Cardiovascular Toxicology

, Volume 11, Issue 3, pp 215–225 | Cite as

Cardioprotective Effects of Hesperetin against Doxorubicin-Induced Oxidative Stress and DNA Damage in Rat

  • P. P. Trivedi
  • S. Kushwaha
  • D. N. Tripathi
  • G. B. JenaEmail author


Doxorubicin is a widely used chemotherapeutic agent; however, its clinical uses are limited due to its cardiotoxicity associated with an induction of oxidative stress. This study was aimed to investigate the protective effect of hesperetin against doxorubicin-induced cardiotoxicity in rats. Doxorubicin was administered at the dosage of 4 mg/kg bw/week, ip for a period of 5 consecutive weeks. Hesperetin was administered at the dosages of 25, 50 and 100 mg/kg bw, po by gavage for 5 consecutive days in a week for 5 weeks. The animals were killed 1 week after the last injection of doxorubicin. Hesperetin at the doses of 50 and 100 mg/kg bw significantly reduced MDA and increased GSH levels in the doxorubicin-treated animals. Further, hesperetin significantly reduced doxorubicin-induced DNA damage as well as apoptosis at 25, 50, and 100 mg/kg bw as evident from the comet and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assays, respectively. Thus, hesperetin ameliorated doxorubicin-induced cardiotoxicity by reducing oxidative stress, abnormal cellular morphology and DNA damage in rat. Moreover, nuclear factor-kappa B, p38, and caspase-3 play a role in the hesperetin-mediated protection against doxorubicin-induced cardiotoxicity. This study indicates the protective effect of hesperetin against doxorubicin-induced cardiotoxicity.


Doxorubicin Hesperetin Heart Oxidative stress Apoptosis 





Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling


Nuclear factor-kappa B




Carboxy methyl cellulose


Hematoxylin and eosin


Ethidium bromide




Normal melting point agarose


Low melting point agarose


Ethylenediamine tetraacetic acid


Hank’s balanced salt solution




Tail length


Tail moment


Olive tail moment


% DNA in comet tail




Reduced glutathione



We wish to acknowledge the financial assistance received from National Institute of Pharmaceutical Education and Research (NIPER), Mohali, to undertake this study. The authors would also like to acknowledge Intas Pharmaceuticals Ltd., Ahmedabad, Gujarat, for benevolently granting the gift sample of doxorubicin.

