigations, biochemical analysis, and DNA damage evaluation. The results showed that ribavirin increased the incidence of abortion during the organogenesis period and caused a marked decrease in corrected body weight, uterus, and placenta weight of dams during the fetal developmental period: ribavirin-induced intra-uterine growth retardation, fetal morphological abnormalities, and decreased number of implants/litter. In addition, ribavirin produced histopathological alterations of the placenta and fetal liver tissue, marked elevation of malonaldehyde level, a significant decrease in the activity of antioxidants, and triggered DNA damage. In conclusion, we found that ribavirin has an embryotoxic effect via the generation of free radicals that cause placental DNA damage and altered placental function that affects normal embryo/fetal development.


Introduction
Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a water-soluble, guanosine nucleoside analog that mimics other purines, including inosine and adenosine [1].It is a broad-spectrum direct antiviral agent (DAA) authorized in 1986 [2].Ribavirin demonstrated antiviral efficacy against DNA and RNA viruses in numerous animal infection models [1].In addition, ribavirin has been utilized to treat Lassa fever virus infection and other viruses [3].However, ribavirin given to adult female rats resulted in severe ovary damage.These genotoxic effects of ribavirin may induce infertility in pregnant rats throughout the reproductive phases [4], and ribavirin at doses of 60 and 90 mg/kg/day resulted in statistically significant and/or dose-related increases in fetal resorptions, malformations, and decreased postnatal survival [5].
The placenta serves as a link between the pregnant rat and the fetus.The placenta is the most essential conduit for material exchange between maternal and fetal blood.Therefore, any disruption in the placental cells' secretory processes delays fetal growth and development [6].
Ribavirin that passes through the placenta is changed to ribavirin-5-phosphate inside the fetal cells, impacting the DNA transcription within the embryo.In addition, ribavirin impacts active cells, which shows why ribavirin is teratogenic to the fetus [7].
Drug-induced histopathological alterations of the placenta in pregnant rats are significant in safety evaluation to comprehend the cause of teratogenicity and developmental harm.In developmental toxicology, the relationship between pregnancy and embryonic toxicity is still a major concern [8].Nevertheless, the placenta has not yet been considered important in identifying mothers and embryos or fetuses at risk [9].
The current study is being conducted to assess the embryotoxic capability of orally administrated 82.3 mg/kg ribavirin on pregnant rats at two different gestation periods, organogenesis period (5 th -13 th GD) and fetal developmental period (13 th -19 th GD).In addition, to assess ribavirin's embryotoxic potential on pregnancy outcome and fetal growth, investigate the mediated pathway by which ribavirin affects embryo/fetal development and the histological alterations, as well as oxidative stress and comet assay in the mother's placenta and fetal liver, were all investigated.

Materials and methods
Ribavirin was purchased from the Marcyrl Company for Pharmaceutical Industries.The drug's medication name is Hepatovirin.Ribavirin capsules were diluted in distilled water and then taken orally at a therapeutic dosage of 82.3 mg/kg/body weight based on the therapeutic dosage [10].The human therapeutic dosage is 400 mg twice daily, which equals 800 mg.

