FOETUSES ABNORMALITIES CAUSED BYTEGRETOLDRUG ON THEIR PARENTS OF THE ALBINO RAT

The current study was performed to illustrate the harmful effects of different doses of Tegretol (CBZ) which has active ingredient ( Carbamazepine; CBZ ) on the prenatal foetuses of rat at 20 th day of gestation. Tegretol (CBZ) noticeably reduced the body weight &length of foetuses in addition the percentage of dead foetuses increased; the early mortality of foetuscs which became resorbed besides late foetuses mortality (stillbirth) during the gestation phase. Tegretol (CBZ) administration caused delay of ossification of some bones and shortness of others .Some histo-pathological alterations were founded in livers, kidneys, and lungs of foetuses tissues which parentally administered with different doses of Tegretol (CBZ). 2 The doses of the according to the value of LD50 for oral administration in rats by Novartis Pharmaceuticals Corporation 2014&Safty Data Sheet.The oral LD 50 in rats(3850-4025).The recentdosesare(98mg/kg; 1/8LD 50 )in G1 ,(16mg/kg; 1/50LD 50 )inG2,(8mg/kg;1/100LD 50 )in G3 ,and (5mg/kg;1/150LD 50 )in G4. The experimental were into 5 groups; Control group (C): was orally. Group one (G1): administered with (1/8 LD50; 98 mg/kg) orally). Group two (G2): was given (1/50 of LD50; 16 mg/kg). Group three (G3): was administered with (1/100 of LD50; 8 mg/kg). Group four (G4): (1/150 ofLD50; 5 mg/kg). Males and females were administered with tegretol (CBZ) for seven days before mating, they do copulation with each other.Pregnant females continued receiving Tegretol (CBZ) at 20 th day of gestation. more than studying the safe side of the drug doses on animals. We had to choose high doses for our study which could be okay for the rat dams and also cause malformations in their foetuses. Our results show that oral dose of Tegretol (CBZ) to rat had a notable effect on the weight of the foetuses compared with that of control. The weight of the foetuses that were antenatally exposed to Tegretol (CBZ) was reduced when compared to control foetuses. As well, most of these foetuses were not alive when they were delivered. This study helped us to regulate the way of administration and dose of Tegretol (CBZ) which can be tolerated by rats and also sured that Tegretol (CBZ) causes low weight in foetuses which parentally exposed to the drug. This result can be useful to clinicians to explain the decreased birth weight of infants of mothers were on Tegretol (CBZ) administration during pregnancy.The present study focused on the effect of Tegretol (CBZ) on the morphology, mortality , skeletal system and the histology of the liver ,kidneys & lung of thefoetuses at 20 th day of gestation.Tegretol showed that Tegretol which has the active ingredient (Carbamazepine; CBZ) causes high percentage of foetal mortality in all treated groups. As well as causes growth retardation for the foetuses. Haematoma; dark red patches scattered on the different parts of the body of foetuses at 20 th day of gestation parerntally treated with Tegretol. This investigation indicated that most elements of foetuses skeleton showed more or less, complete ossificationTegretol (CBZ)causes histological alterations in foetal liver, kidneys and lungs structures of the foetuses at 20 th of gestationin all groups.

