ROLE OF PUMPKIN SEEDS ON HEPATORENAL DYSFUNCTION INDUCED BY AZATHIOPRINE IN ADULT MALE ALBINO RATS

ROLE OF PUMPKIN SEEDS ON HEPATORENAL DYSFUNCTION INDUCED BY AZATHIOPRINE IN ADULT MALE ALBINO RATS

By

Aziza K. Omer, Gehan A. Youssef, Asmaa F. Yousf

and AzzaH. Abd El-Wahab

Physiology Department, Faculty of Medicine (Girls), Al-Azhar University

ABSTRACT

Background: Pumpkin contains several phyto-constituents belonging to the categories of alkaloids, flavonoids, and palmitic, oleic and linoleic acids. Various important medicinal properties including anti-diabetic, antioxidant, anti-carcinogenic, anti-inflammatory and others have been well documented. Azathioprine (AZA), known as Imuran is animmunosuppressive drug. It is widely used in many diseases. A major drawback is the occurrence of side-effects, especially hepatorenal dysfunction.

Objective: Assessing the association between the daily oral dose of pumpkin supplementation as a strategy for amelioration of the side-effects of AZA on liver functions, renal functions and some inflammatory markers and tumour necrosis factor-α.

Material and Methods: Twenty four adult male albino rats, weighing 150–180 g, were used. They were divided into four equal groups: Group I (control), group II treated with (4 ml/kg. b. wt.) of pumpkin seeds oil per day for four weeks by oral gavage, group III were injected intraperitoneally with AZA (10mg/kg b. wt.) 3 times with an interval of 48 h in between, and group IV was treated by oral gavages with 4 ml/kg. b. wt of pumpkin seeds oil for ten consecutive days followed by AZA treatment i.p. at dose 10 mg/kg b. wt. 3 times with an interval of 48 h in between, after which pumpkin seeds oil administration alone was continued for another 2 weeks. Serum ALT, AST, ALP, total bilirubin, urea, creatinine, CRP and TNF-α were detected.

Results: Pumpkin seeds oil supplementation with AZA treatment significantly decreased the levels of serum liver transaminases (ALT and AST), ALP and total bilirubin in addition to serum urea, creatinine as well as CRP and TNF-α compared to AZA group.

Conclusions: Pumpkin seeds oil has the ability to ameliorate the biochemical pathways of the side-effects of AZA on liver and kidney.

Key words: Azathioprine, Pumpkin seed oil, anti-inflammatory activity, antioxidant.

INTRODUCTION

     The azathioprine is effective immuno-suppressant and anti-cancer agent and is prescribed increasingly to treat inflam-matory diseases ( Hawwa et al., 2008). AZA is also used in the therapy of organ transplant patients to prevent rejection following transplantation (Bendre et al., 2005). However, AZA use has been complicated by a high incidence of serious adverse drug reactions including hepatotoxicity and elevation of reactive oxygen species leading to mitochondrial injury and cell death due to necrosis (Shanmugarajan and Devaki, 2008).

      Pumpkin seeds oil is rich in many antioxidants and beneficial nutritional supplements such as essential fatty acid-omega 6, omega 9, vitamin A and vitamin E, squalene, carotenoids, tocopherols, phytoestrogenes, phytosterols, polyphe-nols, hydrocarbon, triterpenoids and selemium (Al-Okbi et al., 2014-b). Pumpkin seeds oil includes fatty acids:palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic  (C18:2) (Abouseif, 2014).

MATERIAL AND METHODS

     Azathioprine (Imuran), from Novartis Company, was dissolved in normal saline (0.9% NaCl) and injected i.p. with a volume of 0.1 ml, in a dose equivalent to human dose (10 mg/kg b. wt.) according to Paget's formula (Paget, 1964). Each dose was injected 3 times with an interval of 48 h (Bendre et al., 2005).

     Pumpkin seeds oil (PSO), from Emtenan Markets, was applied to the animal by oral gavages with a dose of 4 ml/ Kg b. wt. /day (Eraslan et al., 2013).

     The present study was carried out in animal house of faculty of Medicine for Girls, Al-Azhar university on twenty four adult male albino rats weighing 150–180 g. Rats were caged in 40x60x40 cm cages (three rats per cage). They were housed in at room temperature range of 25 ± 5 °C, regular light/ dark cycle, and fed chow and water.

Rats were divided into 4 equal groups:

● Group I: Control group.

● Group II: Rats was treated orally with 4 ml/kg b. wt. of PSO per day for four weeks.  Pumpkin seeds oil was applied to the animal by oral gavages.