Experimental design
A total of 18 pregnant Wistar rats (Rattus norvegicus) (165-185 g), 11-13 weeks old, and five mature Wistar male rats (170-190 g) were procured from the Central Animal House of Veterinary Faculty Cairo University.The animal handling and experimental work was carried out according to the guidelines of The Institutional Animal Care and Use Committee of the Faculty of Science, Cairo University (Approval no: CUIF/23/20).Animals/experimental groups were kept/ housed in (a 12:12 hr LD) illumination cycle at an ambient temperature (of 25 ± 2°C).Animals have free access to food and water ad libitum.Vaginal smears were taken for five days to observe those in the proestrus after one week of residence and inspected under a light microscope [11].For the mating procedure, each 2 of the 18 female rats with recorded estrus cycles was taken at the proestrus phase and placed with a single male rat all night in a regulated environmental condition of warmth, humidity, and light until all the 18 female rats became pregnant.Day one of gestation was deemed to have begun when sperm were discovered in the vaginal smear the following morning [12].The weight of the pregnant rats was measured and separated into three groups randomly (six each): Group 1: 1 ml of distilled water was administered by gavage to the control group.
Group 2: 82.3 mg/kg/body weight/day of ribavirin was orally administered to the treated group during the organogenesis period (5 th −13 th ) gestation day (GD).
Group 3: 82.3 mg/kg/body weight/day of ribavirin was orally administered to the treated group during the fetal developmental period (13 th −19 th ) gestation day (GD).
Pregnant rats were weighed once daily throughout the study, and mortality and behavior change (salivation, lethargy) were recorded.On day 20 of the gestation period, the weights of pregnant rats were recorded.Pregnant rats were sedated with sodium pentobarbital 100 mg/kg I.P and cut open to expose the two uterine horns.
• Weights of the uteri were recorded, and the corrected body weight (20 th GD weight of pregnant rat -uterine weightzero day of pregnant rat weight) was calculated.
• The percent of the post-implantation loss was measured as (implantation sites count -live fetuses count/implantation sites count × 100) [13,14] and is employed to estimate the percentage of embryonic loss at implantation or the abortifacient effect.• Fetuses were removed from the uteri, washed, weighed, and length was measured.

Histopathological investigation
Each rat's fetal liver and placenta were removed and fixed in formaldehyde 10%, embedded in paraffin and 5 µm slides were prepared.Then, tissue segments were dehydrated in alcohol, rinsed in xylol, and embedded in molten paraplast.Finally, Hematoxylin and eosin were utilized to dye 5-μm segments in the end [15].The histological sections were inspected using a bright field microscope (model: B120C) and with a digital camera, 5MP USB 2.0 Color CMOS Digital Eyepiece Microscope Camera (model: MD500) manufactured by AmScope in the United States, A division of United Scope LLC.

Comet assay for genotoxicity assessment
Comet assay/Single cell gel electrophoresis (SCGE) was carried out as explained [21] using hepatic and placental tissues.First, making 10% tissue solution, specimens were homogenized in cooled homogenizer buffer (pH 7.5) with 75 mM NaCl and 24 mM Na2EDTA (pH 13).Next, fluorescence microscopy with a closed-circuit digital camera at 400× magnification was utilized to examine the prepared slides.After that, the software created by Kinetic Imaging Ltd. for Comet five image analysis was utilized to analyze 50 cells for each specimen to gauge the level of DNA damage.

Statistical analysis
Data were analyzed for normality.All parameters showed normal distribution (P > 0.05).
Corrected body weight, uterus weight, and post-implantation loss index were analyzed using ANOVA followed by Tukey multiple comparisons.The other parameters were analyzed using an independent sample ttest.SPSS software (Ver, 19.0;IBM SPSS) was used to run all parameters.Inputs are presented as mean ± standard error of the mean.A statistically significant difference was defined as P < 0.05.

Effect of ribavirin on pregnant rats
No outward symptoms of harm were exhibited in pregnant rats following oral administration of 82.3 mg/kg ribavirin in both groups (2 and 3).The effects of oral ribavirin intake on the outcome of pregnancies are reviewed in (Table 1).No mortalities were noted.There was a noticeable change in the number of fetuses and % of post-implantation loss index.Full resorption was recorded in pregnant rats treated with 82.3 mg/kg ribavirin in the organogenesis period.The corrected body weight and uterine weight were significantly reduced compared to the control group's.On GD 20, the uterus of the control pregnant rats demonstrated the typical distribution of the implanted fetuses in the two horns.The uterus of pregnant rats given 82.3 mg/kg ribavirin (5 th -13 th GD) revealed total resorption and a post-implantation loss index of 100%, contrary to the control group, Whereas the uterus of pregnant rats administrated with 82.3 mg/kg ribavirin (13 th -19 th GD) displayed an uneven distribution of fetuses in the two uteri horns and a decrease in the number of fetuses (Figure 1).

