الفهرس 
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Abstract
The present study aims to evaluate the toxic and teratogenic effects of intragastric administration of sofosbuvir (SOF) and /or daclatasvir (DCV) on the developing mice fetuses. These drugs are a novel group of antiviral medications called nucleosides analogues which commonly used in Egypt as antiviral drugs for treatment of hepatitis c virus (HCV).
The used low and high therapeutic doses of both sofosbuvir (SOF) and daclatasvir (DCV) were calculated for mice and were found to be (80 & 160 mg/kg of body weight) and (8 & 16 mg/kg of body weight) respectively. Firstly, adult albino male and female mice were used to evaluate the histopathological changes on the testicular and ovarian tissue after oral administration of low and high doses of SOF and /or DCV for ten successive days. Then, the pregnant mice were divided into four groups; the control group (C) and three experimental groups (G1, G2 & G3). Each experimental group was divided into two sub-groups for treatment with low and high doses of sofosbuvir and/or daclatasvir from day 6 to day 12 of gestation. All females sacrificed on day 15 and day 18 of gestation and the following parameters are concerned.
1) Histopathological examination of the testes and ovaries of adult males and females.
2) Determination the average increase in the maternal body weight
3) Determination the total number of alive and dead fetuses.
4) The external abnormalities of fetuses, fetal weight and fetal length and skeletal examination were also studied.
5) Histopathological examination of the liver and kidney of the developing fetuses.
6) Ultrastructural observations of the liver and kidney of fetuses of the third group maternally treated with high dose of both drugs (G3b).
The present study showed that :
1) Treatment of adult males and females with low and high doses of SOF and/or DCV drugs revealed histopathological changes in the testicular and ovarian tissues, which were more severe in groups treated with the high dose of DCV and also in both drugs.
A- The testis
The most obvious histological changes appeared in the testis of adult males were in the shape and size of the most seminiferous tubules, where some tubules appeared shrunken while others appeared with irregular basement membrane and complete degeneration of the germinal epithelium. These observations appeared more pronounced in G2b treated with high doses of DCV. In addition, the males treated with both drugs showed disorganization of the germinal epithelium with marked sloughing or obliteration of the tubule lumen. Also, there was a marked reduction in the numbers of spermatogenic cells especially spermatozoa with a large numbers of vacuoles scattered between them. These results were evidenced at the level of high doses.
B- The ovary
Histopathological observations of the ovarian sections of females administrated with low doses of SOF (G1a) revealed well developed ovarian follicles, normal blood vessels and normal stromal cells. In females treated with high doses of SOF (G1b), many atretic follicles were detected within the ovarian stroma and this is a degenerative process of follicles characterized by cessation of mitosis in granulosa cells and death of the oocyte. The histopathological changes became more severe with the high doses of DCV (G2b) and both SOF and DCV (G3b). These changes included congestion and reduction in the numbers of the ovarian follicles. Other changes included vacuolation of the ovarian stromal cells and the granulosa cell layers were activated. Micronuclei formation was observed in the oocytes of pre-ovulatory follicles. Further changes were the disorientation of corona radiata, disruption and thinning of the zona pellucida. Also, the granulosa cells showed very massive apoptosis throwing its residue into the follicular antrum.
2) The mean increase in the maternal body weight of pregnant mothers was statistically insignificant in mothers of the first group treated with low and high doses of SOF. The high doses of DCV (G2b) and both drugs (G3b) caused highly significantly reduction in the maternal body weight gain during gestation and body weight and length of their fetuses.
