Effect of age on the sildenafil impact on the histological and ultra-structure of the liver in male albino rat

Background: Sildenafil is one of PDE-5(phospho-diestrase enzyme) inhibitors. It is used as an antihypertensive drug. Sildenafil is mainly metabolized in the liver, and there are cautions regarding the dose when prescribing sildenafil to children, and patients with hepatic impairment. Materials and method: 24 male albino rats (150-350gm weight) were used. Animals were divided into four groups (6 rats each). The rats of treated groups (2, 3 and 4) received (1.43mg/kg body weight) of sildenafil citrate. The Control group received equal volume of distilled water daily during the period of the experiment. At the end of treatment after 30 days, liver samples were collected, processed and stained with (Haematoxylin & Eosin stain) for histological examination. For, ultrastructure studies, specimens were cut into small pieces, processed and ultra thin sections were cut, mounted on the copper grids. Sections were examined by JEOL100SX transmission electron microscope connected to a digital camera. Result: The pre-pubertal rats showed congested central vein and hepatic artery. Areas of inter-cellular vacuolization were observed. Adult rats showed congested central vein, hepatic artery and portal veins with severe inflammatory cell infiltration. Most of hepatocytes exhibited vacuolated cytoplasm and pyknotic nuclei. The senile rats showed congestion in both portal and central veins and increased number of von-kupffer cells. Areas of hemorrhage and necrosis were observed between hepatocytes. Most hepatocytes were degenerated with reduction of number. Conclusion: Sildenafil Citrate drug may cause serious effect on the liver, this effect becomes worse with advance in age.


Introduction
Sildenafil is one of PDE-5(phospho-diestrase enzyme) inhibitors. It is used as an antihypertensive drug. Sildenafil is a selective inhibitor of PDE-5, the enzyme which metabolizes cGMP in the corpus cavernosum of the penis. cGMP is a mediator of smooth muscle relaxation and vasodilatation of blood vessels in the penis. By this action, sildenafil can prolong the duration of penile erection [1].
PDE-5 enzyme is also located in the pulmonary artery smooth muscle [2] Sildenafil increases the potency of cGMP and nitric oxide which is potent short-acting pulmonary vasodilator. Studies on rat neonates with hypoxia,treated by sildenafil,revealed increased pulmonary cGMP levels, reducedinflammatory response and fibrin deposition [3].
Sildenafil is mainly metabolized in the liver, and there are cautions regarding the dose when prescribing sildenafil to children, and patients with hepatic impairment [4]. The hepatic structural changes induced by sildenafil administration are not yet well identified. The present paper aims to clarify the possible structural alterations in the hepatic tissues following experimental sildenafil administration with respect to age.

Sildenafil citrate administration
The rats of treated groups (2, 3 and 4) received (1.43mg/kg body weight) of sildenafil citrate, after being dissolved in distilled water, every day for 30 days, through oro-gastric feeding tube. The giving doses were similar to the recommended human oral doses (100 mg) [5]. The Control group received equal volume of distilled water daily during the period of the experiment through oro-gastric feeding tube. Sildenafil citrate was supplied from Pfizer Inc. (Egypt), was stored at 2-4°C and protected from sunlight.

Histological analysis
At the end of treatment after 30 days, rats of each group were anaesthetized with diethyl ether, vertical ventral midline incision was made in the abdominal wall in order to get the liver samples. The samples were preserved in 10% formalin, for 24 hours, processed for paraffin sections, were stained with (Haematoxylin & Eosin stain) for microscopical examination.

Tissue preparation for ultrastructural study
The specimens were cut into small pieces 1mm3. They were immediately fixed in cold 3.5% glutaraldehyde in 0.1 M phosphate buffer (PH: 7.2), then fixed with 1% osmium tetraoxide (OsO4), processed and embedded in epon. Semithin sections (1µm thick) stained with toluidine blue stain and examined by the light microscope. Ultrathin sections (50-70nm thick) were cut, mounted on the copper grids. Then stained with uranyl acetate and lead citrate [6]. Sections were examined by JEOL-100SX transmission electron microscope connected to a digital camera.