We brought Virgin males and females of age 8-12 weeks and About 200g weight were supplied by TheodorBilharzResearchInstitute (Giza,Egypt) .Rats were adapted for 7 days with :12-hour of light& dark cycle. All experiments introduced in this study were in compliance withthe international guidelines.Adult males were kept with adult females overnight. In the next morning pregnancy was exacted by the presence of vaginal plug [McClain and Becker, 1975]. Each pregnant female was kept in a separate cage.Males and females were administered withTegretol (CBZ) for a week (7 days) before mating, then pregnant females sustain administration with Tegretol (CBZ). At the 20 th day of her gestation, the uteri were obtained by caesarean sections. For each pregnant female, the number of foetuses bulge in each horn, living / dead foetuses. Early and late resorptions were detected due to their size. For morphological studies: Weights of control and Tegretol (CBZ) administered pregnant rat and the percentages of survival of their foetuses in all groups were recorded at the end of the experiment (20 th day of gestation). The percentage of body weight gain was calculated. Weights of gravid uterine and carcass were also recorded.The pregnant female of all groups were sacrificed on the morning of day 20 th of gestation. The lengths and the weights of the 20 th day foetuses were recorded.
The mortality ratewas also recorded and then the morphological examination for any external malformations has been noted. For the skeletal malformationsstudying: Newly picked foetuses were fixed in 95% ethyl alcohol and handled to double staining; (alizarin red &alcian blue). Next to thecompleteelimination of the soft tissues by 2% (KOH), the specimens were put in increasing grades of glycerine, till bones disclosed [Falkeholmetal. 2001]. The bony tissues were take red color and the cartilaginous were stained with blue color. The transparent specimens were saved in 100% glycerin for examination and photography.For histological examination: Liver, kidney and lung tissues were obtained from the 20 th dayfoetusesof the control and parentally administered with Tegretol (CBZ)groups. Samples were set in 10% formal-saline for a day, then treated due to [Banchroft et al., 1996], dehydrated then cleared and inserted in paraffin wax, after that 5µm sections were cut and stained with hematoxylin& eosin then examined.For Statistical analysis: The statistical analysis was carried out using one-way ANOVA using SPSS, ver. 25(IBM Corp. Released 2013). Data was treated as a complete randomization design according to [Steel et al.;(1997)]. The significance level was set at < 0.05

B) Foetusesmortality:
Tegretol (CBZ)administeration enhanced the ratio of dead foetuses and decreased the quantity of alivefoetuses as showed in Also thedied at the end of gestation stage andcontinued as stillbirth, the early died of foetusesthatbecame resorbed at the remnant of gestation period. Group (1) showeda verysmall uterus with some resorbed embryos. Group (2) produced less embryosenclosed in a small uterus and some resorbed. (G3& G4) which took lower doses of Tegretol (CBZ) exposed larger uteri contain in more embryos than (G1) & (G2) (Fig.3). 402

Ossification of the skull:
Examining the skull at 20 th day of gestation of the control foetuses of albino rat showed; complete ossification of its components (Figs 4, 5, & 6 C). There is malformation in the skull of foetuses parentally administered with different doses of Tegretol (CBZ). As, incomplete ossification of the nasal, frontal, supra-occipital, parietal, interparietal, zygomatic process of squamosal, tympanic bulla, squamosal, periotic, supra-occipital, palatine, pterygoid&ethmoid bones (

2) Appendicular Skeleton: The Pectoral Girdle and Fore Limb:
The pectoral girdle of controlfoetuses at the 20 th day of gestation comprises a well ossified scapula and clavicle stained with alizarin red and cartilaginous supra-scapula stained with alcian blue. The fore limb of control foetuses at the 20 th day of gestation consists of ossified humerus, radius, ulna and phalanges with five digits, as well as cartilaginous carpalia and meta-carpalia ( Fig. 9C) The components of the pectoral girdle and fore limb of foetuses obtained from parentally administered with different doses of Tegretol are manifested by decrease in size, length and the level of ossification in reference to the control group ( Fig.9G1-G4) .At thehigh doses of Tegretolthe pectoral girdle and fore limbs of all foetuses of -G1‖ and -G2‖ showed acute lack of ossification (Fig.9G1&G2).. Moreover, the phalanges and the meta-carpalia of fore limbs G1&G2 appeared arched in shape.