3) This study revealed that most elements of the skull of 15&18-days old fetuses showed moderate to severe malformations especially in G2b and G3b. The vertebral column of 15-days old fetuses in G2 and G3 revealed that there was a complete delay in ossification in all regions of the vertebral column. While, the ossification of the centra of some cervical vertebrae was absent especially at the high dose in G2 and G3 in 18-days old fetuses. In addition, the high doses of drugs caused some unossified caudal vertebrae especially in G2b and G3b.The whole sternum and xiphoid cartilage became shorter than the control one especially in groups treated with both drugs. The cartilaginous portion of the ribs exhibited less blue coloration than the control denoting reduction in its chondrification. In addition, the scapula and clavicle were shorter and less thickened in all treated groups. The long bones of the fore and hind limbs were shorter in length and decreased in thickness compared to the control. In such case, the density of red coloration was less than the control. All these observations detected in 15 and 18-days old fetuses in G2 and G3.
4) Treatment of pregnant mice with these drugs revealed histopathological changes, which appeared more severe in mice fetuses given DCV and with both drugs especially at the level of high doses.
(A) The liver
Light microscopical examination of the liver of 15 and 18-days old fetuses maternally treated with the low and high doses of SOF and /or DCV revealed marked signs of degenerative changes of the hepatic tissue. These alterations were more pronounced especially in the case of fetuses maternally treated with DCV drugs. These effects progressed directly with the increment of the used dose as that reported in G2b and G3b. The severe destructive features were represented by the highly dilated blood vessels and spaces in the liver tissue were seen occluded with red blood cells denoting a kind of hemorrhage in the liver tissue. In addition, great destruction was shown in the liver parenchyma that represented by complete deteriorated hepatocytes with highly vacuolated cytoplasm and the liver cells appeared structurless with ill-defined cellular boundaries and pyknotic or chromatolytic nuclei and also marked hyperplasia of sinusoidal kupffer cells,.
Electron microscope examination of fetal liver of 18-days old fetuses maternally treated with high doses of both drugs (SOF and DCV) revealed significant alterations in the hepatic tissue. The cytoplasm of degenerated hepatocytes detected with the numerous large electron transparent coated vacuoles. Also, the mitochondria exhibited condensed opaque matrices without any internal organization and the blood sinusoids revealed dilations. In addition the nuclei appeared with different shape, size and irregular nuclear membranes. Small gapes were detected between the two nuclear membranes. The nucleus showed patches of heterochromatin, however the euchromatin was rare or absent.
(B) The kidney
Histological examination of the kidney of 15&18-days old fetuses maternally treated with low and high doses of SOF and/or DCV showed moderate to severe histopathological alterations. The atrophied renal corpuscles and degenerated glomeruli were detected. The epithelia of the visceral and parietal layers of some Bowman’s capsule were degenerated and the urinary spaces were dilated. Also, the deteriorated renal cells were observed containing large vacuoles, disrupted cytoplasm and pyknotic nuclei. Some destructed cells showed disintegrated cytoplasm. These changes were increased from the low to the high dose in all treated groups.
Electron microscopical examination of sections of the fetal kidney maternally treated with high doses of the two drugs showed slightly congested glomeruli. The visceral podocyte cell detected with marked enlargement of the nucleus, heavy chromatin condensation at the nuclear envelope and disrupted foot processes and filtration slit. The cells of proximal convoluted tubule revealed variable degrees of pathological alterations. These cells showed thick basement membrane, lack microvilli, disintegrated cytoplasm, degenerated mitochondria and karyolitic nuclei with disintegrated nuclear envelope. The distal convoluted tubules showed various signs of pathological alterations. Some tubules were collapsed with very narrow slit like lumina. The cytoplasm revealed electron dense bodies, polymorphic damaged swollen mitochondria, and numerous smooth endoplasmic vesicles. Also, the nucleus exhibits irregular and indented outline.
from the findings of the present study and the aforementioned studies, we conclude that both antiviral drugs SOF and DCV used to treat hepatitis C virus, showed that using them in concentrations higher than the therapeutic dose resulted in unwanted adverse effects on developing mouse fetuses, especially in the second and third groups. This may be due to the toxicity of the mother, malnutrition or poisoning of the fetus through the passage of the drug and its metabolites through the mother’s placenta. It was recommended that to take awareness in the usage of these drugs during pregnancy period. In addition, further studies are of course required for understanding the mechanism of malformations.