Hepato Renal Toxicity In Rats Biology Essay

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The present investigation was designed to evaluate the aqueous extract of unripe fruits of carica papaya linn on hepato-renal toxicity in rats model. The extract was given intraperitonially at different doses (400 and 800 mg / kg, p.o) against paracetamol(APAP)(2g/kg, i.p., once only) induced hepatocellular damage in rats. We have determined an effective dose of the aqueous extract of unripe fruits of carica papaya through their therapeutic potential and further evaluated them individually against toxicant induced biochemical, molecular alterations and oxidative stress consequences in rats. The administration of toxicant altered blood biochemical variables and significantly increased the level of serum marker enzymes such as AST and ALT. Hepatic and renal lipid per oxidation was increased significantly, whereas substantial depletion was observed in reduced glutathione level after toxicant administration. A slight elevation was found in protein content, while a noteworthy fall was observed in the activities of adenosine triphosphatase and glucose-6-phosphatase after induction of toxicity. The treatment with the aqueous extract of unripe fruits of carica papaya at different doses (400 and 800mg/kg) significantly improved hepatic and renal antioxidant status and suppressed malondialdehyde formation. Thus, it may be concluded that the aqueous extract of unripe fruits of carica papaya can be used to reduce the hepatic and renal damage and may serve as an alternative medicine in hepatic etiology. It is found that the dose of 800 mg/kg of aqueous extract of Unripe Fruits of Carica Papaya is effective.

INTRODUCTION

The liver plays a significant role in the body as the organ responsible for metabolism of toxic substances that enter the body1. It is susceptible to persistent attack by offending agents. Liver contains an important detoxification system, which is supported by over 1000 identified unique vital chemical processes. Any injury to liver can result in many disorders ranging from transient elevation in liver enzymes to life threatening liver cirrhosis and hepatic failure. The common causative agents of liver injuries are toxic chemicals (e.g. APAP, alfatoxin etc), therapeutic drugs (e.g. antibiotics, antitubeular drugs etc), alcohol and microbial agents (e.g. hepatitis virus, leptospira, malarial parasite) 2.

The discovery and development of medicine from galenical to genomical focuses the potential role of Ayurveda. It is a traditional Indian medicinal system being practiced for thousands of years. Considerable research on pharmacognosy, chemistry, pharmacology and clinical therapeutics has been carried out on Ayurvedic medicinal plants The World Health Organization estimates that about three quarters of the world's population currently use herbs and other forms of the traditional medicines to treat their diseases, as they are cheap and easily available 3.

In recent year of usage of herbal drugs for the treatment of liver diseases has increased all over the world 4.The World Health Organization estimated that 80% of the populations of developing countries rely on traditional medicines, mostly plant drugs, for their primary health care needs 5.

Carica papaya (family Caricaceae) is cultivated in tropical and subtropical regions all around the world as fruit due to its palatable taste, nutritive value and easy digestion 6.The latex from unripe papaya fruits contains a mixture of cysteine endopeptidases such as papain, chymopapain A and B, papaya endopeptidase II, papaya endopeptidase IV, omega endopeptidase 7.carica papaya leaves extract also used to treat dengue fever 8. Carica papaya L. (Caricaceae) is an important medicinal plant in Indian traditional medicine 9, 10. The seeds of Carica papaya are reported to possess emmenagogue, abortifacient and antifertility properties 10 and anti inflammatory effects 11. Also Aqueous extract of Carica papaya leaves exhibits anti-tumor activity 12.

METHODS AND PREPARATION

COLLECTION OF Carica papaya:

Carica papaya unripe fruits were purchased from the local market (Gwalior, M.P.). The fruits were dried in shade and powdered (250 g) were soxhlet extracted with D.H2O (4 lit) for 18h. The extract was filtered and evaporated in vacuum to give powdered. An aqueous suspension of crude extract was administered to the animals orally according to their weight.

Selection and maintenance of experimental animals:

Male Sprague Dawley albino rats (150 ± 10 g b.w.) were used in this study. The rats were placed in plastic cages of 36x35x19 cm in dimensions. Animals were housed in the Jiwaji University under standard husbandry condition of temperature (24 ± 2°C), light (photocycle of 14 h light and 10 h dark) and relative humidity (60 % to 70 %). The animals were fed on standard pellet diet (Pranav agro industries, New Delhi) and water ad libitum. Animals were treated and cared for in accordance with the guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Govt. of India.

Chemicals and reagents:

All chemicals were of analytical grade and were procured from Sigma Ltd. (USA) and E-Merck (Germany). The other reagents used in the study were procured from Loba and Ranbaxy.

METHODOLOGY

Animals were divided into 5 groups of 6 each.

Group 1: served as normal control group and received distilled water alone.

Group 2: served as experimental control group and APAP(2 g/kg o.p. once only).

Group 3: aqueous suspension of carica papaya at dose levels of 400 mg/kg with APAP.