Conflict of interest



  1. 1.
    Kim, J. C., Kim, K. H., & Chung, M. K. (1999). Testicular cytotoxicity of DA-125, a new anthracycline anticancer agent, in rats. Reproductive Toxicology, 13, 391–397.PubMedCrossRefGoogle Scholar
  2. 2.
    Alkreathy, H., Damanhouri, Z. A., Ahmed, N., Slevin, M., Ali, S. S., & Osman, A. M. (2010). Aged garlic extract protects against doxorubicin-induced cardiotoxicity in rats. Food and Chemical Toxicology, 48, 951–956.PubMedCrossRefGoogle Scholar
  3. 3.
    Ludke, A. R., Al-Shudiefat, A. A., Dhingra, S., Jassal, D. S., & Singal, P. K. (2009). A concise description of cardioprotective strategies in doxorubicin-induced cardiotoxicity. Canadian Journal of Physiology and Pharmacology, 87, 756–763.PubMedCrossRefGoogle Scholar
  4. 4.
    Fan, G. C., Zhou, X., Wang, X., Song, G., Qian, J., Nicolaou, P., et al. (2008). Heat shock protein 20 interacting with phosphorylated Akt reduces doxorubicin-triggered oxidative stress and cardiotoxicity. Circulation Research, 103, 1270–1279.PubMedCrossRefGoogle Scholar
  5. 5.
    Simoncikova, P., Ravingerova, T., & Barancik, M. (2008). The effect of chronic doxorubicin treatment on mitogen-activated protein kinases and heat stress proteins in rat hearts. Physiological Research, 57(Suppl 2), S97–S102.PubMedGoogle Scholar
  6. 6.
    Liu, Z., Song, X. D., Xin, Y., Wang, X. J., Yu, H., Bai, Y. Y., et al. (2009). Protective effect of chrysoeriol against doxorubicin-induced cardiotoxicity in vitro. Chinese Medical Journal, 122, 2652–2656.PubMedGoogle Scholar
  7. 7.
    Chandran, K., Aggarwal, D., Migrino, R. Q., Joseph, J., McAllister, D., Konorev, E. A., et al. (2009). Doxorubicin inactivates myocardial cytochrome c oxidase in rats: Cardioprotection by Mito-Q. Biophysical Journal, 96, 1388–1398.PubMedCrossRefGoogle Scholar
  8. 8.
    Li, L., Pan, Q., Han, W., Liu, Z., Li, L., & Hu, X. (2007). Schisandrin B prevents doxorubicin-induced cardiotoxicity via enhancing glutathione redox cycling. Clinical Cancer Research, 13, 6753–6760.PubMedCrossRefGoogle Scholar
  9. 9.
    Van Dalen, E. C., van der Pal, H. J., Caron, H. N., & Kremer, L. C. (2009). Different dosage schedules for reducing cardiotoxicity in cancer patients receiving anthracycline chemotherapy. Cochrane Database of Systematic Reviews, 4, CD005008.Google Scholar
  10. 10.
    Aiken, M. J., Suhag, V., Garcia, C. A., Acio, E., Moreau, S., Priebat, D. A., et al. (2009). Doxorubicin-induced cardiac toxicity and cardiac rest gated blood pool imaging. Clinical Nuclear Medicine, 34, 762–767.PubMedCrossRefGoogle Scholar
  11. 11.
    Gil-Izquierdo, A., Gil, M. I., Ferreres, F., & Tomas-Barberan, F. A. (2001). In vitro availability of flavonoids and other phenolics in orange juice. Journal of Agricultural and Food Chemistry, 49, 1035–1041.PubMedCrossRefGoogle Scholar
  12. 12.
    Ameer, B., Weintraub, R. A., Johnson, J. V., Yost, R. A., & Rouseff, R. L. (1996). Flavanone absorption after naringin, hesperidin, and citrus administration. Clinical Pharmacology and Therapeutics, 60, 34–40.PubMedCrossRefGoogle Scholar
  13. 13.
    Lee, N. K., Choi, S. H., Park, S. H., Park, E. K., & Kim, D. H. (2004). Antiallergic activity of hesperidin is activated by intestinal microflora. Pharmacology, 71, 174–180.PubMedCrossRefGoogle Scholar
  14. 14.
    Manach, C., Morand, C., Gil-Izquierdo, A., Bouteloup-Demange, C., & Remesy, C. (2003). Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. European Journal of Clinical Nutrition, 57, 235–242.PubMedCrossRefGoogle Scholar
  15. 15.
    Galati, E. M., Trovato, A., Kirjavainen, S., Forestieri, A. M., Rossitto, A., & Monforte, M. T. (1996). Biological effects of hesperidin, a Citrus flavonoid. (Note III): Antihypertensive and diuretic activity in rat. Farmaco, 51, 219–221.PubMedGoogle Scholar
  16. 16.
    Garg, A., Garg, S., Zaneveld, L. J., & Singla, A. K. (2001). Chemistry and pharmacology of the Citrus bioflavonoid hesperidin. Phytotherapy Research, 15, 655–669.PubMedCrossRefGoogle Scholar
