Protective Effect of Vitamins E & C and Silymarin Against Paracetamol Toxicity on the Liver of Adult male Albino Rat

INTRODUCTION

Liver diseases are serious health problems and is still considered as a great challenge to deal with in the practice of modern medicine.¹ paracetamol or N-acetyl-p-aminophenol) (APAP) is a derivative of para-aminophenol. It is widely used as a safe analgesic and antipyretic drug. It is metabolized in the liver to excretable glucuronide and sulphate conjugates. High doses of paracetamol can cause nephrotoxicity² and hepatotoxicity mainly due to the formation of a highly reactive metabolite, namely N-acetyl-p-benzoquinone imine (NAPQI).³ Paracetamol-related hepatotoxicity is one of the commonest causes of liver failure. Most of such cases are the result of ingestion of large paracetamol doses. ⁴

Vitamin E is a naturally occurring lipid soluble antioxidant scavenging free radicals. ⁵ Free radical damage was implicated in a number of degenerative states including carcinogenesis, arthritis, platelet hyperaggregability, ischemia and reperfusion of injury, cataract and lung injury caused by pollutants. ⁶

Vitamin C is a widely known vitamin abundant in different kinds of fruits and vegetables and is available as an OTC (over the counter) supplement.⁷ It plays an important physiological role in cells as a reducing agent, antioxidant and free radical scavenger. It protects the cells from reactive oxygen species and hence prevents oxidative damage to the different biological macromolecules, such as proteins, DNA, and lipids. ⁸

Silymarin is a flavonoid extracted from the seed of Silybum marianum Gaetrn (milk thistle plant) which is a member of the aster family (Asteraceae).  Owing to the plant’s antioxidant role, its seeds have been used to treat a range of liver and gallbladder disorders. Silymarin was proved to inhibit fibrogenesis and stabilize the lipid structures in the hepatocellular membrane.⁹

The aim of this work is to study the possible protective effects of vitamins E, C and silymarin, on the hepatotoxicity induced by paracetamol.

PATIENTS AND METHODS

Experimental animals:

Forty adult male albino rats were used in this experiment. Their weight ranged from 200-250gm. They were housed in a plastic cage in an environmentally controlled animal facility (room temperature, 12 h light: 12 h dark cycle). The rats were kept under the same circumstances and fed on normal diet and water throughout the experiment. The experiment was conducted for

a period of 14 days. The experiment was done in Faculty of Medicine, Al-Azhar University.

Chemical materials:

Paracetamol was obtained from Pharco Company, in the form of tablets (500mg/ tab). Silymarin was obtained from Sedico Company, in the form of packs. (140mg/pack). Vitamin E was Obtained from Pharco Company, in the form of gelatinous capsules. (400mg/cap). Vitamin C: Obtained from Memphis company, in the form of ampule. (1000mg/amp).

The rats were divided in a random manner into four groups, (each group consisted of ten rats); Group I (Control group): The rats were given 10 ml 0.9% NaCL orally daily for 14 days; Group II: The rats were given paracetamol (PC) from the 8^th to the 14^th day of the experiment, in a dose of 1g/kg orally by gastric tube once daily. ¹⁰ Group III: The rats were given silymarin + paracetamol as follows: Silymarin, in a dose of 25mg/kg orally by gastric tube, once daily for 14 days. ¹¹ and paracetamol (1g/kg orally by gastric tube once daily from the 8^th to the 14^th day of the experiment. Group IV:The rats were given vitamins E and C together with paracetamol as follows:

Vitamins E and C (VEC) for two weeks and paracetamol was given orally (1g/kg) by gastric tube once daily from the 8^th to the 14^th day of the experiment. The dose of vitamin E was 50 mg/kg.¹² The dose of vitamin C was 100mg/kg, by intraperitoneal injection once daily.¹³

After 48 hours from the last day of the experiment, the animals were sacrificed under ether anesthesia. Then the livers were carefully extracted and processed for light microscopy (LM) (hematoxylin and eosin stain) and transmission electron microscopy (EM).

RESULTS

Light Microscopic Examination

Group I (Control group):

The hepatocytes of different sections appeared normal in their structure. They were arranged in cords and radiating from the central vein towards the periphery. The blood sinusoids appeared separating the hepatic cords. They were lined with endothelial cells that appeared flat with appearance of few Kuppfer cells. The cytoplasm of the hepatocytes appeared acidophilic with rounded and vesicular nuclei (Fig.1).

Group II:

There was loss of the normal architecture of the hepatic cords. The hepatocytes appeared with vacuolations in the cytoplasm of most of them. Some nuclei of the hepatocytes appeared became eccentric and other nuclei appeared small and pyknotic. Some hepatocytes showed areas of focal necrosis. There were marked cellular infiltration. The central vein appeared dilated and congested. (Fig. 2 and 3)

Group III:

Restoration of the normal hepatic architecture was obvious. The arrangement of the hepatic cords all around the central vein was normal. The hepatic cords appeared radiating from the central vein towards the periphery. Most of the hepatocytes appeared normal with acidophilic cytoplasm. The nuclei appeared rounded and vesicular with intact nuclear membrane. In some sections the central vein appeared congested and dilated (Fig. 4).

