Pharmacological Evaluation of Glutamine Against Isoniazid Induced Hepatotoxicity in Laboratory Rat

Certain medicinal agents when taken in overdoses and sometimes even when taken within therapeutic range may cause the damage of liver. Some chemicals used in laboratories and industries, herbal remedies and natural chemicals (such as microcystins) may also induce hepatotoxicity. There are more than 900 drugs involved in liver injury and it is the most common reason for withdrawal of the drug from the market (Pandit et al., 2012). Hepatotoxins often cause subclinical injury to liver which manifests only as abnormal liver enzyme tests.

Liver physiology

The liver is the largest organ of the human body, weighs approximately 1500 g, located in the upper right corner of the abdomen. It is closely associated with the small intestine and processes the nutrient-rich venous blood that leaves the digestive tract. It performs over 500 metabolic functions, resulting in synthesis of products that are released into the blood stream (e.g. glucose derived from glycogenesis, plasma proteins, clotting factors and urea), or that are excreted to the intestinal tract (bile). Many products including glycogen, fat and fat-soluble vitamins are stored in liver parenchyma. Almost all blood that enters the liver via the portal tract originates from the gastrointestinal tract as well as from the spleen, pancreas and gallbladder. A second blood supply to the liver comes from the hepatic artery, branching directly from the celiac trunk and descending aorta. The portal vein supplies venous blood under low pressure conditions to the liver, while the hepatic artery supplies high-pressured arterial blood. Since the capillary bed of the gastrointestinal tract already extracts most O2, portal venous blood has a low O2 content. Blood from the hepatic artery on the other hand, originates directly from the aorta and is, therefore, saturated with O2. Blood from both vessels joins in the capillary bed of the liver and leaves via central veins to the inferior caval vein. The basic anatomy of liver is shown in figure 1.

       
           
       
Figure 1. Anatomical view of liver

The major blood vessels, portal vein and hepatic artery, lymphatics, nerves and hepatic bile duct communicate with the liver at a common site, the hilus. From the hilus, they branch and rebranch within the liver to form a system that travels together in a conduit structure, the portal canal (Fig. 2.8). From this portal canal, after numerous branching, the portal vein finally drains into the sinusoids, which is the capillary system of the liver. Here, in the sinusoids, blood from the portal vein joins with blood flow from end arterial branches of the hepatic artery. Once passed through the sinusoids, blood enters the collecting branch of the central vein, and finally leaves the liver via the hepatic vein. The hexagonal structure with, in most cases, three portal canals in its corners draining into one central vein, is defined as a lobule (figure 2).

       
           
       
Figure 2. Blood vessel network in liver and liver lobules

The lobule largely consists of hepatocytes which are arranged as interconnected plates, usually one or two hepatocytes thick. The space between the plates forms the sinusoid. Amore functional unit of the liver forms the acinus. In the acinus, the portal canal forms the center and the central veins the corners. The functional acinus can be divided into three zones: 1) the periportal zone, which is the circular zone directly around the portal canal, 2) the central zone, the circular area around the central vein, and 3) a midzonal area, which is the zone between the periportal and pericentral zone.

Liver diseases:

Liver diseases can be classified in many different ways.

• Viral hepatitis Viral hepatitis is an infection that causes liver inflammation and damage. Several different viruses including hepatitis A, B, C, D and E cause viral hepatitis. The hepatitis A and E viruses typically cause acute infections, whereas the hepatitis B, C, and D viruses can cause acute and chronic infections. Hepatitis A, B, C viruses can result in acute disease with symptoms of nausea, abdominal pain, fatigue, malaise, and jaundice. Chronic infection can develop into cirrhosis, liver failure and hepatocellular carcinoma.
• Cirrhosis Cirrhosis is an abnormal liver condition in which liver tissue is replaced by fibrous scar tissue as well as regenerative nodules in response to chronic liver injury. It leads to development of portal hyprtension and end stage liver disease. Worldwide the incident of liver cirrhosis is high. About 2.8 million people were affected with cirrhosis and resulted in 1.3 million deaths in 2015. Generally, liver cirrhosis cannot be reversed and liver transplantation is the only curative available for established cirrhosis.
• Hepatocelluar carcinoma (HCC) HCC is a primary malignancy of the liver. Chronic liver diasease and liver cirrhosis subsequently can develops into HCC. Now it is the third leading cause of cancer death worldwide. The incidence of HCC is highest in asia and Africa, where the endemic prevalence of the hepatitis B & C is high.
• Non-alcoholic liver disease (NAFLD) NAFLD is one of the types of fatty liver and most common form of chronic liver disease, affecting 80 to 100 million people in United States. It is a major hepatic disorder in patients with metabolic syndrome. Studies indicated that insulin resistance, abnormal lipid metabolism and degradation of cytokinesis/adipokines are profoundly involved in the pathogenesis of NAFLD.