● Group III: Rats was injected i.p. with AZA (10mg/kg b. wt.) 3 times with an interval of 48 h in between.

● Group IV: Rats was treated by oral gavages with 4 ml/kg. b. wt of PSO for ten consecutive days, then AZA treatment was started concomitantly with PSO i.p. at a dose of 10 mg/kg b. wt. 3 times with an interval of 48 h in between, after which PSO administration alone was continued for another 2 weeks.

     At the end of the experiment, animals were fasted for 12 hours before collection of blood samples. Blood was collected from retro–orbital sinus by heparinized capillary tubes under ether anesthesia (Simmons and Brick, 1970). The blood samples were collected in centrifuge tubes and allowed to clot for an hour at room temperature, and then centrifuged at 3000 rpm for 15 minutes. Sera were separated and stored at-20° C (using Co REVCO refrigerator).

The separated sera were analyzed for estimation of:

- Liver transaminases (AST and ALT- Young, 1995).

- Alkaline phosphatase (ALP- Young et al., 1972).

- Serum urea and creatinine (Schirmeister et al., 1964 and Jung et al. (1975).

- CRP (Hirschfield and Pepys, 2003). 

- TNF-α was estimated by ELISA (Englmann et al., 1990).

Statistical Analysis of results was done using statistic package for social science version 12 (SPSS, 12) for windows. Results were expressed as Mean ± Standard deviation (SD) and statistically analyzed using one-way analysis of variance (ANOVA) for a completely randomized design and Tukey multiple comparison tests. The level of significance was taken at P value of <0.05.

RESULTS

     Azathioprine administration resulted in deterioration of both liver and renal functions as well as enhancement of the inflammatory state as manifested by the significant elevation of in serum ALT, AST, ALP, bilirubin, urea, creatinine, CRP and TNF-α when compared to control group. Pumpkin administration alone did not cause any significant change in all the tested parameters when compared to control group. Pumpkin administration to AZA- treated rats resulted in improvement in liver and kidney functions and in the inflammatory state disturbed by AZA. There was a significant decrease in serum liver functions (serum ALT, AST, ALP and bilirubin) and renal functions (serum urea and creatinine) as well as the inflammatory markers (CRP and TNF-α) when compared to AZA group. Moreover, pumpkin administration to AZA treated rats could return serum creatinine, CRP and TNF-α to normal as there was a non significant difference if compared to pumpkin group (Table 1).

Table (1): The effect of Pumpkin seeds oil treatment on serum ALT, AST, ALP, bilirubin, urea, creatinine, CRP and TNF- α of azathioprine administered rats.

a: significant values versus control group.                                             b: significant values versus AZA group.

c: significant values versus pumpkin group.

DISCUSSION

      Treatment with AZA exhibited a significant increase of AST, ALT, ALP, bilirubin, urea and creatinine compared to control group.  These results were in agreement with those of El-Beshbishy et al. (2010) who attributed the occurrence of hepatic enzyme elevation to endogenous protein breakdown due to possible increase in tissue wasting. Shanmugarajan and Devaki (2008) noticed an increase in serum levels of AST, ALT, ALP, lactate dehydrogenase (LDH), and gamma glutamyl trans-peptidase (GGT) in serum 24 h after AZA treatment. They described that AZA administered rats displayed declined levels of endogenous antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione (GSH)], along with elevated levels of malondialdehyde (MDA)].

     The results of the present study also demonstrated that AZA induced deterioration in renal functions in the form of significant increase in serum urea and creatinine. These results were hand in hand with Shin et al. (2006) who described that AZA could induce acute interstitial nephritis.  Bir et al. (2006) also reported that AZA could be a cause of rapidly progressive renal failure. AZA treatment could induce a variety of DNA modifications such as chromatid damage, DNA strand breaks and DNA–protein cross links. Also, it is required as an active DNA mismatch repair system and, while the effects of purine starvation have been suggested, DNA damage seems to be the main mechanism for the cytotoxic effects of thiopurines (Karran and Attard, 2008).

     Pumpkin seed oil is suggested to be healthy addition towards human diet and have protential suitability for food and industrial applications because it is being rich in unsaturated fatty acids especially linoleic acid, oleic acid, tocopherols, and with very oxidative stability (Abouseif, 2014). PSO is an excellent source of protein, zinc, manganese and phosphorus. It contains a high amount of tryptophan, an essential amino acid involved in the synthesis of key brain chemical serotonin (Amin et al., 2013 and Galaly et al., 2014).