Effect of ribavirin on fetal growth
The group treated with 82.3 mg/kg ribavirin (13 th −19 th GD), compared to the control group, showed a reduction in the fetal weight and length (Table 1).Ribavirin administration caused multiple hematomas on various fetal parts (facial, limb and abdominal bleeding) (Figure 2).

Impact of ribavirin on the histology of the placenta and fetal liver
The placental tissue of the control group exhibited a basal zone with normal giant cells and spongiotrophoblast (Figure 3a,b).The labyrinth zone showed regular morphology of blood vessels coated with epithelial cells.Trophoblastic trabeculae (T) comprise trophoblasts and syncytiotrophoblast.The fetal capillaries are covered by endothelial cells comprising fetal erythroblasts and maternal sinusoids comprising maternal erythrocytes (Figure 3c,d).
Ribavirin administration during the fetal developmental period caused placental histopathological changes.The basal zone showed glycogen cyst degeneration, congestion, and degenerated necrotic area.Also, the spongiotrophoblast showed pyknotic.There are also degenerated giant cells with pyknotic nuclei (Figure 3e,f).While the labyrinth zone showed disorientation of its structure, dilated maternal sinusoid & necrotic change, and the blood vessels lined with a degenerated endothelial coating (Figure 3g,h).
Control fetal liver showed a normal histologic arrangement of hepatocytes around the central vein, megakaryocyte, sinusoid, and erythroblast (Figure 4a,b).On the other hand, the fetal liver tissues maternally treated with ribavirin (13 th − 19 th GD) showed disorganization and degenerative changes, congested central vein dilated, sinusoid, necrotic area, vacuolation, hemorrhagic area, and degenerated hepatic cells (Figure 4c,d).

Effect of ribavirin on the oxidative state
Ribavirin intake from (13 th −19 th GD) induced in the placenta a significant decrease in SOD, CAT, and GSH levels and a significant increase in MDA levels compared to the control (Table 2).While in the fetal liver, it induced a significant increase in MDA, SOD, CAT, and GSH in comparison with control (Table 3).

Assessment of genotoxicity of ribavirin by comet assay
Ribavirin significantly increased DNA deterioration in the placenta and fetal liver of the treated  groups, measured by % DNA damage, % DNA in the tail, tail moment, and tail length (Tables 4  and 5, Figure 5).The treated placenta (Figure 5b), showed significant DNA fragmentation induced by ribavirin indicated by % DNA damage, % DNA in the tail, tail moment, tail length compared to the control (Figure 5a).As well as the treated fetal liver (Figure 5d) showed significant DNA fragmentation induced by ribavirin, indicated by % DNA damage, % DNA in the tail, tail moment, and tail length compared to the control (Figure 5c).