The Pelvic Girdle and Hind Limb:
The pelvic girdle of control foetuses is consisted of 3 well ossified bones which are (ilium, ischium and pubis). The pubic symphysis remains cartilaginous in nature. The hind limb of controlfoetuses consists of femur, tibia, fibula, and tarsals, metatarsals and phalanges  1) The liver: the liver from control group at 20 th day of gestation cleared that the hepatic lobule is the unitofanatomical structure of the liver, the connective tissue is dividing the liver into many lobules which structure is incomplete. The hepatic lobulewas consisted of an epithelial parenchyma and a community of blood sinusoid. The parenchyma was made up of: hepatic cells organized in irregular and branching inter-connected plates & the hepatic cordswhich arranged in a radial direction around the central vein which has a circular outline in general, being limited by a thin cover of endothelial cells ( Fig. 11; C). The hepatic strands are intervenedby many of blood sinusoids. Two types of cells could be seen on the wall of blood sinuso id, defined by endothelial flat cells, with flattened nuclei and scanty cytoplasm, and Kupffer cells having dark nuclei and branching pseudopodial process and a clear vacuolar appearance ( Fig. 11; C). Hepatocytes are large in size, and are polygonal in shape with granular cytoplasm. The nuclei are large and located in the center. Groups of cells which darkly stained are of frequent occurrence in the hepatic tissue they reveals the different types of blood forming cells named, erythroblasts, lymphocytes and mega-karyocytes ( Fig. 11; C).
The liver sections obtained fromTegretol-parentally administered foetusesof rats at different doses as; G1 was given (1/8 LD50; 98 mg/kg); G2 (1/50 LD50; 16 mg/kg); G3 (1/100 LD50; 8 mg/kg) and G4 (1/150 LD50; 5 mg/kg) showed clear degenerative alterations (Fig.11 G1-G4). In general, the hepatic cells were pale stained and between the noticeable signs of damage encountered is the general destruction of the normal structural organization of the hepatic lobules. This also inferred the similarity the characteristic plate /cord-like arrangement of the normal liver cells. The hepatocyte injury was clearly manifested by noticeable cytoplasmic vacuolization. Such vacuolization may be attributed to either lipolytic (fatty) or hydropic degeneration ( Fig.11 G1 & G3). The central&portal veins were much swollen. They were inflamed with dull blood with clear marks of hemorrhage (Fig.11G&G3). Some cells showed histological features of necrosis; the nucleus of each necrotic cell displayedpyknotic, being smaller, condensed, and deeply stained with haemato-xylin ( Fig.11G1-G4). Additionally, most of these nuclei are showingclear signs of nuclear pyknosis, karyorrhexis, pluskaryolysis (Fig.11G3). In some samples, severe necrosis of liver was noticedaround the central vein accompanied with acomplete loss of the cellular outlines & of nuclear staining (Fig.11G1&G3). The endothelial lining of the central vein was completely eroded causing perceptible hemorrhage in the whole tissue ( Fig.11 G1& G3). As well congestion blood vessels appears in the central& portal veins ( Fig. 11 G2 &G4). The blood sinusoids are dilated, with eroded lining and hypertrophied Kupffer cells were pushed into the internal lumina (Fig.11G2&G4).
408  (Fig.12G1-G4). In many cases, some glomeruli lost its normal circular shape and became irregular and decreases in size (Fig.12G2, G3&G4). In the other cases, the parietal and visceral layers of Bowman's capsule may fuse together (Figs.12G1&G3); these two cases are known as glomerulonephritis. A large number of the epithelial cells that lining the convoluted [proximal & distal] and collecting tubules became swollen and project inwards forming a conical shaped appearance and the lamina of the tubules became small.This case is identified as cloudy swelling (Fig.12G1&G2). Cells of the renal tubules showed necrosis; these cells detach in the lumen of the tubules, their nuclei are pyknotized or completely disappeared (Fig.12G1-G4). Other cells are fused together in homologous mass ( Fig. 12G1-G4). Few cells undergo fragmentation, a case which is known as tubulorrhexis ( Fig.12G1-G4). 409