     The present investigation demonstrated that PSO reduced the toxic effect of AZA on liver male rats, and this might be due to high content of beta-carotene: Makni et al. (2008) discussed the anti- oxidant and hepatoprotective effect of active groups treated with pumpkin. Beta-carotene had been proved to be a powerful antioxidant and profound protective action against oxidative stress (Oboh, 2005).

     The pumpkin seeds oil has a powerful antioxidant and protective actions against tissue damage because of the role of antioxidant vitamins like 13- carotene in neutralization of free radicals and overtly aggressive oxygen species (Nkosi et al., 2006). Beta-carotene was among the most efficient substance known for quenching the excitation energy of single oxygen, and also for trapping certain organic free radicals. It has a direct inhibitory effect on liver microsomal enzymes (Ardabili et al., 2011). Moreover, the experimental study of Abouseif (2014) showed that the natural plant components found in pumpkin could improve the liver against alcohol-induced liver toxicity and oxidative stress.

     Renal functions improved according to the present study as serum urea and creatinine both significantly decreased with PSO and AZA- treated group in comparison to AZA group. These results were agreed with Makni et al. (2010) who found that pumpkin seeds ameliorated the antioxidant enzymes activities in nephropathic kidneys of rats. They also noticed that the histopathological changes were less prominent in pumpkin seeds treated rats than nephropathic kidneys. This improvement in the antioxidant enzyme system caused by pumpkin could be the cause in the manifested improvement of renal functions as observed in the present study.

      In the present study, it was noticed that TNF-α and CRP significantly decreased in combined pumpkin and AZA group compared to AZA group. These results were in agreement with Xanthopoulou et al. (2009) who showed that pumpkin seeds oil has anti-inflammatory activity which depends on their total phenolic content. The anti-inflammatory activity of pumpkin was due to their inhibitory activity against lipid peroxidation catalyzed by lipoxyganase. Sedigheh et al. (2011) who showed that pumpkin powder (1 and 2 g/kg for 4 weeks) supplementation in male diabetic rate revealed a significant reduction in CRP level. It is speculated that the anti-inflammatory effects of this plant is related to its anti-oxidant compounds such as flavonoids. It is also established that quercetin, from the class of flavonoids, provides protection against free radicals, chelate metal ion transporters, and also inhibit oxidases including lipoxygenase.

      Al-Okbi et al. (2014-a) demonstrated that PSO produced a significant reduction in plasma level of TNF-α. They suggested that the hypolipidemic, antioxidant and anti-inflammatory effects of pumpkin seed oil in their study are attributed to the presence of its bioactive constituents. El-Mosallamy et al. (2012) reported that PSO exhibited anti-inflammatory effects as pumpkin seeds are rich in linoleic and oleic unsaturated fatty acids. Linoleic and lionlenic acids compete with arachidonate for oxidative enzymes, thereby reducing the production of arachidonate cyclo-oxygenase products. It has been shown that a diet rich in C-linolenic acid has action similar to no steroidal anti-inflammatory agents in reducing the production of prostaglandin E2 and leukotriene B4 generated during inflammation.

     Singh et al. (2005) and Calder et al. (2009) attributed the anti-inflammatory effect of PSO to the presence of vitamin E, which is one of the important components of PSO, as it has a potent antioxidant with anti-inflammatory properties. Vitamin E has effects on inflammatory properties. Vitamin E has effects on inflammatory processes due to the antioxidant function of α-tocopherol. α-Tocopherol exerts anti-inflammatory effects through a number of different mechanisms, for example, by decreasing levels of CRP and pro-inflammatory cytokines and by inhibiting the activity of protein kinase C, an important cell-signaling molecule, and other enzymes such as syslooxygenase-2.

     Gammone et al. (2015) attributed the anti-inflammatory effect of PSO to the presence of beta-carotene which is one of the important components of pumpkin seed oil reducing the expression of IL-1a, VCAM-1 and E-selectin.

CONCLUSION

     Pumpkin seeds oil was effective in preventing the toxic effect of AZA as an immune suppressant drugs on liver and kidney of male rats. Much additional studies are needed before the authors might confidently make recommendations regarding dietary pumpkin in the prevention of toxic effect of immune suppressant drugs. Further clinical studies are also required to assess the safety and benefits of pumpkin seeds oil in human beings.

REFERENCES

1. Abouseif, H.S. (2014): Ameliorative effect of pumpkin oil (Cucurbitapepo L.) against alcohol-induced hepatotoxicity and oxidative stress in albino rats. Beni-Suef University Journal of Basic and Applied Sciences, 3 (3): 178–185.