  17. 17.
    Aranganathan, S., Selvam, J. P., & Nalini, N. (2008). Effect of hesperetin, a citrus flavonoid, on bacterial enzymes and carcinogen-induced aberrant crypt foci in colon cancer rats: A dose-dependent study. Journal of Pharmacy and Pharmacology, 60, 1385–1392.PubMedCrossRefGoogle Scholar
  18. 18.
    Choi, E. J., & Ahn, W. S. (2008). Neuroprotective effects of chronic hesperetin administration in mice. Archives of Pharmacal Research, 31, 1457–1462.PubMedCrossRefGoogle Scholar
  19. 19.
    Dimpfel, W. (2006). Different anticonvulsive effects of hesperidin and its aglycone hesperetin on electrical activity in the rat hippocampus in vitro. Journal of Pharmacy and Pharmacology, 58, 375–379.PubMedCrossRefGoogle Scholar
  20. 20.
    Guthrie, N., & Carroll, K. K. (1998). Inhibition of mammary cancer by citrus flavonoids. Advances in Experimental Medicine and Biology, 439, 227–236.PubMedCrossRefGoogle Scholar
  21. 21.
    Miyake, Y., Yamamoto, K., Tsujihara, N., & Osawa, T. (1998). Protective effects of lemon flavonoids on oxidative stress in diabetic Rats. Lipids, 33, 689–695.PubMedCrossRefGoogle Scholar
  22. 22.
    Saija, A., Scalese, M., Lanza, M., Marzullo, D., Bonina, F., & Castelli, F. (1995). Flavonoids as antioxidant agents: Importance of their interaction with biomembranes. Free Radical Biology and Medicine, 19, 481–486.PubMedCrossRefGoogle Scholar
  23. 23.
    Nagi, M. N., & Mansour, M. A. (2000). Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: A possible mechanism of protection. Pharmacological Research, 41, 283–289.PubMedCrossRefGoogle Scholar
  24. 24.
    Aranganathan, S., Selvam, J. P., & Nalini, N. (2009). Hesperetin exerts dose dependent chemopreventive effect against 1, 2-dimethyl hydrazine induced rat colon carcinogenesis. Investigational New Drugs, 27, 203–213.PubMedCrossRefGoogle Scholar
  25. 25.
    Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95, 351–358.PubMedCrossRefGoogle Scholar
  26. 26.
    Moron, M. S., Depierre, J. W., & Mannervik, B. (1979). Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta, 582, 67–78.PubMedGoogle Scholar
  27. 27.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.PubMedGoogle Scholar
  28. 28.
    Vikram, A., Tripathi, D. N., Ramarao, P., & Jena, G. B. (2008). Intervention of d-glucose ameliorates the toxicity of streptozotocin in accessory sex organs of rat. Toxicology and Applied Pharmacology, 226, 84–93.PubMedCrossRefGoogle Scholar
  29. 29.
    Kasamatsu, T., Kohda, K., & Kawazoe, Y. (1996). Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay. Mutation Research, 369, 1–6.PubMedCrossRefGoogle Scholar
  30. 30.
    Speit, G., & Hartmann, A. (1999). The comet assay (single-cell gel test). A sensitive genotoxicity test for the detection of DNA damage and repair. Methods in Molecular Biology, 113, 203–212.PubMedGoogle Scholar
  31. 31.
    Singh, N. P. (2000). A simple method for accurate estimation of apoptotic cells. Experimental Cell Research, 256, 328–337.PubMedCrossRefGoogle Scholar
  32. 32.
    Lebrecht, D., Geist, A., Ketelsen, U. P., Haberstroh, J., Setzer, B., & Walker, U. A. (2007). Dexrazoxane prevents doxorubicin-induced long-term cardiotoxicity and protects myocardial mitochondria from genetic and functional lesions in rats. British Journal of Psychiatry, 151, 771–778.Google Scholar
  33. 33.
    Simunek, T., Sterba, M., Popelova, O., Adamcova, M., Hrdina, R., & Gersl, V. (2009). Anthracycline-induced cardiotoxicity: Overview of studies examining the roles of oxidative stress and free cellular iron. Pharmacological Reports, 61, 154–171.PubMedGoogle Scholar
  34. 34.
    Su, Y. W., Liang, C., Jin, H. F., Tang, X. Y., Han, W., Chai, L. J., et al. (2009). Hydrogen sulfide regulates cardiac function and structure in adriamycin-induced cardiomyopathy. Circulation Journal, 73, 741–749.PubMedCrossRefGoogle Scholar
  35. 35.