Discussion
Ribavirin is a guanosine nucleoside analog that demonstrated teratogenic/embryogenic effects in several animal types of research.In our study, oral administration of ribavirin to pregnant rats at both examined periods did not induce any obvious external symptoms of maternal.This finding was consistence with the observations of Ferm et al. [22], where he gave pregnant rats ribavirin in a single oral dose of either 25.0 or 37.5 mg/kg on the 9 th GD, and no effect was observed on mothers.The same finding was observed by Johnson [5], who administrated ribavirin orally to pregnant rats in dosages of 0, 0.1, 1, and 10 mg/kg starting on day six and finishing on day 15 of pregnancy.Also, this finding parallels Kochhar et al. [23], who used a single i.p dose of ribavirin 10, 25, 50, 100, 200 from (9 th − 12 th GD) on pregnant mice.
In the present study, the administration of ribavirin drastically decreased the corrected body weight of female rats in the organogenesis group.However, there was no significant change in the corrected body weight during the fetal developmental period compared with the control group.The Same observations were   seen by Johnson [5], where the corrected body weight of pregnant rats during ribavirin (10 mg/ kg) administration from day 6 th through 15 th treatment period was less than in any other group.Ribavirin intake could provoke peculiar changes in regulating midbrain dopaminergic system activity, thus influencing/diminishing physiological (novelty-induced motor activity) response and appetite that rely on it Petković et al. [24].Also, the decreased uterine weight, placenta weight, and the number of delivered fetuses observed and recorded in the treated groups reduced the corrected body weight of the pregnant rats compared to the control group.
The outcomes of our research revealed a considerable reduction in the placenta and uterine weights of rats administrated with 82.3 mg/kg ribavirin in both treated groups as opposed to the control group, with unequal distribution of embryos on the two horns of the uterus.The same finding of unequal allocation of embryos was found by Khalaf and Kamees [7], where they orally administrated ribavirin at a dosage of 16 mg/kg twice daily to pregnant rats from 5 th -10 th GD.
A small placenta limits uterine blood supply to the placenta and transfers to the fetuses, which is a significant cause of fetal growth retardation [25].Histopathologically, the labyrinth and basal zones in the treated group had an excessive number of giant and apoptotic cells; a significant reduction in glycogen cell islands was seen, as well as hypoplasia of the labyrinth and basal zones.This outcome was parallel with Furukawa et al. [26], who orally administered the antifungal drug ketoconazole at 0 and 25 mg/kg/day throughout 12 th −14 th GDs, and the placentas were collected on 15 th , 17 th , and 21st GDs.A rise in thickness was observed in the labyrinth and basal zones on the 17 th and 21st GDs, while on the 15 th GD, the old zone had already witnessed the alteration.In addition, the mitotic figures of the trophoblasts' labyrinth zone were dramatically increased on the 15 th GD.Furthermore, some placentas showed numerous cystic dilatations of maternal sinusoids on the 15 th , 17 th , and 21st GD.Lastly, spongiotrophoblasts and glycogen cell clusters rose in the basal zone on the 17 th and 21st GDs [26].
Ribavirin (82.3 mg/kg) caused full resorption during the organogenesis period.While induced hematoma (dark red spots) on different fetal parts during fetal development.This outcome was in parallel with Ferm et al. [22], who orally administrated ribavirin to pregnant rats at doses of 25, 37.5 mg/kg on the ninth day of gestation and showed that a high dose of 37.5 mg/kg enhanced the frequency of embryonic resorption along with the frequency and severity of facial malformations in survivors.Kochhar et al. [23] used a single IP dose of ribavirin 10, 25, 50, 100, and 200 from 9 th − 12 th GD on pregnant mice.The lowest dose, 10 mg/kg, did not reveal any embryotoxic effect.However, all doses over 25 mg/kg produced various developmental effects (ribs, cleft palate, limb, tail reduction), growth retardation, and high frequency of resorption; resorption increased by increasing the dose.In another study that agrees with our result Johnson [5], who used lower doses of ribavirin 0, 0.1, 1, 10 mg/kg to pregnant rats from 6 th − 15 th GD, discovered that resorptions were more noticeable at higher dosage levels.Kilham and Ferm [27] showed ribavirin's effects on fetal development when administered to female hamsters in a single intraperitoneal dosage of 1.25 to 4.2 mg/kg on 8 th GD, ribavirin was effective in generating resorptions along with malformations, and the most prevalent anomalies were limb malformation.
Ribavirin causes hemolytic anemia in pregnant women, resulting in a deficiency of oxygen flow and ischemia in the placenta that alters the fetus's development [28].As for how ribavirin directly impacts the fetus, it does so by passing through the placenta, where Kochhar et al. [23]  demonstrated the possibility of passing 1-5 μg to the fetus per 50-100 mg/kg of ribavirin administered to female mice.Furthermore, because ribavirin is a guanosine analog, it transverse the placenta via Equilibrate Nucleoside Transporters (ENTs) [29], which is a type of the live cells' plasma membranes' integral protein and transports nucleobases required for DNA and RNA biosynthesis, where ENTs contribute to the absorption of ribavirin by a placental cell line which is capable of absorbing a variety of nutrients including glucose, and amino acids and this line is known as BeWo [30].