Quantitative analysis
The hepatocytes were measured in a fixed field in serial sections by using Leica Quin 500" image analyzer computer system (England). Evaluation of hepatocytes degeneration was assessed as the following: mild 0-25% of hepatocytes are degenerated, Moderate 25-50%, sever 50-75% [7]. The degree of infiltration with the inflammatory cells was calculated according to the grading system: 0, absent; I, mild; 2, moderate; 3 sever [8].

Statistical analysis
The analysis was carried out through computer software SPSS program (statistical package for social science) version 10. The data were collected as the mean±SD. The significance level considered was P≤0.05.

Control rats
Histological examination of liver section in this groups showed classical lobules with radiating and anastomosing cords of hepatocytes arising from the central vein. The cords of hepatocytes were separated by hepatic sinusoids which were lined by von-kupffer cells. The portal triad showed a clear branch of portal vein, hepatic artery and bile duct (Figures 1 and 5A).

Sildenafil treated rats Prepubertal rats
The liver lobular architecture was maintained in this group. The central vein and hepatic artery were congested. There were moderate inflammatory cell infiltration around the central vein. Few areas of intercellular vacuolization were observed. The bile ducts showed mild hyperplasia. Some hepatocytes exhibited pyknotic nuclei (Figures 2 and 5B).

Adult rats
The hepatic lobular architecture was preserved in this group. The central vein, hepatic artery and portal veins were congested. There was moderate inflammatory cell infiltration (lymphocytes and neutrophils) around the portal vein. Areas of intercellular vacuolization were observed in liver parenchyma. The bile ducts showed severe hyperplasia. Most hepatocytes exhibited vacuolated cytoplasm and pyknotic nuclei, with intercellular areas of hemorrhage and necrosis (Figures 3 and 5C).

Senile rats
This group showed marked congestion in both portal and central veins and increased number of von-kupffer cells. Areas of hemorrhage and necrosis were observed between hepatocytes and around central vein. Most hepatocytes degenerated

Control rats
Transmission electron micrograph sections in the liver of control rats showed hepatocytes with spherical nucleus, mitochondria, glycogen granules, rough endoplasmic reticulum. Von-Kupher cells and blood cell were seen (Figure 6).  with reduction of number. Focal intercellular vacuolization was observed in liver parenchyma (Figures 4 and 5D).

Sildenafil treated rats In prepubertal rat
In this group hepatocytes appeared with spherical nucleus and dispersed chromatin, mitochondria, dilated rough endoplasmic reticulum, lysosomes and phagosomes; Von-Kupher cells was identified (Figure 7).

Adult rat
Transmission electron micrograph of the liver of adult treated rats showed some hepatocytes, with two spherical nuclei, dispersed chromatin and irregular nuclear membrane, swollen mitochondria, dilated rough endoplasmic reticulum, lysosomes and phagosomes. Cytoplasmic vacuoles were noticed (Figure 8).

Senile rats
Transmission electron micrograph of the liver of senile treated rats showed hepatocytes, with irregular indented nucleus with dispersed chromatin, irregular nuclear membrane, swollen mitochondria with unclear cristae, dilated rough endoplasmic reticulum, lysosomes and phagosomes. Sever inter cellular vacuolization was noticed (Figure 9).

Morphometric results and statistical analysis
In comparison with the control group, the area % of degenerated hepatocytes showed a significant increase in the treated groups especially the senile rats (Graph 1).