Alcoholic liver disease (ALD) Alcohol is the single most significant cause of liver disease in the western world and affects millions of people worldwide each year. The progression of ALD is well characterized and is actually a spectrum of liver diseases, ranging from steatosis to inflammation and necrosis, to fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Histological alcoholic hepatitis is characterized by ballooning degeneration of hepatocytes with polymorphonulcear cell infilteration, moderate to severe fatty infilteration, Mallory bodies sclerosing hyaline necrosis and increased fibrosis with perivenular fibrosis (Arteel G et al., 2003). Jaundice Jaundice/Icterus refers to the yellow tinge of the skin, sclera and mucous membrane that is caused by increase bilirubin concentration in blood. Levels of serum bilirubin are normally below 1.0 mg/dL and levels over 1.5 mg/dL typically results in jaundice.

Epidemiology of hepatotoxicity:

The true incidence of drug-related liver disease is unknown because just a minority of the actual cases is reported to the regulatory agencies. Worldwide data regarding the prevalence of hepatotoxicity is not available till now, but a study in France over a 3- year period showed the vulnerability of hepatotoxicity (Sgro et al., 2002). According to this study, the annual incidence of hepatic reactions to drugs was 139 cases per 1 million people, which was 16 times higher than the number reported to the French reporting system of Adverse Drug Reactions. In addition, the lack of an accurate diagnosis is an important limitation (Larrey, 2002). Approximately 50% of the reactions reported to regulatory authorities have been found to be unrelated to the incriminated drug when evaluated carefully later (Aithal et al., 1999). Nevertheless, available data clearly indicate that the incidence of drug-induced hepatotoxicity is increasing (Dossing& Andreasen, 1982; Friis & Andreasen, 1992). This is probably due to a combination of factors including greater exposure to drugs, a better knowledge of their toxic effects on the liver and a more rigorous exclusion of alternative causes of liver damage thanks to the availability of new specific tests for diagnosing viral hepatitis. Cause of hepatotoxicity may result not only from direct toxicity of the primary compound, but also from a reactive metabolite or from an immunologically mediated response affecting hepatocytes, biliary epithelial cells and/or liver vasculature (Saukkonen et al., 2006; Deng et al., 2009; Singh et al., 2011).

Isoniazid and Rifampicin-induced hepatotoxicity:

Isoniazid (INH) and rifampicin (RFP) are the first-line antitubercular drugs. The single or combined use of isoniazid and rifampicin can cause liver injury, leading to liver failure, accounting for 5%–22% of acute liver failure cases (Devarbhavi, et al., 2013). Natural medicinal ingredients have the characteristics of multi-level, multi-target, and multi-channel comprehensive regulation and have unique advantages in improving patients’ symptoms, reducing the risk of liver injury, delaying the progress of liver injury, and enhancing the repair ability of the body (Hong, et al., 2015). In recent years, natural medicinal ingredients have shown a good protective effect on liver injury caused by INH and RFP. This paper reviewed the molecular mechanisms of INH and RFPinduced liver injury and natural medicinal ingredients’ preventive and therapeutic effects on liver injury. We hope this review article will serve as an educational resource for researchers interested in developing new drugs against INH and RFP-induced liver injury. The pathogenesis of isoniazid-induced liver injury (INH-ILI) has not been fully elucidated. The mechanisms of INH-ILI mainly involve oxidative stress, mitochondrial dysfunction, drug metabolic enzymes, protoporphyrin IX accumulation, endoplasmic reticulum stress, bile transport imbalance, and immune response. It has been suggested that the oxidative stress injury caused by INH is because of the dysregulated compensatory activation of the nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) antioxidant stress system and the reactive oxygen species (ROS) accumulation (Figure 1).