Pregnant rats and their fetuses have a strong connection through the placenta.Thus drugsor chemicals can induce aberrant fetal development, resulting in fetal resorption.In addition, mitotic suppression, apoptosis, deterioration, and/or destruction of trophoblasts caused by direct placental damage or nonspecific effects caused by an abnormally adverse pregnancy environment also inhibits placental growth, resulting in a decreased placenta weight [9].
Apoptosis is a type of planned cell death where a cell deliberately kills itself -however, a disruption in this pathway in the placenta results in placental malfunction [ 31] .Placental dysfunction results in an insufficient amount of nutrients and oxygen to enable normal fetal development, leading to fetal blood flow reallocation [32,33].
Our work revealed significant fetal growth retardation in the fetal developmental treated group compared to the control group.This result corresponded with Ferm et al. [22]; Kochhar et al. [23], who studied the ribavirin effect on female hamsters and rats in different routes and dosages with corresponding controls.
These findings can be explained by the direct action of the parent compound or its metabolites via the active role of the placental transfer of compounds through the placental barrier.The direct action of a compound on embryonic tissues leads to cytotoxic effects and cell death [9] .ENT1 protein produced in placental microvillous membranes also appears to have a key role in ribavirin absorption [34] .Another factor limiting the development and growth of the embryo is the development of decidual cells.During pregnancy, the placenta develops fast for a brief period due to increased blood flow [9] .A decrease in viable fetuses' frequency may occur due to incomplete placental development and deterioration of both the trophoblasts and decidual cells, which are essential for providing nutrients to the embryo, or early fetal loss and upsurge in the incidence of fetal resorption [35] .As a result, fetal weight loss may ensue from the suppression of DNA transcription during the early phases of embryonic development [36].
In the current study, ribavirin's administration during gestation significantly elevated oxidative stress of pregnant rats' placenta and fetal liver.This outcome was in parallel with Khalaf and Khamees [7], who orally administrated ribavirin at 16 mg/kg twice daily to female rats from 5 th -10 th GD.The treated group had apparent hepatocyte degradation and an increased number of macrophages.The explanation for the drug's effect on the liver of the fetus might be the result of ribavirin-inducing oxidative damage and free radicals.Since this impacts female rats' pancreatic tissue, the synthesis of pancreatic lipase reduces, resulting in a decrease in fatty substance absorption and, as a result, a decrease in the uptake of fatsoluble vitamins, such as (A, K, D, E, K), this causes a drop in its levels in the pregnant rat blood and failure to reach the fetus in appropriate quantities, which are required throughout the fetal development process, which results in the fetuses being undersized and the appearance of certain tissue abnormalities [7].
One of several speculative processes proposed as potential treatments for fetal toxicity is the activation of biochemical or metabolic alterations (with negative effects on the fetus) in the mother's system.For example, the development of oxidative stress in the embryonic system is a critical mechanism of teratogenic agent-induced fetal toxicity [37] .Oxidative stress can induce cell death, intermediate oxidative stress induces apoptosis, and more severe stress induces necrosis [38,39].However, Wells et al. [40] found that just 5% of the maternal level of most antioxidant enzymes is present in the embryo.This result may be linked to findings showing that low levels of antioxidant enzyme activity increase as development progresses and that the fetus might not be able to adapt well to oxidative disturbances early in organogenesis, leading to teratogenicity [41].
Excessive amounts of reactive oxygen species (ROS) metabolites are recognized to cause fatal series reactions, which include oxidation and structural damage, which are essential for cellular integrity and viability.Such free radicals can potentially harm membranes and macromolecules by three fundamental mechanisms: deoxyribonucleic acid (DNA) fragmentation, protein oxidation, and lipid peroxidation [42].Oxidative damage is believed to be among the principal mechanisms in almost all drug toxicity.
This research showed a significant rise in % DNA damage, % DNA in the tail, tail moment, and tail length in pregnant rats' placenta and fetal liver compared to the control.These findings agreed with Kochhar et al. [23], who attempted to determine the drug concentration reaching the embryo by approximating the metabolic inhibition produced by the known concern of ribavirin.It was found that the lowest dose of 10 mg/kg of ribavirin reduced DNA synthesis by about 20% of the control level; increasing the dose to 50, 100, and 200 mg/kg increased DNA inhibition up to 73%.
The quantity of ribavirin that has passed through the placenta is changed to ribavirin-5-phosphate inside the fetus's cells, which impacts the performance of Inosine monophosphate dehydrogenase (IMPDH) that is involved in the production of nitrogenous base guanosine monophosphate.So ribavirin impacts the transcription of DNA within the fetus [23] .In addition, ribavirin impacts active cells, which shows why ribavirin is teratogenic to the fetus; since the cells are constantly and perpetually dividing, this implies that they must generate new genetic information, so inhibiting DNA synthesis as a result of action on IMPDH, cumulative impact manifested as teratogens in distinct fetal areas [22].This outcome is compatible with Salman et al. [43] when they investigated the effects of ribavirin at various dosages of 10, 20, and 50 mg/kg on rat fetuses and their skeletons.