Lungs:
Pulmonaryalveolusis a cup-shaped structure cell. The mouth of each alveolus opens inside the lumen of the respiratory bronchiole, an alveolar duct or sac. An Inter-alveolar septum is found among neighbouringalveoli. This septum is consisted of thelining cells of closest alveoli and the structures introduced between the alveoli. Capillaries inhabit a major part of the septum, & theyare shared by the lining cells of the next alveoli. Terminal bronchioles are outlined by asimple columnar ciliated & non-ciliated epithelialcells. Among the alveoli, the wall of the respiratory bronchiole is crinkled by cuboidal epithelium. The walls of the alveolar ducts are composed of pulmonary sacs and alveoli and only a few squamous to low cuboidal epithelial cells prevailing. The alveolar ducts conductsto a blindalveolar sacs that are thin-walled structures which are popper with pulmonary alveoli (Fig. 13C).
410 .As our main objective was to obtain the developmental malformations of Tegretol; with the active ingredient CBZ in the rat'sfoetuses more than studying the safe side of the drug doses on animals. We had to choose high doses for our study which could be okay for the rat dams and also cause malformations in their foetuses. Our results show that oral dose of Tegretol (CBZ) to rat had a notable effect on the weight of the foetuses compared with that of control. The weight of the foetuses that were antenatally exposed to Tegretol (CBZ) was reduced when compared to control foetuses. As well, most of these foetuses were not alive when they were delivered.

411
This study helped us to regulate the way of administration and dose of Tegretol (CBZ) which can be tolerated by rats and also sured that Tegretol (CBZ) causes low weight in foetuses which parentally exposed to the drug. This result can be useful to clinicians to explain the decreased birth weight of infants of mothers were on Tegretol (CBZ) administration during pregnancy.The present study focused on the effect of Tegretol (CBZ) on the morphology, mortality , skeletal system and the histology of the liver ,kidneys & lung of thefoetuses at 20 th day of gestation.Tegretol (CBZ administrated orally with The doses (98mg/kg; 1/8 LD50),(16mg/kg; 1/50 LD50), (8mg/kg ;1/100 LD50),(5mg/kg;1/150 LD50) to the male and female for 7 days before mating then the pregnant female continuous to administrated with tegretol at 20 th day of gestation.Our study also showed that administration of Tegretol ( Suchestonet al.;1986 revealed that toxic effects of high CBZ dose caused intrauterine growth retardation which was cleared by decrease in bodyweight and length reduction of foetuses and they also noted that decreased foetus length may be due to retarded long bone ossification and there was a relevance between decreased long bone ossification centers and foetus low weight.In studies of developmental toxicity, a decrease in foetal body weight is in usual considered as a result of prenatal growth retardation [Chahoud&Paumgartten; 2005]. In the current work, Tegretolalso increased the number/percentage of deadand resorbed fetuses in the administered groups as compared to control group. These results are in agreement with [Waters etal. 1994] found 3 times more abnormal outcomes between offspring of epileptic women who treated with anticonvulsants, when compared to control. These included the neonatal death and congenital anomalies. The higher rate of poor outcome was found in pregnant mothers treated with phenol-barbitone . ] found no effect of (CBZ) on the foetal growth.
The effect of Tegretol (CBZ); in our study on foetalgrowth, organogenesis, and intrauterine growth retardation and malformations inskeleton in compare with foetuses of control group. These alterations were more severe in groups administered with high doses of Tegretol than in groups administered with the low doses. As regard, the axial skeleton, our results revealed that Tegretolconsiderably decreasedlength and size of the skull, reduced the ossification of skull components and bones of the lower jaw. Also, Tegretolclearly decreased ossification of atlas, axis, and cervical, sacral and caudal vertebrae. Additionally , Tegretol diminished the length and size of ribs, sternebrae and xiphoid that also showed loss of ossification. The undesirable effects of Tegretol on the appendicular skeleton includes reduction in length and thickness of humerus, radius and ulna which also showed low ossification. In our study, cesarean sections have been performed on the 20 th day of gestation and the skeletal abnormalities noticed infoetuses of the Tegretol (CBZ)-administered females should be deduced with caution, and also studies are essential to complete this assessment. Even so, excessive oxidative stress is highly associated to embryo toxicity and embryonic stage are highly having a tendency to tissue and organ damage. Additionally [Wells etal In histopathological studies, significant hepatic necrosis & loss of hepato-cytes, especially around the central vein, were found in the foetuses administered (CBZ) maternally, and these effects were same to acetaminophen -induced liver injury [Antoineet al; 2009].Because Cy-ps are generally expressed around the central vein of the liver, this observation suggested that Cy-ps may be occupied in (CBZ)-induced liver injury.
On other hand Tegretol (CBZ) in our study not only caused hepatotoxicity but also caused damage for the foetus's kidney .The current study displayed that Tegretol produced adverse injuries in renal tissues of parentally administeredfoetuses. The histopathological examination revealed severe deterioration in glomerulus, hemorrhage, congestion in the inter-tubular capillaries, pyknosis of nuclei, glomerulus atrophy, cytoplasm vacuolation, hydropic degeneration, cloudy swelling, fragmentation, homology and necrosis of renal tubule cells. The foetuses kidneys were looked affected in all animals parentally administered with Tegretol (CBZ) drug; Another effect is separation of epithelial lining cells from the basement membrane of renal tubules and the collecting ducts may be result of the toxicity of drug and this result is in harmony with [Van de Water etal.;1994] that found separation of the epithelial lining from the basement membrane due to tubules epithelial lining cells death & this may resulting from damage of the cells significance to loss cytoskeleton and destruction of plasma membrane. This result prove the findings of previous studies [Robbins etal.;1994] that stated the congestion is a result of acute inflammation causing blood flow alters within the vessels and arise relax and extend of blood vessels then cause accumulation of blood inside vessels. In addition, [Garveretal.; said that the congestion caused by the concentration of the drug in critical sits of cells, which causes higher levels of red blood serum than simple plasma drug levels do . In the present work a damage effect of Tegretol (CBZ) on the lung of foetuses under experiment ,as it caused oedema, corrugation of the bronchiolar wall, degeneration, necrosis, hemorrhage, pyknosis of nuclei in the lining epithelium of respiratory bronchiole and air spaces, dilation and severe congestion of blood vessels. The epithelial cells of respiratory and terminal bronchioles detached in their lumen.