       
           
       
Figure 3. The mechanisms of oxidative stress injury caused by INH (Compensatory Nrf2/ARE antioxidative stress system inhibition, production of lipid peroxides, activation of NF-?B signaling pathway and the JNK/Bax pathway play important roles in INH-induced oxidative stress injury) (Zhuang X et al., 2022)

Symptoms of hepatotoxicity:

Response of hepatotoxicity depends on the concentration of toxicant which may be either the parent compound, or toxic metabolite, differential expression of enzymes and concentration of gradients of co-factors in blood across the acinus (Kedderis, 1996). In hepatotoxicity, symptoms depend on the extent of liver damage. pruritus, severe abdominal pain, nausea or vomiting, weakness, severe fatigue, continuous bleeding, skin rashes, generalized itching, swelling of the feet and/or legs, abnormal and rapid weight gain in a short period of time, dark urine and light-colored stool (Bleibel et al., 2007; Chang & Schaino, 2007; Singh et al., 2011).

Treatment of hepatotoxicity:

If the drug causing hepatotoxicity is withdrawn earlier, normal liver function returns. At that time the patient may require supportive treatment. Treatment depends on the causative agent, the degree of liver dysfunction, and the age as well as general health of the patient. But if the toxicity reaches a certain limit, there may be no treatment of liver function in normal medicines. the individual. There is no effective treatment other than removal from the exposure to the causative agent or stopping the causative medication, and providing general supportive care. The best way is to discontinue the use of any medicinal drug that may put excess stress on the liver and use an alternate medication that helps to diminish or manage the side effects of hepatotoxicity. Alternatively, the dosage of current drug may be changed (Singh et al., 2011). Withdrawal of use of alcohol may also reduce the risk of hepatotoxicity. Prompt use of N-acetylcysteine after acetaminophen overdose (Polson, 2005) and i.v. administration of carnitine for valproate-induced hepatotoxicity (Bohan et al., 2001) has been reported for the treatment of acute liver injury. Diuretics or water-pills work to prevent or treat fluid accumulation in the body. Some common diuretics like furosemide and hydrochlorothiazide may also be prescribed to eliminate the drug from the body. Cholestyramine and ursodeoxycholic acid may be used for alleviation of pruritus. Nutrient supplements like taurine, methionine, Sadenosylmethionine, arginine, polyenylphosphatidylcholine, ?-lipoic acid, vitamin B, antioxidant vitamins [A,C,E] and methylsulfonylmethane that support phase I and phase II activities also serve as hepatoprotective agents(Singh et al., 2011).

A. Liver diseases –

1. Hepatitis: Inflammation of the liver, which can be caused by viruses (hepatitis A, B, C, etc.), alcohol abuse, autoimmune diseases, or certain medications and toxins.

2. Fatty Liver Disease: Characterized by the accumulation of fat in the liver cells. Non-alcoholic fatty liver disease (NAFLD) occurs in individuals who do not consume excessive alcohol, while alcoholic fatty liver disease is caused by alcohol abuse.

3. Cirrhosis: A late stage of scarring (fibrosis) of the liver caused by many forms of liver diseases and conditions, such as hepatitis and chronic alcoholism. Cirrhosis can lead to liver failure and complications such as portal hypertension, ascites, and hepatic encephalopathy.

4. Liver Cancer: Primary liver cancer, also known as hepatocellular carcinoma (HCC), originates in the liver cells. Secondary liver cancer, or metastatic liver cancer, occurs when cancer from other parts of the body spreads to the liver.

 5. Autoimmune Liver Diseases: Conditions in which the body's immune system attacks the liver, leading to inflammation and damage. Examples include autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis.

6. Liver Failure: A severe deterioration of liver function, which can occur suddenly (acute liver failure) or gradually over time (chronic liver failure). Causes include viral infections, drug-induced liver injury, alcoholic liver disease, and cirrhosis.

7. Hemochromatosis: A genetic disorder characterized by excessive iron absorption and accumulation in the liver and other organs, leading to liver damage and dysfunction

8. Wilson's Disease: An inherited disorder that causes copper to accumulate in various organs, including the liver, brain, and eyes, resulting in liver damage and neurological symptoms.

9. Biliary Tract Disorders: Conditions affecting the bile ducts, such as cholangitis (inflammation of the bile ducts), gallstones, and biliary atresia (congenital absence or obstruction of the bile ducts).

10. Liver Abscess: A pus-filled cavity within the liver, often caused by bacterial infection or parasites. (https://www.mayoclinic.org/diseases-conditions/liverproblems/symptoms-causes/syc-20374502)

Causes & Symptoms of liver diseases:

1. Hepatitis:

• Causes: Viral infections (hepatitis A, B, C, D, and E), alcohol abuse, autoimmune disorders, medications, toxins, and metabolic disorders.

