Effect of Phenylhydrazine-induced hemolytic anemia on morphological changes of renal cortex in adult mice: Protective effect of crocin.

Document Type : Basic Sciences


1 1Department of Basic Science, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 2Department of Basic Science, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

3 3Razi Vaccine & Serum Research Institute, Agricultural Research, Educationa and Extension Organisation, Karaj, Iran


BACKGROUND: Hemolytic anemia and resultant hypoxia can cause a dysfunction in the body functions. Iron delivered from hemolysis of erythrocytes can also stimulate oxidative stress. OBJECTIVES: In this study, we aimed to investigate the protective effect of crocin on renal quality in animals that were exposed against hemolytic anemia induced by phenilhydrazine. METHODS: Forty-nine male and adult mice (20 – 25 g) were grouped within 7 groups. First group was control and treated with normal saline, and test groups were nominated as 2, 3, and 4 and were treated with phenyl hydrazine 2, 4, and 6 mg/100g/48 h (i.p.). Animals in groups 5, 6, and 7 were treated with crocin (200 mg/k/day, i.p.) in addition of 2, 4, and 6 mg/100g/48 h phenyl hydrazine. After 35 days, the blood samples were collected directly from the heart and after centrifugation (5 min and 3000 rpm), the serum samples were subjected to evaluate the Malondialehyde (MAD) level and total antioxidant capacity (TAC), and renal were fixed in 10% formal saline for Paraffin sections. After tissue processing sample staining with H&E method. RESULTS: This study showed that hemolytic anemia effects in proximal tubule of renal cortex, elso showed that the phenylhydrazine administration resulted in a dose-dependent declining of TAC and the crocin administration slightly was able to protect from TAC reduction. Moreover, PHN increased MDA level in serum in a dose-dependent manner, while co-administration of crocin with PHN significantly (P<0.05) reduced the rate of lipid peroxidation.while those groups which were treated with crocin showed a remarkable improvement in testis parameters. CONCLUSIONS: These results indicate a protective effect of Crocin against the anemia-related hypoxia on renal parameters. The protective capacity of Crocin might relate to its known antioxidant power.


