Effect of Oyster mushroom in Paracetamol Induced Toxicity of Liver in Wistar albino Rats

Backgroud: Liver is an important metabolic organ. It has wide range of functions including detoxification, storage of glycogen, vitamins A, D and B12, production of several coagulation factors, growth factors such as insulin-like growth factor-1 (IGF-1), angiotensinogen, and biochemicals necessary for digestion (bile). Its damage occurs due to its multidimensional functions, various xenobiotics and oxidative stress leading to distortion of all of its functions. Oyster mushroom which is excellently edible and nutritious has got free radical scavenging activity, and so may be considered as a hepatoprotective agent. Objective: To observe the hepatoprotective effect of Oyster mushroom (Pleurotus florida) against paracetamol induced liver damage in Wistar albino rats. Materials and Methods: This experimental study was carried out in the Department of Physiology, Sir Salimullah Medical College (SSMC), Dhaka from 1st July 2009 to 30th June 2010. Thirty four Wistar albino rats, aged 90 to 120 days, weighing between 150 to 210 grams were used for the study. After acclimatization for 14 days, they were divided into two groups –– control group (Group A) and experimental group (Group B, mushroom-pretreated and paracetamol-treated group). Control group was again subdivided into Group A1 (baseline control group) and Group A2 (paracetamol-treated control group). Animals of all groups received basal diet for 30 consecutive days. In addition, Group A1 rats received propylene glycol (2 mL/kg body weight orally) only on 30th day, Group A2 rats received single dose of paracetamol suspension (750 mg/kg body weight orally) only on 30th day and Group B rats received mushroom extract (200 mg/kg body weight orally) for 30 consecutive days and paracetamol suspension (750 mg/kg body weight orally) only on 30th day. All the animals were sacrificed on 31st day. Then liver specimens were collected. Histology of liver was done by using standard laboratory procedure. Statistical analysis was done by one way ANOVA test by using SPSS version 15.0. Result: In this study, histological examination of liver reveals abnormal histological findings in 100% of rats in paracetamol-treated group (Group A2), almost normal structure in 80% of rats and mild histological changes in 20% rats in mushroom-pretreated and paracetamol-treated group (Group B). Conclusion: The present study reveals the hepatoprotective effect of Oyster mushroom (Pleurotus florida) against paracetamol induced liver damage in Wistar albino rats.