• Symptoms: Fatigue, jaundice (yellowing of the skin and eyes), dark urine, abdominal pain, nausea, vomiting, fever, loss of appetite, and joint pain.

2. Fatty Liver Disease:

• Causes: Non-alcoholic fatty liver disease (NAFLD) is often associated with obesity, type 2 diabetes, insulin resistance, high cholesterol, and metabolic syndrome. Alcoholic fatty liver disease results from excessive alcohol consumption.

 • Symptoms: Often asymptomatic in the early stages. As the disease progresses, symptoms may include fatigue, weakness, abdominal discomfort, and hepatomegaly (enlarged liver).

3. Cirrhosis:

• Causes: Chronic hepatitis B or C infection, alcoholic liver disease, nonalcoholic fatty liver disease, autoimmune hepatitis, and other chronic liver diseases.

• Symptoms: Fatigue, weakness, jaundice, easy bruising and bleeding, ascites (abdominal fluid buildup), edema (swelling), hepatomegaly, splenomegaly (enlarged spleen), confusion, and portal hypertension-related complications (e.g., esophageal varices, hepatic encephalopathy).

4. Liver Cancer (Hepatocellular Carcinoma):

• Causes: Chronic viral hepatitis (especially hepatitis B and C), cirrhosis, alcohol abuse, non-alcoholic fatty liver disease, exposure to aflatoxins, and certain genetic conditions (e.g., hemochromatosis, Wilson's disease).

• Symptoms: Often asymptomatic in the early stages. As the cancer progresses, symptoms may include abdominal pain, weight loss, loss of appetite, jaundice, abdominal mass, and general malaise.

5. Autoimmune Liver Diseases:

• Causes: Exact causes are unknown but may involve genetic predisposition, environmental factors, and autoimmune responses.

• Symptoms: Autoimmune hepatitis may present with fatigue, jaundice, abdominal discomfort, joint pain, and other nonspecific symptoms. Primary biliary cholangitis may present with pruritus (itching), fatigue, jaundice, and hepatomegaly.

6. Liver Failure:

• Causes: Acute liver failure can result from acute viral hepatitis, drug-induced liver injury, acetaminophen overdose, autoimmune hepatitis, and other acute liver insults. Chronic liver failure can result fromprogressive liver diseases such as cirrhosis.

• Symptoms: Acute liver failure may present with jaundice, confusion, coma, coagulopathy, ascites, and hepatic encephalopathy. Chronic liver failure symptoms are similar to those of cirrhosis, including fatigue, jaundice, ascites, and hepatic encephalopathy.

Hepatotoxicity:

Hepatotoxicity refers to liver damage or injury caused by exposure to various toxic substances. These substances can include medications, chemicals, herbal supplements, environmental toxins, and even some natural compounds. Hepatotoxicity can manifest as acute liver injury, chronic liver diseases, or even fulminant hepatic failure, depending on the severity and duration of exposure. (Kaplowitz N et al., 2005). There are several mechanisms through which hepatotoxicity can occur: Direct Cellular Injury: Some substances can directly damage liver cells (hepatocytes), leading to cell death and inflammation. This can occur through chemical reactions or the generation of toxic metabolites (Jaeschke H et al., 2013).

Oxidative Stress: Certain substances can induce oxidative stress in the liver, leading to the production of reactive oxygen species (ROS) and free radicals. Prolonged oxidative stress can damage cellular components and impair liver function. (Jaeschke H et al., 2013).

1. Immune-Mediated Reactions: In some cases, the immune system may mistakenly target liver cells, leading to autoimmune hepatitis or other immune-mediated liver diseases. This can result in chronic inflammation and tissue damage. (Krawitt E. L. et al., 2006)

2. Metabolic Disturbances: Disruption of normal metabolic pathways in the liver can also contribute to hepatotoxicity. For example, some medications may interfere with the synthesis or metabolism of essential molecules, leading to liver dysfunction. (James et al., 2005) Hepatotoxicity can present with a range of symptoms, including jaundice (yellowing of the skin and eyes), abdominal pain, nausea, vomiting, fatigue, and changes in urine color. In severe cases, it can lead to liver failure, which is a life-threatening condition requiring urgent medical attention.