    Iqbal, M., Dubey, K., Anwer, T., Ashish, A., & Pillai, K. K. (2008). Protective effects of telmisartan against acute doxorubicin-induced cardiotoxicity in rats. Pharmacological Reports, 60, 382–390.PubMedGoogle Scholar
  36. 36.
    L’Ecuyer, T., Sanjeev, S., Thomas, R., Novak, R., Das, L., Campbell, W., et al. (2006). DNA damage is an early event in doxorubicin-induced cardiac myocyte death. American Journal of Physiology, 291, H1273–H1280.PubMedGoogle Scholar
  37. 37.
    Chao, H. H., Liu, J. C., Hong, H. J., Lin, J. W., Chen, C. H., & Cheng, T. H. (2011). L-carnitine reduces doxorubicin-induced apoptosis through a prostacyclin-mediated pathway in neonatal rat cardiomyocytes. International Journal of Cardiology, 146, 145–152.PubMedCrossRefGoogle Scholar
  38. 38.
    Li, N., & Karin, M. (1999). Is NF-kappaB the sensor of oxidative stress? The Faseb Journal, 13, 1137–1143.PubMedGoogle Scholar
  39. 39.
    Wang, S., Kotamraju, S., Konorev, E., Kalivendi, S., Joseph, J., & Kalyanaraman, B. (2002). Activation of nuclear factor-kappaB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: The role of hydrogen peroxide. Biochemical Journal, 367, 729–740.PubMedCrossRefGoogle Scholar
  40. 40.
    Liu, J., Mao, W., Ding, B., & Liang, C. S. (2008). ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes. American Journal of Physiology, 295, H1956–H1965.PubMedGoogle Scholar
  41. 41.
    Lou, H., Danelisen, I., & Singal, P. K. (2005). Involvement of mitogen-activated protein kinases in adriamycin-induced cardiomyopathy. American Journal of Physiology, 288, H1925–H1930.PubMedGoogle Scholar
  42. 42.
    Kim, J. Y., Jung, K. J., Choi, J. S., & Chung, H. Y. (2006). Modulation of the age-related nuclear factor-kappaB (NF-kappaB) pathway by hesperetin. Aging Cell, 5, 401–411.PubMedCrossRefGoogle Scholar
  43. 43.
    Sardao, V. A., Oliveira, P. J., Holy, J., Oliveira, C. R., & Wallace, K. B. (2009). Morphological alterations induced by doxorubicin on H9c2 myoblasts: Nuclear, mitochondrial, and cytoskeletal targets. Cell Biology and Toxicology, 25, 227–243.PubMedCrossRefGoogle Scholar
  44. 44.
    Zhang, S. H., Wang, W. Q., & Wang, J. L. (2009). Protective effect of tetrahydroxystilbene glucoside on cardiotoxicity induced by doxorubicin in vitro and in vivo. Acta Pharmacologica Sinica, 30, 1479–1487.PubMedCrossRefGoogle Scholar
  45. 45.
    Hwang, S. L., & Yen, G. C. (2008). Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells. Journal of Agricultural and Food Chemistry, 56, 859–864.PubMedCrossRefGoogle Scholar
  46. 46.
    Choi, E. J., & Kim, G. H. (2011). Anti-/pro-apoptotic effects of hesperetin against 7, 12-dimetylbenz(a)anthracene-induced alteration in animals. Oncology Reports, 25, 545–550.PubMedCrossRefGoogle Scholar
  47. 47.
    Ali, M. M., Agha, F. G., El-Sammad, N. M., & Hassan, S. K. (2009). Modulation of anticancer drug-induced P-glycoprotein expression by naringin. Zeitschrift für Naturforschung Section C, 64, 109–116.Google Scholar
  48. 48.
    Hsiao, Y. C., Hsieh, Y. S., Kuo, W. H., Chiou, H. L., Yang, S. F., Chiang, W. L., et al. (2007). The tumor-growth inhibitory activity of flavanone and 2′-OH flavanone in vitro and in vivo through induction of cell cycle arrest and suppression of cyclins and CDKs. Journal of Biomedical Science, 14, 107–119.PubMedCrossRefGoogle Scholar
  49. 49.
    Choi, E. J. (2007). Hesperetin induced G1-phase cell cycle arrest in human breast cancer MCF-7 cells: Involvement of CDK4 and p21. Nutrition and Cancer, 59, 115–119.PubMedCrossRefGoogle Scholar
  50. 50.
    Masoodi, T. A., & Alhamdanz, A. H. (2010). Inhibitory effect of flavonoids on mutant H-Rasp protein. Bioinformation, 5, 11–15.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • P. P. Trivedi
    • 1
  • S. Kushwaha
    • 1
  • D. N. Tripathi
    • 1
  • G. B. Jena
    • 1
    Email author
  1. 1.Department of Pharmacology and ToxicologyNational Institute of Pharmaceutical Education and ResearchS.A.S. NagarIndia

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