2. Al-Okbi, S.Y.; Mohamed, D.A.; Hamed, T.E. and Esmail, R.S.H. (2014-a): Rice bran oil and pumpkin seed oil alleviate oxidative injury and fatty liver in rats fed high fructose diet. Pol. J. Food Nutr. Sci., 64 (2): 127–133.

3. Al-Okbi, S.Y.; Mohamed, D.A.; Kandil, E.; Ahmed, E.K. and Mohammed, S.E. (2014-b): Functional ingredients and cardiovascular protective effect of pumpkin seed oils. Grasas Y Aceites, 65 (1): 1-11.

4. Amin, K.A.; Galaly, S.R.; Hozayen, W.G. and Ramadan, S.M.(2013):Effects of Orlistat and Herbal Mixture Extract on RenalFunction and Oxidative Stress Biomarkers in a Rat Model of HighFat Diet. International Journal of Biochemistry Research & Review,4 (2): 173-192.

5. Amin, A. and Hamza, A.A. (2005): Hepatoprotective effects of hibiscus, rosmari-nus and salvia on azathioprine-induced toxicity in rat. Life Sci., 77(3): 266–278.

6. Ardabili, A.G.; Farhoosh, R. and Khodaparast, M.H.H. (2011): Chemical composition and physicochemical properties of pumpkin seeds (cucurbitapepo subsp. pepo var. styriaka) grown in Iran. Journal of Agriculture Science Technology, 13: 1053-1063.

7. Bendre, S.V.; Shaddock, J.G.; Patton, R.E.; Dobrovolsky, V.N.; Albertini, R.J. and Heflich, R.H. (2005): Lymphocyte Hprt mutant frequency and sperm toxicity in C57BL/6 mice treated chronically with azathioprine. Mutat. Res., 578 (1–2):1–14.

8. Bir, K.1.; Herzenberg, A.M. and Carette, S. (2006): Azathioprine induced acute interstitial nephritis as the cause of rapidly progressive renal failure in a patient with Wegener's granulomatosis.J Rheumatol., 33(1):185-187.

9. Calder, P.C.; Albers, R.; Antoine, J.M.; Blum, S.; Bourdet, S.R.; Ferns, G.A.; Folkerts, G.; Friedmann, P.S.; Frost, G.S.; Guarne,r F.; Lّvik, M.; Macfarlane, S.; Meyer, P.D.; M'Rabet, L.; Serafini, M.; Van, E. W.; Van, L. J.; Vas, D.W.; Vidry, S.; Winklhofer, R. B.M. and Zhao, J. (2009): Inflammatory disease processes and interactions with nutrition. National Center for Biotechnology Information, U.S. National Library of Medicine, 1: S1-45.

10. El-Beshbishy, H.A.; Mohammed, A.; Autifi, B.; Amr, D. and Mariee, A.D. (2010): Protective effect of green tea extract against azathioprine hepatotoxicity. Asian Journal of Traditional Medicines, 5 (1): 1-11

11. El-Mosallamy, A.E.M.K.; Sleem, A.A.; Abdel-Salam, O.M.E.; Shaffie, N. and Kenawy, S.A. (2012): Antihypertensive and cardioprotective effects of pumpkin seed oil. Journal of Medicinal Food, 15 (2): 180–189.

12. Englmann, H.; Liabak, N.B.; Sundan, A.; Waage, A. and Espevik, T. (1990): Develop-ment of immunoassays for detection of soluble tumor necrosis factor receptors. J Biol Chem., 265:1531-1536.

13. Eraslan, G.; Kanbur, M.;  Aslan, O. and Karabacak, M. (2013): The antioxidant effects of pumpkin seed oil on subacuteaflatoxin poisoning in mice. Environmental Toxicology, 28(12): 681–688.

14. Galaly, S.R.; Hozayen, W.G.; Amin, K.A. and  Ramadan, S.M. (2014): Effects of orlistat and herbal mixture extract on brain, testesfunctions and oxidative stress biomarkers in a rat model of high fatdiet. Beni-Suef University Journal of Basic and Applied Sciences, 3(2): 93–105.

15. Gammone, M.A.; Riccioni, G. and D'Orazio, N. (2015): Carotenoids: potential allies of cardiovascular health? National Center for Biotechnology Information, 6 (59):26762.