Discussion
As liver has the sole role in detoxification and excretion of drugs, administration of injurious materials, it is the first organ to be affected through the portal circulation [9]. Although several studies have demonstrated some side-effects of the sildenafil drug on liver, the present study is the first one that reveals morphological changes both at cellular and ultra-structural  levels with respect to the age of the rat.
Sildenafil is mainly metabolized in the liver, and there are major considerations when prescribing sildenafil dose to neonates, children, and patients with hepatic impairment. In the first days of neonatal life, the clearance of sildenafil is about one-third of that seen in adults with increased half-life of 48-56 hours. This could be explained by immaturity of the cytochrome P450 system, which becomes mature in the first week of life [10].
The present study revealed that liver lobular architecture was intact in prepubertal treated group. The central vein, hepatic artery were congested. There were moderate inflammatory cell infiltration (lymphocytes and neutrophils) around the central vein. Only a few areas of intercellular vacuolization were observed. The bile ducts showed mild hyperplesia. Some hepatocytes exhibited pyknotic nuclei. Similar findings reported that exposure of prepubertal rats to Ribnacin drug induced hepatocyte necrosis and mononuclear cell infiltration in the portal tract [11]. It was documented that the hepatocellular injury due to any factor is mediated through free-radicle from lipid peroxidation [12].
The current study revealed that ingestion of sildenafil in adult rats caused congestion of the central vein, hepatic artery and portal veins. There was moderate inflammatory cell infiltration (lymphocytes and neutrophils) around the portal vein. Areas of intercellular vacuolization were observed in liver parenchyma. The bile ducts showed severe hyperplasia. Most of hepatocytes showed vacuolated cytoplasm with pyknotic nuclei, with intercellular areas of hemorrhage and necrosis. Transmission electron micrograph of the liver of adult treated rats showed hepatocytes, with dispersed chromatin and irregular nuclear membrane, swollen mitochondria, rough endoplasmic reticulum, lysosomes and phagosomes. Cytoplasmic vacuoles were noticed.
Similar results have been reported after injection of sildenafil (1μg/g body wt.) in adult rats for 15 days, in the form of central vein congestion and cytoplasmic vacuolization [13]. It was documented that cytoplasmic vacuolization is one of the primary responses to any cell injury. It occurs as a result of increased permeability of cell membranes leading to increase in the intracellular water content [14].
Also, injection of 9mg/kg of sildenafil in adult rabbit induced hepatocytes nuclear changes. This change may be due to cell injury [15]. karyolysis was observed in sildenafil overdoses. Nuclear changes caused by chromatin condensation while karyolysis resulted from chromatin matter dissolution of a necrotic cells [16]. The presence of inflammatory cells in the liver after sildenafil exposure may indicate an effect of this drug on the antioxidant defense which may cause generation of reactive oxygen species and inflammatory response [17]. In addition, sildenafil can cause lysosomal enzymes leakage and cytoplasmic degeneration. The degeneration may be due tocell membrane disturbance and massive influx of water and sodium ions [18]. * * doi: 10.7243/2055-091X- [3][4][5] This study revealed that the treated senile rats showed marked congestion in both portal and central veins, increased number of von-kupffer cells. Areas of hemorrhage and necrosis were observed between hepatocytes and around central vein. Most hepatocytes were degenerated with reduction in number. Intercellular vacuolization were observed in between liver cells.
In human, a case of sildenafil-induced liver disorder was reported in 49 year-old male after 4 weeks of drug initiation in dose of 50 mg/day. He presented with pain in the right hypochondrium, hepatomegaly and elevated liver enzymes. All other investigations were normal including anti bodies to hepatitis B, hepatitis and hepatitis C. Liver enzymes returned to normal values after sildenafil withdrawal in 20 days [19].
The sever hepatic changes in senile rats may be explained according to the fact that there is a reduction in drugs clearance with age especially those undergo transformation by liver microsomal mono-oxygenase [20]. In 1968, Kato and Tanaka were the first to report a significant age-related reduction in the activities of the microsomal mono-oxygenase hepatic enzyme in male rats in vitro [21].
Studies in humans showed that aging is associated with reduction in hepatic drug metabolism. Cytochrome P450 in human decreases 30% after the age of 70 years. Reduced drug metabolism may be also due to reduction of liver voume and the blood flow [22]. The apoptotic potential of the liver decrease with advance of age. So, the injured and pre-neo plastic cells may increase in liver with advanced age. This may explain the brown atrophy and hepatocellular neoplasms with old ages [23].
Also, aging is associated with increase of gene expression associated with fibrosis, stress of the cell and inflammation. This gene is normally associated with activation of macrophages [24]. Activated macrophages and inflammatory cells are included in some liver diseases like cirrhosis, hepatitis, endotoxin-induced liver lesion [25].
This study provides evidence that Sildenafil Citrate drug may cause seroius effect on the liver, which becomes worse with advance in age. Further studies are needed to assess whether these morphological changes are transitory or irreversible.