Effect of Oyster mushroom in Paracetamol Induced Toxicity of Liver in Wistar albino Rats
Paracetamol is an antipyretic and analgesic drug which is widely used to cure fever, headache and other pains and is readily available without prescription.When taken in at toxic dose, it may cause hepatotoxicity by overproduction of reactive oxygen species (ROS) such as superoxide radicals, hydrogen radicals and hydroxyl radicals during the formation of N-acetyl pbenzoquinoneimine (NAPQI) by cytochrome p450. 3,4xcess levels of ROS can attack biological molecules such as DNA, protein, phospholipids, which leads to lipid peroxidation and depletion of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GP x ). 5,6mage to the liver by hepatotoxic agents is of grave consequence as chronic liver injury leads to fibrosis, end stage cirrhosis and hepatocellular carcinoma.As a result there is increasing need to search of an agent which could protect the liver from such damage.
Mushrooms have been used in folk medicine throughout the ancient time.Now-a-days, mushrooms are widely used as a nice vegetable. 7shrooms are a good source of vitamins and minerals and are preferred in many countries because of its special flavor and aroma. 8Oyster mushroom (Pleurotus ostreatus) can suppress damage of the liver by its hepatoprotective effect. 9,10stological architecture of the liver of toxic animals revealed extensive damage, characterized by the disruption of the normal structure of the hepatocyte, damaged cell membranes, degenerated nuclei, disintegrated central vein and damaged hepatic sinusoids. 9,11Use of mushroom extract may protect the liver from damage. 9,12,13tioxidant activity or the inhibition of the generation of free radicals is important in providing protection against hepatic damage.In absence of reliable liver protective drugs in modern medicine, Ayurveda recommended some medicinal preparations for the treatment of liver disorder.Some researchers showed that some mushrooms such as Lentinus edodes, Grifola frondosa and Tricholoma lobayense have hepatoprotective activity. 14But in Bangladesh, no study has been done yet to evaluate the hepatoprotective effect of mushroom.Oyster mushroom has highly nutritive and medicinal value, is cultivated and harvested in Bangladesh all over the year.It is reasonably cheap, available and relatively safe in comparison to other mushrooms.Therefore, the present study was designed to observe the hepatoprotective effect of Oyster mushroom (Pleurotus florida) in experimental animals after inducing hepatotoxicity by paracetamol.The findings of this study may be helpful to make this mushroom acceptable among the people as a source of functional food for the prevention of liver diseases.After acclimatization for 14 days, all animals were divided into two groups ---control group (Group A) and mushroom-pretreated and paracetamol-treated group (Group B).Control group was again subdivided into Group A 1 (baseline control group) and Group A 2 (paracetamol-treated control group).Group A 1 consisted of 10 rats, Group A 2 consisted of 14 rats and Group B consisted of 10 rats.Animals of all groups received basal diet for 30 consecutive days.In addition, Group A 1 rats received propylene glycol (2 mL/kg body weight orally) only on the 30 th day; Group A 2 rats received single dose of paracetamol suspension (750 mg/kg body weight orally) only on the 30 th day. 15roup B rats received mushroom extract (200 mg/kg body weight orally) for 30 consecutive days and paracetamol suspension (750 mg/kg body weight orally) only on the 30 th day. 15During the study period, one rat of paracetamol-treated control group died.It might be due to toxic effect of paracetamol.On the 31 st day final body weights of all rats (FBW) were measured and then the rats were sacrificed.Then liver samples were collected.After washing in ice-cold saline these were preserved in 10% formalin for subsequent histological processing which was done by using standard laboratory procedure in the department of Pathology of SSMC.

Materials and Methods
Statistical analysis was done by one way ANOVA, Fisher's exact test and Bonferroni test by using SPSS version 15.0.

Preparation of paracetamol solution
1 g of paracetamol (active ingredient) was dissolved in 9 mL of propylene glycol.After thorough mixture a homogeneous solution was obtained, giving strength of 111.11 mg of paracetamol per mL of solution. 16

Preparation of mushroom extract
Oyster mushroom was collected from National Mushroom Development and Extension Center (NMDEC), Savar, Dhaka.Fresh mushroom was dried in the sun and finally in the oven and then crushed into powder with a mechanical grinder.Mushroom powder was extracted with ethanol, filtered and evaporated to obtain mushroom extract. 9

Results
Table I  Table II shows the liver weight in different groups of rats and also comparison between groups.The mean (± SD) of liver weights of rats were 3.57 ± 0.40, 4.22 ± 0.55 and 3.88 ± 0.42 g in groups A 1 , A 2 and B respectively.The liver weight was higher in group A 2 when compared to that of group A 1 and B; though it was statistically significant (p<0.01) between groups A 1 vs A 2 , but non-significant between groups A 2 vs B. Again, liver weight of group B was higher than that of group A 1 , but the difference was non-significant.

Histological observation of liver in rats of different groups
In Group A 1 (baseline control group) hepatic structure was normal (Fig 1).In Group A 2 (paracetamol-treated group) moderate histological changes (presence of centrilobular necrosis, disorganization of hepatic sinusoids, infiltration of lymphocytes and Kupffer cells, fatty changes and ballooning degeneration) were found (Figs 2, 3 and 4).In Group B (mushroom pretreated and paracetamol-treated group) normal histological findings were observed in 8 rats (Fig 5), but presence of minimal fatty change, ballooning degeneration, infiltration of lymphocytes and centrilobular necrosis were found in 2 rats.
Table IIIA shows the distribution of rats by histological changes in liver.In this study, histological examination of liver revealed normal findings in 100% of rats in Group A 1 and abnormal histological findings were observed in 100% of rats in Group A 2 .Eighty per cent of rats in Group B showed almost normal structure whereas 20% showed mild histological changes in liver.
Though the percentage of abnormal histological changes was higher in Group B than that of Group A 1 but the difference was statistically non-significant.
Table IIIB shows comparison of histological findings in liver.
Table IIIA