16. Hawwa, A.F.; Millership, J.S.; Collier, P.S.; Vandenbroeck, K.; McCarthy, A.; Dempsey, S.; Cairns, C.; Collins, J.; Rodgers, C. and McElnay, J.C.(2008): Pharmacogenomic studies of the anticancer and immuno-suppressive thiopurinesmercaptopurine and azathioprine. Br. J. Clin. Pharmacol., 66 (4): 517–528.

17. Hirschfield, G.M. and Pepys, M.B. (2003): C-reactive protein andcardiovascular disease: new insights from an old molecule. QJM, 96(11): 793-807.

18. Jung, D.; Biggs, H.; Erikson, J. and Ledyard, P.U. (1975): New colorimetric reaction for end-point, continuous-flow, and kinetic measurement of urea. Clin. Chem., 21:1136–1140.

19. Karran, P. and Attard, N. (2008):Thiopurines in current medical practice molecular mechanisms and contributions to therapy-related cancer. Nat. Rev. Cancer, 8: 24–36.

20. Makni, M.; Fetoui, H.; Gargouri, N.K.; Garoui, E.M.; Jaber, H.; Makni, J.; Boudawara, T. and Zeghal, N. (2008): Hypolipidemic and hepatoprotective effects of flax and pumpkin seed mixture rich in ω3and ω-6 fatty acids in hypercholesterolemic rats. Elsevier, Foodand Chemical Toxicology, 46 (12): 3714–3720.

21. Makni, M.; Sefi, M., Fetoui, H.; Garoui, M.; Gargouri, N.K; Boudawara, T. and Zeghal, N. (2010): Flax and Pumpkin seeds mixture ameliorates diabetic nephropathy in rats. Food Chem Toxicol., 48(8-9): 2407-12.

22. Nkosi, C.Z.; Opoku, A.R and  Terblanche, S.E. (2006): In vitro antioxidant activity of pumpkin seed (cucurbitapepo) protein isolate and its in vitro on alanine transaminase and aspartate transaminase in acetaminophen induced liver injury in low protein fed rats. Phytother. Res., 20(9): 780-3.

23. Oboh, G. (2005): Hepatoprotective property of ethanolic and aqueous extracts of fluted pumpkin (Telfairiaoccidentalis) leaves against garlic-induced oxidative stress. J Med Food, 8 (4): 560–563.

24. Paget M. (1964): Hormonal exploration in "cortico-adrenal disorders". J. Sci. Med lille., 82:631-48.

25. Sahasranaman, S.; Howard, D. and Roy, S. (2008): Clinical pharmacology and pharma-cogenetics of thiopurines. Eur. J. Clin. Pharmacol., 64 (8): 753-67.

26. Schirmeister, J.; Willmann, H. and Kiefer, H. (1964): Critical evaluation of plasma creatinine as a test of glomerulus filtrate. Verh Dtsch Ges Inn Med., 70: 678-681.    

27. Sedigheh, A.S.; Moshtaghian, J. and Mahbubeh, S. (2011): Hypoglycaemic and hypolipidemic effects of pumpkin (cucurbitapepo) on alloxan-induced diabetic rats. African Journal of Pharmacy and Pharmacology, 5 (23): 2620–2626.

28. Shanmugarajan, T.S. and Devaki, T. (2008): Ficushispida Linn. leaf extract possesses antioxidant potential and abrogates azathioprine induced prooxidant and antioxidant imbalance in rat liver. International Journal of Pharma-cology, 4 (5): 376-381.

29. Shin, J.I.; Lee, J.S. and Jeong, H.J. (2006): Azathioprine and tubulointerstitial nephritis in HSP.J Rheumatol.; 33 (12): 2551.

30. Simmons, M. I. and Brick J. O. (1970): The laboratory mouse: selection and management. Pbl. Englewood cliffs N J: Prentice – Hall. New Jersey, pp. 11-14.

31. Singh, U.; Devaraj, S. and Jialal, I. (2005): Vitamin E, oxidative stress, and inflammation. Annual Review of Nutrition, 25: 151-174.

32. Xanthopoulou, M.; Nomikos, T.; Fragopoulou, E. and Antonopoulou, S. (2009): Antioxidant and lipoxygenase inhibi-tory activities of pumpkin seed extracts. Food Res Int 42 (5-6): 641- 646. 

33. Young, D.S. (1995): Effect of drugs on clinical laboratory test, 4^thedition Pbl. AACC press Washington, page 50.

34. Young, D.S.; Thomas, D.W.; Friedman, R.B. and Pestaner, L.C. (1972): Clinical chemistry, 18(10):1041-1303.