MATERIALS AND METHODS:

Materials:

1. Animals: Sprague Dawley rats weighing 180-220 g were purchased from National Institute of Biosciences, Pune. The animals were housed in polypropylene cages and maintained under environmental condition of temperature 25±1 ºC and relative humidity of 45-55 % under 12h light: 12 dark cycle. The animals had free access to food pellet (Nav Maharashtra Chakan oil mills Ltd., Pune) and water ad libitum. All the experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of constituted under the guidelines of Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA).

       
           
       
Kit Used:

       
           
       
4. Preparation of drug solution, storage, volume, and route of administration:

1. L-Glutamine:

Preparation of test drug solution:

Drug solution of L-Glutamine was prepared by using distilled water a vehicle.

Storage of drug solution:

L-Glutamine powder was stored in a desiccator. A fresh drug solution was prepared for each day’s work. The solution was kept in airtight amber-colored bottles and stored at room temperature until ready for use.

The volume of drug administration:

The volume of L-Glutamine solution to be administered was calculated based upon the body weight of animals.

Route of administration:

In the pylorus ligation- and acetic-induced peptic ulcer model, the solution of L- Glutamine was administered per oral (p.o.) route.

2. Silymarin:

Preparation of test drug solution:

Drug solution of Silymarin was prepared by using distilled water as a vehicle

Storage of drug solution:

Silymarin powder was stored in a desiccator. A fresh drug solution was prepared for each day’s work. The solution was kept in airtight amber-colored bottles and stored at room temperature until ready for use.

The volume of drug administration

The volume of Silymarin solution to be administered was calculated based upon the body weight of animals.

Route of administration:

The Silymarin solution was administered per oral (p.o.) route.

3. Isoniazid-rifampicin-induced hepatotoxicity in laboratory animals:

1. Experimental designs:

The animals were divided randomly into groups with six rats per group as follows:

• Group I: Normal group

The rats treated with vehicle (distilled water, 10 mg/kg, p.o.) for 21 days and received saline (250 mg/kg, p.o.) for 21 days.

• Group II: Vehicle control

The rats were administered a vehicle (distilled water, 10 mg/kg, p.o.) 2 h prior to oral administration of Isoniazid + Rifampicin (100 mg/kg, p.o.) for 21 days.

• Group III: Silymarin (25) treated group

The rats treated with Silymarin (25 mg/kg, p.o.) 2 h prior to oral administration of Isoniazid + Rifampicin (100 mg/kg, p.o.) for 21 days.

• Group IV: L-Glutamine (50) treated group

The rats treated with L-Glutamine at a dose of 50 mg/kg, p.o 2 h prior to oral administration of Isoniazid + Rifampicin (100 mg/kg, p.o.) for 21 days.

• Group V: L-Glutamine (100) treated group

The rats treated with L-Glutamine at a dose of 100 mg/kg, p.o 2 h prior to oral administration of Isoniazid + Rifampicin (100 mg/kg, p.o.) for 21 days.

• Group VI: L-Glutamine (200) treated group

The rats treated with L-Glutamine at a dose of 200 mg/kg, p.o 2 h prior to oral administration of Isoniazid + Rifampicin (100 mg/kg, p.o.) for 21 days.

2. Treatment of L-Glutamine and Silymarin:

The different doses of L-Glutamine (50, 100 and 200 mg/kg) and Silymarin (25 mg/kg) were calculated based on the animal’s body weight were administered per oral for 21 days.

3. Parameter for assessment of the effect of L-Glutamine Isoniazid-rifampicin- induced hepatotoxicity in rats:

• Body weight
• Liver Weight
• Liver weight /Body Weight
• AST
• ALT
• ALP
• Albumin
• Total bilirubin
• Direct bilirubin
• Anti-oxidant activity (Total protein, MDA, nitric oxide, GSH and SOD) in hepatic tissue
• Histopathology of hepatic tissue

4. Parameter for assessment of effect of L-Glutamine on Isoniazid-rifampicin- induced hepatotoxicity in rats:

1. In-vivo parameters:

Serum parameters:

The serum was separated by centrifugation using an Eppendorf cryocentrifuge (model no. 5810, Eppendorf, Hamburg, Germany), maintained at 4 ºC and run at a speed of 7000 rpm for 15 min.

The levels of serum aspartate transaminase (AST) and alanine transaminase (ALT), alkaline phosphatase (ALP), Albumin, Total bilirubin and Direct bilirubin were measured by a spectrophotometer (UV–visible spectrophotometer, Jasco V-530, Tokyo, Japan) using commercially available reagent kits according to procedure provided by manufacturer (Pathozyme Diagnostics, India).