El-Gaafarawi&Aboel
The mechanism of lung injury by the long term use of (CBZ) has been assumed to be an immune mediated hypersensitivity, according to the in vitro lymphocyte stimulation studies [Houwerzijlet al.; 1977]. The mechanisms of acute (CBZ) -toxicity conversely are unknown.In acute processes, three phases can be well-known in sequence: the so-called initial, exudative & proliferative stages. [Kitson&Wauchob; 1988] described pulmonaryoedema after a massive overdose of (CBZ). They recommended that, in massive overdose, the anti-diuretic effect of the drug may be sufficient to cause pulmonary oedema, a hypothesis supported by their low protein levels in blood. Although pulmonary toxicity is un-common; broncho spasm, pulmonary oedema , interstitial pneumonitis, bronchiolitis 414 obliteransorganising pneumonia, and pulmonary nodules have all been attributed [Milesi-Lecatet al;1997 , Wilschutet al;1997] The procedure of lung damage is believed to be an immune-mediated hyper-sensitivity reaction. [MauriHellwegetal.;1995].This may partly clarify the infrequency of the condition in lung transplantation, as this patient populace are on high dose immuno suppressant manner , in this way a combination of cyclosporin, azathioprine &prednisolone.CBZ ;induced hyper sensitivity condition has been reported in associate to re-activation of viral infection, a common trouble in transplantation[Aiharaet al.

;2003]
Histological examination through lung biopsy showed intra luminal fibrosis of the distal air spaces with foamy alveolar macrophages, signifying Bronchiolitis ObliteransOrganising Pneumonia (BOOP). Later than stopping of (CBZ) administration; all the abnormalities in gamma-globulins gradually improved without any medication. BOOP may be a result of various causes such as; drugs, acute respiratory infections, radiation treatment or emerge idiopathically.

Conclusions:-
This work showed that Tegretol which has the active ingredient (Carbamazepine; CBZ) causes high percentage of foetal mortality in all treated groups. As well as causes growth retardation for the foetuses. Haematoma; dark red patches scattered on the different parts of the body of foetuses at 20 th day of gestation parerntally treated with Tegretol. This investigation indicated that most elements of foetuses skeleton showed more or less, complete ossificationTegretol (CBZ)causes histological alterations in foetal liver, kidneys and lungs structures of the foetuses at 20 th of gestationin all groups.