Group 4: aqueous suspension of carica papaya at dose levels of 800 mg/kg with APAP.

Group 5: The standard drug silymarin (50mg/kg body weight p.o.).

Animals in all the groups were sacrificed 24hrs after the last treatment and the serum, tissue and molecular parameters were investigated.

Statistical Analysis:

Numerical data were subjected to statistical analysis in order to test the significance of differences between means of various groups. One-way analysis of variance (ANOVA) was done to compare the mean levels of various parameters of the different experimental groups and the F values were computed at Pï‚£0.05 13. The test of choice was the student't'-test. The tabulated figures are presented as mean ± standard deviation (SD). The calculated P value has been included within the tables.

Results

Serum transaminases:

AST and ALT activities were increased in the APAP exposed rats. The extract restored the elevated level of transaminases at dose dependant manner however maximum protection was observed at 800 mg/kg. Significant variations between the different experimental groups were also indicated by a high F ratio.

Lipid peroxidation:

Lipid peroxidation was assessed in terms of the thiobarbutaric acid reactive substances (TBARS). A 2 fold increase of TBARS was observed in the liver and kidney with toxicant administration, Therapeutic agents showed marked reversal in the lipid peroxidation level at dose dependant manner however, highest dose 800 mg/kg produced a statistically significant diminution (P < 0.05).

Reduced glutathione:

Reduced glutathione is presumed to be an important endogenous defense against per oxidative destruction of cellular membranes. Significantly low levels of GSH were observed in the liver and kidney of APAP rats when compared to that of control. Therapy with aqueous extract restored the GSH level significantly at both doses (400 and 800 mg/ kg). A significant F ratio was also observed when compared with different experimental groups (P < 0.05).

Adenosine triphosphatase:

The membrane bound enzyme adenosine triphosphatase is known to be intimately associated with the intracellular ion regulation and transcellular ion transport. A significant depletion was found in the enzymatic activity of adenosine triphosphatase after acetaminophen toxicity in both the organs. These demonstrate that the activity of adenosine triphosphatase was significantly restored by the post-treatment of aqueous extract of papaya fruits. Highest dose of extract (800 mg/kg) was found to be more effective, which is biostatistically analyzed by Student's't' test and can be were compared with silymarin.

Protein:

Single exposure of acetaminophen caused significant (P < 0.05) increase in protein content of liver and kidney but therapeutic agents was not showed remarkable protective effect in both the organs.

SERUM

GROUP

AST(IU/L)

ALT(IU/L)

SERUM PROTEIN(mg/100g)

1.

68.2 ± 3.57

45.00 ± 3.27

38 ± 3.22

2.

203 ± 12.31#

300.0 ± 33.12#

57 ± 4.77#

3.

121.8 ± 9.20*

179.1 ± 21.42*

45 ± 3.59 *

4.

119.5 ± 9.05*

169 ± 19.73*

42.7 ± 2.76*

5.

75 ± 4.84*

60.00 ± 3.56*

35 ± 2.43*

LIVER

GROUP

LPO (n mole of MDA/mg protein)

GSH(µ mole/g)

ATPase(mg Po/100g/min)

TISSUE PROTEIN (mg/100mg)

G6PASE(µ mole Po/min/g liver)

1.

0.29 ± 0.01

7.50 ± 0.41

2000.0 ± 114.7

15.21 ± 0.88

5.38 ± 0.31

2.

1.9 ± 0.13#

4.10 ± 0.22#

910 ± 50.51#

21.0 ± 2.09#

3.23 ± 0.30#

3.

0.7 ± 0.07 *

6.93 ± 0.39 *

1821.4 ± 119.19*

17.01 ± 1.05 *

4.73 ± 0.38*

4.

0.53 ± 0.05*

7.05 ± 0.35*

1935.68 ± 106.69*

16.08 ± 0.92*

5.06 ± 0.47*

5.

0.3 ± 0.03*

7.1 ± 0.37*

2095 ± 119.92*

15.77 ± 0.79*

5.25 ± 0.48*

KIDNEY

GROUP

LPO (n mole of MDA/mg protein)

GSH (µ mole/g)

ATPase(mg Po/100g/min)

TISSUE PROTEIN (mg/100mg)

1.

0.3 ± 0.02

7.3 ± 0.37

2200.0 ± 111.53

14.30± 0.91

2.

1.7 ± 0.14#

4.5 ± 0.30#

878 ± 84.28#

19 ± 1.05#

3.

1.06 ± 0.06*

7.0 ± 0.38*

1900 ±105.10*

16.99 ± 1.04

4.

0.92 ± 0.09*

7.1 ± 0.42*

2010 ±128.33*

17.78 ± 0.95

5.

0.44 ± 0.02*

7.2 ± 0.40*

2100 ±109.52*

14.80 ± 0.84*

N=Normal, APAP=Acetaminophen, CP = Carica Papaya Sy=Silymarin, Data are mean ± S.E., N=6.