2. Ex-vivo parameters:

Tissue Parameters:

Hepatic tissue homogenate preparation:

• All animals were sacrificed at the end of study i.e., 15th day and hepatic tissue was immediately isolated.
• Tissue homogenate was prepared with 0.1M Tris-HCl buffer (pH 7.4) and supernatant of homogenate was employed to estimate total protein, superoxide dismutase (SOD), reduced glutathione (GSH), lipid peroxidation (MDA content), and nitric oxide content

Determination of Lipid Peroxidation (MDA content):

• It was estimated using the method described by Slater and Sawyer (1971).
• 2.0 ml of the tissue homogenate (supernatant) was added to 2.0 ml of freshly prepared 10% w/v trichloroacetic acid (TCA) and the mixture was allowed to stand in an ice bath for 15 minutes.
• After 15 minutes, the precipitate was separated by centrifugation and 2.0 ml of clear supernatant solution was mixed with 2.0 ml of freshly prepared thiobarbituric acid (TBA).
• The resulting solution was heated in a boiling water bath for 10 minutes. It was then immediately cooled in an ice bath for 5 minutes. The colour developed was measured at 532nm against reagent blank.
• Different concentrations (0-23 nM) of standard malondialdehyde were taken and processed as above for standard graph.
• The values were expressed as nM of MDA/mg protein.

2. Determination of Superoxide Dismutase (SOD):

• Superoxide dismutase was estimated using the method developed by Misra and Fridovich (1972).
• 0.5 ml of tissue homogenate was diluted with 0.5 ml of distilled water, to which
• 0.25 ml of ice-cold ethanol and 0.15 ml of ice-cold chloroform was added.
• The mixture was mixed well using cyclo mixer for 5 minutes and centrifuged at 2500 rpm. To 0. 5ml of supernatant, 1.5 ml of carbonate buffer and 0.5 ml of EDTA solution were added.
• The reaction was initiated by the addition of 0.4 ml of epinephrine and the change in optical density/minute was measured at 480 nm against reagent blank. SOD activity was expressed as units/mg protein.
• Change in optical density per minute at 50 % inhibition of epinephrine to adrenochrome transition by the enzyme is taken as the enzyme unit. Calibration curve was prepared by using 10-125 units of SOD.

3. Determination of Reduced glutathione (GSH):

• Reduced glutathione was determined by the method described by Moron et al. (1979).
• Equal volumes of tissue homogenate (supernatant) and 20% TCA were mixed. The precipitated fraction was centrifuged and to 0.25 ml of supernatant, 2 ml of DTNB reagent was added. The final volume was made up to 3ml with phosphate buffer.
• The colour developed was read at 412 nm against reagent blank. Different concentrations (10-50 gm) of standard glutathione were taken and processed as above for standard graph.
• The amount of reduced glutathione was expressed as µg of GSH / mg protein.

4. Determination of tissue protein:

• Protein concentration was estimated according to the method of Lowry et al. (1951), using BSA (bovine serum albumin) as a standard.
• Briefly, dilute tissue fraction aliquots (0.1 ml) were taken in test tube. To this, 0.8 ml of 0.1 M sodium hydroxide and 5.0 ml Lowry C reagent was added and the solution was allowed to stand for 15 min.
• Then 0.5 ml of Folin phenol reagent was added and the contents were mixed by vortex mixer.
• Color developed was measure at 660 nm against reagent blank containing distilled water instead of sample.
• Different concentrations (40-200 µg) of BSA were taken and process as above for standard graph. The values were expressed as mg of protein/ gm of wet tissue (mg/gm).

5. Determination of Nitrite:

• Nitrite was estimated in the hepatic tissue homogenate using the Greiss reagent as per method described by Miranda et al., 2001.
• A measure of 500 µl of Greiss reagent (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% napthaylamine diamine dihydrochloric acid in water) was added to 100 µl of post-mitochondrial supernatant and absorbance was measured at 546 nm.
• Nitrite concentration was calculated using a standard curve for sodium nitrite. Nitrite levels were expressed as µg/ml.

2. Histopathological analysis:

On day 15th the all animals were sacrificed and hepatic tissue were collected. Samples of hepatic tissue were kept in the fixative solution (10% formalin) and it processed for 12 hr using isopropyl alcohol, xylene and paraffin embedded for light microscopic study (Nikon E200).

