Effect of abhrak bhasma and silicon dioxide on hepatic and renal glutathione status in rats: hepatoprotection testing against single dose carbon tetrachloride induced hepatotoxicity

Background: Glutathione (GSH) is an important intracellular antioxidant. Intrahepatic GSH levels are depleted in liver diseases. Objectives: In present study, effect of abhrak bhasma (an Ayurvedic drug) and silicon dioxide (SiO2) on hepatic and renal GSH status against CCl4 intoxicated male albino rats were investigated. Methods: Single dose of CCl4 (3.0ml/kg body wt, sc) was used to induce hepatotoxicity. Graded doses (10, 20, 30 and 40mg/ kg body wt) of abhrak bhasma and SiO2 were concurrently given with CCl4. Hepatic and renal GSH content was studied after 24 hrs. Results: Results showed that rats exposed to CCl4 exhibited decreased GSH in liver. It was counteracted and maintained to normal levels by the treatment of abhrak bhasma (minimum protective dose-10mg). SiO2 treatments did not affect GSH activity in liver significantly. Single dose of CCl4 had not influenced GSH content in kidney alone or with any of the doses of abhrak bhasma or SiO2. Conclusion: CCl4 single dose depletes GSH content significantly in liver but not in kidney. These results suggest that single dose treatment of abhrak bhasma (10mg onwards) protects GSH content and thus manages CCl4 induced free radical generation scavenging them.


Introduction
Liver and kidneys play a vital role in the metabolism, detoxification of xenobiotics by biotransformation and protect clearance (Edward & Celia, 1998;Majno & Joris;Nebbia, 2001). Carbon tetrachloride (CCl 4 ) is one of the most commonly used hepatotoxin in the experimental study of liver diseases. CCl 4 induced oxidative stress is associated with increased free radical generation and lipid peroxidation (LPO) (Recknagel et al, 1992;Burk et al, 1984;Kaplowitz et al, 1986;Teli et al., 2014); which leads to fatty degeneration in liver. Oxidative damage induced by free radicals can be prevented by the use of antioxidants. LPO and cellular antioxidant defense have importance in the oxidative stability. Ongoing oxidative processes and decreased intrahepatic glutathione level induced oxidative stress in liver is known (Ljubuncic et al., 2000;Ljubuncic & Bomzon, 2006). Endogenous glutathione, a thiol compound synthesized mainly in liver plays an antioxidant role by reducing reactive oxygen species (ROS) formed during cellular metabolism and protects cells (Parke & Piotrowski, 1996;Deneke, 2000). It has negative correlation with LPO in liver and kidney. Many researchers have focused on natural antioxidants for the treatment of oxidative stress induced complications. Abhrak bhasma is a commonly used Ayurvedic drug in various disorders including hepatitis (Sharma, 1977). In therapy it is useful in antiaging treatment, rejuvenation treatment etc. It has been reported for a strong immune system, rapidly increasing the production of T-Cell phagocytes. In our earlier work abhrak bhasma has protected single dose of CCl 4 induced increased malondialdehyde content in liver (Teli et al., 2014). Thus, to study the possible scavenging activity in vivo; it was planned to study glutathione (GSH) content in single dose of CCl 4 induced hepatotoxicity and associated changes in kidney. SiO 2 was used as silicon control for abhrak bhasma.

Animal
Male albino rats, Rattus norvegicus weighing about 130-140g each were used for experiment. They were bred and maintained in the departmental animal house (Reg. No. 233/CPCSEA) under standard conditions and were given standard pellet diet (prepared by Amrit feeds, Sangli, MS, India). Food and water were provided ad libitum.

Preparation of abhrak bhasma and silicon dioxide
Abhrak bhasma was prepared in the laboratory as described in Rasa Ratna Sammucchaya (Sharma, 1977). SiO 2 treatment was given as silicon control. To study dose dependent effects of abhrak bhasma and SiO 2 on GSH content of liver and kidney, different doses viz. 10, 20, 30 and 40 mg/kg body wt were administered orally with honey. Honey control rats that were used; showed data as normal rat. Therefore, honey control data is not presented.

Preparation of tissue homogenate
The livers and kidneys were perfused with chilled phosphate buffer saline (PBS). They were dissected out, minced and washed with PBS. The minces were then suspended in 0.25M sucrose containing 10mM Tris-HCl buffer (pH 7.0) homogenizing buffer (HB) and washed 3 times with HB. The minces were homogenized with Potter-Elvehjam homogenizer with Teflon piston at 1500 RPM with 8 up and down strokes. The liver and kidney homogenates were centrifuged in refrigerated centrifuge at 4 o C for 10 minutes at 3000 x g. The supernatants were collected and used for GSH estimation.

Estimation of GSH
Biochemical assay of GSH was performed as per method proposed by Grunert & Phillips (1951). Bioassay was conducted in different tubes, one blank and sample tube as per experimental schedule of rats. To blank tube was added 6.0 ml 0.25 M sucrose in homogenate buffer and to sample tubes added 6.0 ml of supernatants. One ml sodium nitroprusside and 1.0 ml sodium cyanide solution in sodium carbonate were added to all tubes. Optical densities were measured at 520 nm against blank.

Statistical analysis
All the experimental data was statistically evaluated and expressed as mean ± standard error for six rats in each group. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by students 't' test. Values P<0.05, P<0.01 and P<0.001 were considered to point out statistical significance. Glutathione is a reducing agent that is in high concentrations in mammalian tissues. It is an antioxidant, rich in cytosol. GSH reacts efficiently with oxidizing substances such as active oxygen species and lipid peroxidation. Several studies have revealed that status of GSH contents is markedly decreased in hepatic injury (Purucker et al., 1995). It is caused by reduced concentrations of liver GSH. In present work, we investigated abhrak bhasma and SiO 2 induced alterations in GSH status of liver and kidney of male albino rat, during hepatotoxicity induced by single dose of CCl 4 at 24 hrs. The results were shown in  GSH content in liver of normal rat exhibited 28.96±0.65 μg/ gm tissue, which was not altered by the administration of 10, 20 and 30 mg abhrak bhasma and remained in its normal range. When treatment of 40mg abhrak bhasma was given to the normal rat, GSH content was marginally increased (1.11 fold). Thus, abhrak bhasma alone with 10mg through 30mg doses are not affecting GSH contents while highest dose used marginally increased the GSH content. Similar doses of SiO 2 when given to the normal rat in same experimental condition, it showed significant elevation in GSH contents (P <0.001) which was noted at all SiO 2 doses studied. As contrast to abhrak bhasma, SiO 2 given alone showed highly significant increase in hepatic GSH, but not in renal GSH con- These studies require need of other parameter studies to resolve hepatoprotective mechanism of action/s of abhrak bhasma against CCl 4 induced toxicity.