P Values # P < 0.05 vs normal control group, * P < 0.05 vs APAP administered group at @ = Significant at 5% level

DISCUSSION

Paracetamol , taken in overdose can cause hepatorenal toxicity14. PCM is activated and converted to toxic metabolite NAPQI (N-acetyl-p-benzoquinoneimine) 15, 16, which is responsible for its toxicity17 that causes oxidative stress and glutathione (GSH) depletion by cytochrome P450 enzymes14, 18. For testing the hepatoprotective agents, PCM-induced hepatotoxicity has already been used19, 20, 21 . Paracetamol is metabolized primarily in the liver and eliminated by conjugation with sulfate and glucuronide, and then excreted by the kidney. paracetamol hepatotoxicity has been caused due to the formation of toxic metabolites, when a part of paracetamol is activated by hepatic cytochrome P-450 to a very reactive significant alkylating metabolite NAPQI22. NAPQI is the irreversibly conjugated with the sulfhydryl groups of glutathione23. Paracetamol-induction increase biochemical parameters (SGOT, SGPT, alkaline phospatase and bilirubin) 24, 25

over doses of paracetamol have caused the elevation of serum levels of ALT and AST 26,27.liver damage by paracetamol is determination of enzyme levels such as ALT and AST is largely used 28 ,29. AST is an enzyme that is localized in hepatic cells takes part in transamination reaction of liver. The AST levels goes up in the circulation when the hepatic cells are damaged, therefore, it can be measured in serum. So high levels of this enzyme indicate liver damage 30 and better parameter to detect liver injury 31. High levels of AST indicate liver damage due to muscle injury, viral hepatitis and cardiac infarction. ALT catalyses the conversion of alanine to pyruvate and glutamate. Therapy with aqueous extract of unripe fruit of papaya at all the doses reversed the activity of transaminases and restored them towards normal value depicting a marked hepatoprotective effect.

Lipid peroxidation is a molecular mechanism of cell injury causes the generation of toxic products like malondialdehyde (MDA) and 4-hydroxynonenal. Toxicity is imputed to generate reactive oxygen species that causes peroxidation of membrane lipids 32 and induces a plethora of alteration in structure and function of cellular membranes. It has been asserted that the destructive process of liver injury is due to acetaminophen. The reactive oxygen species cause induced liver and kidney injury 33 LPO mainly arises from damaged kuffer cells. The increase in MDA levels in liver suggests increase in lipid peroxidation cause tissue damage and failure of anti oxidant defense mechanism to counter excessive free radicals34. In order to elucidate the protection mechanism of papaya against paracetamol induced rat liver and kidney were examined at lipid peroxide levels and antioxidative enzyme activities. In our study, elevations in the levels of end products of lipid peroxidation in liver and kidney of rat treated with paracetamol were observed which is also substantiated by 34,35,36,37. Treatment with extract significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection of papaya is due to its inhibition of lipid peroxidation probably being due to arrest of oxygen reactive species.

Glutathione is a tripeptide, non-enzymatic biological antioxidants demonstrate in the liver which eliminates the free radical species such as alkoxy radicals, hydrogen peroxide, superoxide radicals and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase (GPx) and GST 38. Under conditions of NAPQI formation following toxic paracetamol doses, GSH level may decrease the centrilobular cells, and the peroxide detoxification enzyme, GSH peroxidase, which functions very inefficiently when GSH depletes 39. Glutathione scavenges free radicals produced in the body and deliver protection against lipid peroxidation and damage of cell membrane 40. High level of lipid peroxidation make GSH depleted because high rate of scavenging and antioxidant system loses its control over free radicals. This causes the disruption of cell membranes and finally the cell death. low levels of GSH indicates high rate of lipid peroxidation 41. We examined significant depletion in glutathione level after acetaminophen toxicity which is also substantiated by 36, 42. Treatment with extract of papaya fruit shows recovery.

ATPase is a mitochondrial enzyme and is associated with the intracellular ion regulation and transport. Membrane protein that controls ion gradient across cell organelles and plasma membrane are susceptible to oxidative changes. Increased peroxidation causes membrane leakage, resulting in migration of Na+ along with water in the cell efflux of K+. Studies have showed that activities of Na+/K+ and Ca+2/Mg2+ ATPase were inhibited by the induction of oxidative stress 43. The decreased Na+/K+ ATPase in rat liver and kidney fraction on exposure of paracetamol may cause disturbance in ionic balance. This observation is well correlated with histological changes in hepatic parenchyma described as cloudy swelling, which indicates the degenerative changes and necrosis 44, 45, 46. Treatment with papaya extract protects the animal from the toxic effect of paracetamol.

CONCLUSION

Thus it may be concluded that therapy with aqueous extract of papaya fruit was more effective when compared to individual treatments.

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