Paraffin embedded tissue section cut at 5?m thickness were prepared and staining was done by using hematoxylin and eosin as described by Yukari et al., 2004. Tissue sections were analyzed qualitatively under light microscope (100 ×) for inflammatory influx, congestion, oedema and necrosis etc.

3. Statistical Analysis:

Data analysis was performed using GraphPad Prism 5.0 software (GraphPad, San Diego, USA). Statistical comparisons were made between drug-treated groups and disease control animals (vehicle control). A value of P < 0>

RESULTS

1. Effect of L-Glutamine on INH+RIF-induced alteration in body weight:

       
           
       
       
           
       
Fig 7.1 Effect of L-Glutamine on INH+RIF-induced alteration in body weight

When compared to normal group, administration of INH+RIF did not cause any significant change in body weight in vehicle control group. Treatment of silymarin (25 mg/kg) and L- Glutamine (50, 100 and 200 mg/kg) for 21 days did not show any significant change in body weights of rats.

2. Effect of L-Glutamine on INH+RIF-induced alteration in absolute and relative liver weights:

       
           
       
Fig 7.2 Effect of L-Glutamine on INH+RIF-induced alteration in absolute and relative liver weights

Data were analyzed by One-Way ANOVA followed by Dunnett’s. ###P < 0>

When compared to normal group, administration of INH+RIF cased a significant increase (P < 0>

3. Effect of L-Glutamine on INH+RIF-induced alteration in AST and ALT levels:

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

On 22nd   day, the AST and ALT levels in vehicle control group showed a significant (P < 0>

4. Effect of L-Glutamine on INH+RIF-induced alteration in ALP level:

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

The significant increased (P < 0>

5. Effect of L-Glutamine on INH+RIF-induced alteration in total and direct bilirubin levels:

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

Administration of INH+RIF caused significant increased (P < 0>

6. Effect of L-Glutamine on INH+RIF-induced alteration in albumin level:

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

On 22nd   day, the albumin level in the vehicle control group was found to be significantly (P < 0>

7. Effect of L-Glutamine on INH+RIF-induced alteration in hepatic total protein level:

       
           
       
       
           
       
Fig. 7.7 Effect of L-Glutamine on INH+RIF-induced alteration in hepatic total protein level

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

There was a significant increase (P < 0>

8. Effect of L-Glutamine on INH+RIF-induced alteration in hepatic SOD and GSH level:

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

The hepatic SOD and GSH level in the vehicle control rats was significantly decreased (P < 0>

9. Effect of L-Glutamine on INH+RIF-induced alteration in hepatic MDA and NO level:

       
           
       
       
           
       
Fig. 7.9 Effect of L-Glutamine on INH+RIF-induced alteration in hepatic MDA and NO level

Data were analyzed by One-way ANOVA followed by Dunnett’s test. ###P < 0>

There was significant increase in hepatic MDA and NO levels in vehicle control rats as compared to normal rats. When compared to vehicle control rats, the MDA and NO level in hepatic tissue of silymarin (25 mg/kg) was significantly deceased (P < 0>

 showed significant (P < 0>

10. Effect of INH+RIF on histopathological alteration in hepatic tissue:

Presence of the normal central vein with portal triads without any evidence of necrosis (grade 0), congestion (grade 0), inflammatory infiltration (grade 0) and oedema (grade 0) reflected the normal architecture of liver tissue (Figure A).

Histopathological studies of the liver tissue from INH+RIF-treated rats showed presence of centrilobular necrosis (grade 4) with inflammatory infiltration (grade 4) around the centrilobular veins with periportal degeneration (Figure B).

Liver from silymarin treated rats showed mild necrosis (grade 1), congestion (grade 1), and scant number of inflammatory cells (grade 1) were seen around centrilobular veins. There was no evidence of oedema (Figure C).

Biochemical analysis showed that treatment with L-Glutamine (50 and 100 mg/kg) treated rats did not show any protection against INH+RIF-induced alteration in hepatic parameters. Thus, histopathological analysis of liver was not performed for L-Glutamine (50 and 100 mg/kg) treated group.

Histology of liver tissue from L-Glutamine (200 mg/kg) treated rats showed moderate damage in the liver architecture. Hepatocytes were unremarkable. However, it showed moderate necrosis, congestion and infiltration of inflammatory cells (grade 2) (Figure D).