Alessandro, M.V., Francesco, P., Silvia, L., Cristina, C., Roberto, C., Pierluigi, R.F., Sanchez, M., Migliaccio, G., Migliaccio, A.R. (2012) Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice. Blood. 95: 2559-2568.##
Barton, J.C., Conrad, M.E., Nuby, S., Harrison L. (1978) Effects of iron on the absorption and retention of lead. J Lab Clin Med. 92: 536-547.##
Berger, J. (2007) Phenylhydrazine haematotoxicity. J Appl Biomed. 5: 125-130.##
Brezis, M., Rosen, S., Spokes, K., Silva, P., Epstein F.H. (1986) Substrates induce hypoxic injury to edullary thick limbs of isolated rat kidneys. Am J Physiol. 251: 710-717.##
Bustos-Obregón, E., Esveile, C., Contreras, J., Maurer, I., Sarabia, L. (2006) Effects of Chronic Simulated Hypobaric Hypoxia on Mouse Spermatogenesis. Int J Morphol. 24: 481-488.##
Dobyan, D.C., Nagle, R.B., Bulger, R.E. (1977) Acute tubular necrosis in the rat kidney following unstained hypotension. Lab Invest. 37:  411-422.##
Endre, Z.H., Ratcliffe, P.J., Tange, D.J., Ferguson, P., Radda, G.K., Ledingham, J.G.G. (1989) Erythrocytes alter the pattern of renal hypoxic injury: predominance of proximal tubular injury with moderate hypoxia.  Clin Sci Lond. 76: 19-29.##
Ferrali, M., Ciccoli, L., Signorini, C., Comporti, M. (1990) Iron release and erythrocyte damage in allyl alcohol intoxication in mice. Biochem Pharmacol. 40: 1485-1490.##
Fine, L.G., Bandyopadhay, D., Norman, J.T. (2000) Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia. Kidney Int Suppl. 75: 22-26.##
Friedmann, B., Jost, J., Rating, T., Weller, E., Werle, E., Eckardt, K.U., Bärtsch, P., Mairbäurl, H. (1999) Effects of iron supplementation on total body hemoglobin during endurance training at moderate altitude. Int J Sports Med. 20: 78-85.##
Gorustovich, A.A., Steimetz, T., Giglio, M.J., Guglielmotti, M.B. (2006) A histomorphometric study of alveolar bone modeling and remodeling under experimental anaemia and polycythaemia in rats. Arch Oral Biol. 51: 246-251.##
Grigorovich, N.A. (1966) Pathogenesis of hemolytic anemia caused by phenylhydrazine. Biull Eksp Biol Med. 61: 126-129.##
Hosseinzadeh, H., Modaghegh, M.H., Saffari Z. (2009) Crocus sativus L. (Saffron) extract and its active constituents (crocin and safranal) on ischemia-reperfusion in rat skeletal muscle. Evid Based Complement Alternat Med. 6: 343-350.##
Hosseinzadeh, H., Sadeghnia, H.R., Ziaee, T., Danaee, A. (2005) Protective effect of aqueous saffron extract (Crocus sativus L.) and crocin, its active constituent, on  renal ischemia- reperfusion -induced oxidative damage in rats. J Pharm Pharmaceut Sci. 8: 387-393.##
Kaissling, B., Spiess, S., Rinne, B., Le Hir, M. (1993) Effects of Anemia On Morphology Of Rat Renal Cortex. Am J Physiol. 264: 608-617.##
Konoshima, T., Takasaki, M., Tokuda, H., Morimoto, S., Tanaka, H. (1998) Crocin and Crocetine derivatives inhibit Skin tumor promotion in mice. Phytother Res. 12: 400-404. ##
Lin, J.K., Wang, C.J. (1986) Protection of crocin dyes on the acute hepatic damage induced by aflatoxin B1 and dimethylnitrosamine in rats. Carcinogenesis. 7: 595-599.##
Lipschitz, D.A., Cook, J.D., Finch, C.A. (1974) A Clinical Evaluation of Serum Ferritin as an Index of Iron Stores. N Engl J Med. 290: 1213-1216.##
Mousavi, S.H., Tayarani, N.Z., Parsaee, H. (2010) Protective Effect of Saffron Extract and Crocin on Reactive Oxygen Species-Mediated High Glucose-Induced Toxicity in PC12 Cells. Cell Mol Neurobiol. 30: 185-191.##
Naghizadeh, B., Broshki, M.T., Mofidpour, H. (2006) Study of protective effect of Crocin in cisplatin nephrotoxicity in rat. Iran Biomed J. 9: 281-286.##
Ochiai, T., Ohno, S., Soeda, S., Tanaka, H., Shoyama, Y., Shimeno, H. (2006) Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo. Biochim Biophys Acta. 1770: 578-584.##
Puntarulo, S. (2005) Iron, oxidative stress and human health. Mol Aspects Med. 26: 299-312.##
Ravie, O., Lake, P.E. (1985) The phospholipids-bound fatty acids of fowl and turkey spermatozoa. Anim Report Sci. 9:189-192.##
Sharma, R.K., Agarwal, A. (1996) Role of reactive oxygen species in male infertility. Urol. 48: 835-850.##
Stevens, R.G., Jones, D.Y., Micozzi, M.S., Taylor, P.R. (1988) Body Iron Stores and the Risk of Cancer. N Engl J Med. 319: 1047-1052.##
Thushara, R.M., Hemshekhar, M., Santhosh, M.S., Jnaneshwari, S., Nayaka, S.C., Naveen, S., Kemparaju, K., Girish, K. S. (2013) Crocin, a dietary additive protects platelets from oxidative stress-induced apoptosis and inhibits platelet aggregation. Mol Cell Biochem. 373: 73-83.##
Vakili, A., Einali, M.R., Bandegi A.R. (2012) Protective Effect of Crocin against Cerebral Ischemia in a Dose-Dependent Manner in a Rat Model of Ischemic Stroke. J Stroke Cerebrovasc Dis. 23: 106-113.##
Vankatachalam, M.A., Bernard, D.B., Donohoe, J.F., Levinsky, N.G. (1978) Ischemic  damage  and  repair  in  the  rat  proximal tubule:  differences  among  the  Sl,  S2,  and  S3  segments.  Kidney Int.  14:  31-49. ##
Zheng, Y.Q., Liu, J.X., Wang, J.N. (2006) Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia. Brain Res. 1138: 86-94.##