 Group IV:

Normal structure of the hepatocytes was preserved. The arrangement of the hepatic cords appeared normal. The central vein and its associated sinusoids appeared intact in all sections. Most of the hepatocytes appeared with acidophilic cytoplasm. The nuclei appeared intact, with intact nuclear membrane. Some hepatocytes contain vacuolated cytoplasm (Fig 5).

Fig 1: Photomicrograph of liver of a control rat shows: The central vein (Cv), hepatocytes (H) with the nuclei appear rounded and intact (N) the hepatocytes appeared radiating from the central vein towards the periphery. The hepatic cords appear separated from each other by blood sinusoids (S), lined by kupffer cells (Kc) (Hx & EÍ400).

Fig 2: Photomicrograph of liver of rat treated with paracetamol shows, dilated and congested central vein (Cv) with signs of degeneration in the form of vacuolated liver cells (V), inflammatory cellular infiltration (IC), pyknotic nuclei (Pn), karyolitic nuclei (Kn) (Hx & EÍ400).

Fig 3: Photomicrograph of liver of rat treated with paracetamol shows, marked hepatic vacuolations (V), dilated congested central vein (CV) and small dark pyknotic nuclei (Pn) and karyolitic nuclei (Kn) of hepatocytes(Hx & EÍ400).

Fig 4: Photomicrograph of liver of rat treated with paracetamol and silymarin shows congested central vein (Cv), some hepatocytes (H) appear intact, others are vacuolated (v). Cords of hepatocytes appeared separated by sinusoids(S) (Hx & EÍ400).

Fig 5: Photomicrograph of liver of rat treated with paracetamol and vit. E and C showing preserved hepatic architecture, central vein (CV) appears of normal diameter, not dilated, not congested with its associated sinusoids (S) appearing intact. Most of hepatocytes (H) have acidophilic cytoplasm and intact nuclei (N) and lined by Kuffer cells (kc) (Hx & EÍ400).

Electron microscopic examination

Group I (Control group):

 Normal ultrastructural features of the hepatocytes were observed. The nuclei appeared rounded with condensed chromatin. The cytoplasm and the cell organelles appeared intact. The mitochondria were intact and numerous. (Fig. 6 and Fig. 7).

Group II:

Many abnormal findings were observed. The hepatocytes appeared vacuolated. Some nuclei were without nucleolus with intranuclear and intracytoplasmic vacuolations (cloudy swelling). The cytoplasm of most of the hepatocytes showed few numbers of mitochondria and endoplasmic reticulum. (Fig. 8 and Fig. 9).

Group III:

The hepatocytes appeared normal in their structure. The nucleus appeared rounded with intact membrane. The cytoplasm contains numerous organelles. The mitochondria appeared numerous and intact. The endoplasmic reticulum appeared preserved. (Fig.10 and Fig.11).

Group IV:

The hepatocytes appeared intact. The nuclei appeared normal rounded and contains normal nucleoli and the nuclear membranes appeared intact. The cytoplasm contained numerous and intact mitochondria and endoplasmic reticulum (Fig. 12 and Fig. 13).

Fig 6: Electron photomicrograph of the liver of control rat: The nucleus of the hepatocyte appeared rounded with intact nuclear membrane (Nm) and condensed peripheral chromatin (CPC) and normal nucleolus (Nu). The cytoplasm contains many mitochondria (M) and endoplasmic reticulum (ER), (EM  Í 8000).

Fig 7: Electron photomicrograph of the liver of control rat: A higher magnification of the previous section showing: The hepatocyte with rounded nucleus (N) with nuclear membrane (Nm), mitochondria (M), and endoplasmic reticulum (ER), condensed peripheral chromatin (CPC). (EM  Í 12000).

Fig 8: Electron photomicrograph of the liver of rat treated with paracetamol shows: signs of degeneration: Nucleus (N) with no nucleolus, nuclear membrane (Nm) appeared intact. Few mitochondria. (M), vacuolated hepatocyte, (V) (cloudy swelling)(EM  Í 5000).

Fig 9: Electron photomicrograph of the liver of rat treated with paracetamol shows: Nucleus(N) without nucleolus, congested blood sinusoid (S), Vacuolated hepatocytes (V) (EM  Í 3000).

Fig 10: Electron photomicrograph of the liver of rat treated with paracetamol and silymarin shows: The hepatocyte with rounded nucleus (N) and nuclear membrane (Nm). The cytoplasm contains intact mitochondria (M) preserved endoplasmic reticulum (ER), lipid droplets (L). (EM  Í 8000).