Conclusion
Lastly, we showed that ribavirin is an embryotoxic agent in pregnant rat fetuses during the crucial phases of pregnancy.Rat fetuses had growth retardation and histopathological abnormalities; ribavirin is expected to primarily activate reactive oxygen species, raise the percentage of DNA fragments, and cause pathological effects on the pregnant rat placenta and fetal liver.
Assessing the risk of birth defects associated with exposure to highly active anti-retroviral therapy during organogenesis in rats.

Figure 1 .
Figure 1.Photographs of the uterus of a pregnant rat at the 20 th day of gestation.(a) the control group showed the normal symmetrical distribution of fetuses in the two uteri horns.(b) the treated group with 82.3 mg/kg ribavirin at the organogenesis period (5 th −13 th GD) showing full resorption of fetuses with clear visible embryonic resorption sites.(c) the treated group with 82.3 mg/kg ribavirin at the fetal developmental period (13 th −19 th GD) showing the asymmetrical distribution of fetuses in the two uteri horns and a reduction in the number of fetuses.U, uterus; V, vagina.

Figure 2 .
Figure 2. Photographs of (a) Control show a normal morphology of the fetus.(b -d) the treated group with 82.3 mg/kg ribavirin at fetal developmental period (13 th −19 th GD) showing hematoma at different body parts (arrows).

Figure 3 .
Figure 3. Photomicrographs of sections of pregnant mothers' placenta.Hematoxylin and eosin stain.(a & b) Represents the control basal zone showing normal construction of giant cells (Ga) and spongiotrophoblast (SP).(c & d) Represents the control labyrinth zone showing normal appearance of blood vessels (BV) lined with epithelial cells.Trophoblastic trabeculae (arrowhead) consisting of trophoblasts and syncytiotrophoblast, fetal capillaries (bold arrow) lined by endothelial cells containing fetal erythroblast and maternal sinusoid (notch arrow) containing maternal erythrocytes.(e & f) Represents the basal zone of treated group with 82.3 mg/kg ribavirin (13 th −19 th GD) showing glycogen cyst degeneration (Gly), congestion (Star), degenerated necrotic area (triangle), pyknotic spongiotrophoblast (Bifid arrow), degenerated gain cells with pyknotic nuclei (Ga).(g & h) Represents the labyrinth zone of treated group with 82.3 mg/kg ribavirin (13 th −19 th GD) showing disorientation of labyrinth zone structure dilated maternal sinusoid (MS) and necrotic change.Blood vessel (BV) with degenerated endothelial lining (hollow arrow).

Figure 5 .
Figure 5. Representative photo of DNA damage induced by ribavirin in placenta (b) and fetal liver (d) compared with intact DNA (A) and (c) respectively.

Table 1 .
The effect of ribavirin on pregnancy outcomes.

Table 2 .
The effect of ribavirin on pregnant rats' placenta oxidative state.

Table 3 .
The effect of ribavirin on fetal liver oxidative state.

Table 4 .
The effect of ribavirin on the placental DNA.

Table 5 .
The effect of Ribavirin on the fetal liver DNA.