Discussion
In this study, the final body weight of all the animals were found increased compared to their initial weight, but this was statistically nonsignificant.This finding is in agreement with that of some other researchers. 17The percentage change of body weight from initial body weight to the final body weight was significantly lower (p<0.05) in mushroom-pretreated and paracetamoltreated group compared to that of baseline control group.This is similar to the findings of other investigators. 18The liver weight was significantly higher (p =0.007) in paracetamol-treated rats compared to that of baseline control group, but nonsignificantly higher when compared to that of mushroom-pretreated and paracetamol-treated group.Nonsignificant difference in this value was observed in mushroompretreated and paracetamol-treated rats in comparison to that of baseline control rats.
In this study, moderate histological changes such as centrilobular necrosis, disorganization of hepatic sinusoids, infiltration of lymphocytes and Kupffer cells, fatty changes and ballooning degeneration were observed in paracetamol-treated rats.These findings are also in agreement with those of different investigators of other countries. 19,20On the other hand, only minimal histological changes of liver were noted in 20% rats of mushroom-pretreated and paracetamol-treated rats which are in consistence with findings of different researchers of different countries. 9,13tensive studies on the development of hepatic damage with paracetamol showed centrilobular necrosis in the liver including infiltration of lymphocytes and Kupffer cells, fatty changes and ballooning degeneration in overdose due to increased production of free radicals. 15,19Again, it has been suggested that, hepatotoxicity may be produced by altering liver microsomal membranes in experimental animals. 21The antioxidant activity or the inhibition of the generation of the free radicals is important in providing protection against hepatic damage. 22Many studies have been carried out to find out antioxidants of natural origin.Among the various naturally occurring substances, mushroom may be one of the effective antioxidants due to its free radical scavenging activity. 23Some research workers suggested that the possible antihepatotoxic mechanisms may be related to the suppression of tumor necrosis factor-αα) and free radical scavenging activity.This anti-inflammatory role of mushroom may be helpful in protecting the hepatocyte from damage. 12oreover, it has been suggested that the extract of mushroom probably acts to prevent the fall of glutathione (GSH) through some GSH dependent enzymes.Thus the structural integrity of the hepatic cell membrane is preserved. 24fferent researchers suggested that some active compounds present in Oyster mushroom such as βglucan 17 , phenol and vitamin C 18 scavenge free radicals and thus play the role for antioxidant property of mushroom.These active components of Oyster mushroom increase the activities of some antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GP x ) 17 and these antioxidant activities of mushroom cause reduction of hepatic cell necrosis 12 , stabilization of hepatic cell membrane 13 and may protect the liver architecture by scavenging free radicals.
In the present study, hepatic damage was observed in rats treated with paracetamol as evidenced by moderate changes in liver architecture observed by histological examination.These changes may be due to increased production of free radicals which initiate lipid peroxidation and subsequent cell damage. 19,20stopathological changes found in mushroompretreated and paracetamol-treated groups were less than those of paracetamol-treated rats' liver.It provides a direct evidence of hepatoprotective effect of the extract of Oyster mushroom.However, the exact mechanism involved in the hepatoprotective activity of Oyster mushroom extract against paracetamol induced liver damage in rats cannot be found out from this type of study.According to the suggestions made by different investigators, free radical scavenging activities may be responsible for the effect. 9,12,13om this study it can be concluded that Oyster mushroom (Pleurotus florida) may have some hepatoprotective role, but the active component of mushroom, which is responsible for this effect, is not known.The hepatoprotective effect may be due to its free radical scavenging activity.

Table I :
Initial and final body weight of rats and percentage of change of body weight in different groups (n=33)

Table II
Group A 1 , baseline control group; Group A 2 , Paracetamol-treated control group; Group B, Mushroom pretreated and paracetamoltreated group; ns, non-significant; **, significant at p<0.01