Fig 11: Electron photomicrograph of the liver of rat treated with paracetamol and silymarin shows: The hepatocytes with rounded nucleus (N) and nuclear membrane (Nm) with intact endoplasmic reticulum (ER), intact mitochondria (M) and congested sinusoids (S) with red blood cells (RBC) (EM  Í 5000).

Fig 12: Electron photomicrograph of the liver of rat treated with paracetamol and vit. E and C shows:  Normal rounded nucleus with prominent nucleolus (Nu). the nuclear membrane is preserved (Nm). The cytoplasm contains numerous and intact mitochondria (M) and endoplasmic reticulum (ER). (EM  Í 6000).

Fig 13: Electron photomicrograph of the liver of rat treated with paracetamol and vit. E and C: higher magnification of previous section Showing:  Normal rounded nucleus with prominent nucleolus (Nu) and nuclear membrane (Nm). The cytoplasm contains numerous mitochondria (M) and endoplasmic reticulum (ER). (EM  Í 10000).

DISCUSSION

The liver performs a fundamental role in the regulation of many physiological processes, and its activity is related to different vital functions, such as metabolism, secretion, and storage. It detoxifies endogenous (waste metabolites) and/or exogenous (toxic compounds) substances or organisms and synthesize many useful sustances.¹⁴

The hepatic diseases are of the most common threats to the public health, and they are distributed all over the world .¹⁵

Paracetamol is considered safe at therapeutic doses. Overdoses of paracetamol cause severe nephrotoxicity³ and hepatotoxicity. ¹⁶

In this study, it was found that, vitamins E and C performed a hepatoprotective effect against the harmful effects induced by paracetamol. Many researches were in agreement with that they reported that Vitamin C decreased and treated hepatic damage that was produced by many chemical agents. This is greatly in agreement with the work of Bashandy and Alwasel.¹⁷

Vitamin C is well known to make synergy with many other anti-oxidants. One of the common and proved synergies is between vitamin C and vitamin E.¹⁸

The results proved by the present study showed that livers of rats received paracetamol suffered from loss of normal appearance of hepatocytes with appearance of focal necrosis of hepatocytes. Some hepatic nuclei became eccentric and other nuclei appeared pyknotic and others were karyolitic. There was mitochondrial dysfunction in paracetamol hepatotoxicity.¹⁹

These results are in agreement with results by Reham and Mohammad ²² who found that the livers of the rats were greatly affected with loss of their normal appearance.

Also, in the present study there was marked cellular infiltration and diffuse cytoplasmic vacuolations. It correlates with results found by Manov et al ²⁰ who found necrosis of some hepatocytes, focal chronic inflammatory cellular infiltration, and evidence of vacuolation.

The results of the present study correlate with the results found bySaheed et al ²¹ that noticed vacuolated cytoplasm, area of necrosis and infiltration by inflammatory cells.

Electron microscopic examination of hepatocytes of rat received paracetamol only showed many abnormalities. There was many cytoplasmic vacuolations. Mitochondria and endoplasmic reticula markedly decreased.

These results go in agreement with Rehamand Mohammad ²² who found disintegration and loss of the normal structure of most of cellular contents.

In our study, administration of silymarin showed marked improvement, where the hepatocytes appeared normal with their nuclei appeared intact and rounded. The cytoplasm contains many intact organelles. Mitochondria were numerous and intact with preserved endoplasmic reticula.

Reham and Mohammad ²² found that, liver tissues of mice treated with silymarin and paracetamol appeared intact with intact nuclei and numerous mitochondria. 

Padmanabhan and Jangle ²³ said that silymarin reduced hepatic sinusoidal congestion, cloudy swelling and fatty changes in rats treated with alcohol.

Similar results were reported by Wu et al.²⁴ who showed that silymarin restored normal appearance of the liver tissue specially in the early stages of liver damage.

In the present study light microscopic examination of rat’s liver treated by paracetamol and vitamin-E and vitamin-C showed improvement. The hepatic architecture appeared intact with normal arrangement of the hepatic cords and the central vein.

These results were in full agreement with the results found by Ganesh et al ²⁵ who noticedabsence of any signs of necrosis. The structure of the hepatic lobules appeared normal after hepatotoxicity introduced by paracetamol when rats were treated with vitamin-E.

These results came in agreement with Fatma et al ²⁶when rats given a combination of both vitamins, C and E, 15 minutes prior to daily paracetamol administration. They found marked decrease of vacuolar degeneration and fatty changes.

CONCLUSION

Paracetamol has harmful effects on the hepatic tissues in the form of focal necrosis and loss of normal structure of hepatocytes. Silymarin, vitamins E and vitamin C markedly decreased these effects and restored the normal appearance of the hepatic tissues.