CellBio  Vol.2 No.3 , September 2013
Role of Active Principles of Podophyllum hexandrum in Amelioration of Radiation Mediated Lung Injuries by Reactive Oxygen/Nitrogen Species Reduction
Abstract: Radiation induced reactive oxygen/nitrogen species (ROS/RNS) are reported to cause lung injuries such as pneumonitis and fibrosis which may be fatal at times. Current study is designed to analyse the radioprotective efficacy of P. hexandrum active principles (G-002M) on lungs of mice exposed to high dose of gamma irradiation (7 Gy). Cellular profiles and inflammatory cell infiltrates of irradiated bronchoalveolar lavage fluid (BALF) have shown correlations with lung pathology. Cell counts were determined in BALF of control, 7 Gy radiation exposed and radiation with G-002M pretreated mice. ROS/Nitric Oxide (NO) production was measured by 2,7 dichlorodihydrofluorescein diacetate (DCF-DA) and diaminofluorescein diacetate (DAF-2DA) through microscopy and flow cytometry respectively. Immunostaining of inducible nitric oxide synthase (iNOS) in BALF cells and lung sections was also observed microscopically. iNOS ex- pression was observed in lungs by western blotting. BALF was also processed to estimate total protein, LDH, and phospholipids content. Catalase, reduced Glutathione (GSH), Glutathione reductase (GR) and lipid peroxidation were estimated in lung tissues. Pre-administration of G-002M significantly decreased radiation mediated neutrophils count in BALF of irradiated mice. ROS generation, iNOS expression, total protein, LDH and phospholipids were found less affected in G-002M pretreated group in comparison to radiation alone group. Radiation exposure to mice was found apparently leading to parenchymal fibrosis, an architectural distortion of the lung tissue with edema, infiltration of inflammatory blood cells with increased immunolabeling of iNOS. G-002M pretreatment significantly countered radiation mediated increased lipid peroxidation and decreased GR, catalase and GSH in mice. Current study demonstrates possible role of P. hexandrum (G-002M) in minimizing lung damage induced by radiation mediated ROS/RNS generation.
Cite this paper: Saini, R. , Verma, S. , Singh, A. and Lata Gupta, M. (2013) Role of Active Principles of Podophyllum hexandrum in Amelioration of Radiation Mediated Lung Injuries by Reactive Oxygen/Nitrogen Species Reduction. CellBio, 2, 105-116. doi: 10.4236/cellbio.2013.23012.

[1]   J. C. Lee, P. A. Kinniry, E. Arguiri, M. Serota, S. Kanterakis, S. Chatterjee, et al., “Dietary Curcumin Increases Antioxidant Defenses in Lung, Ameliorates Radiation-Induced Pulmonary Fibrosis, and Improves Survival in Mice,” Radiation Research, Vol. 173, No. 5, 2010, pp. 590-601. doi:10.1667/RR1522.1

[2]   V. Mehta, “Radiation Pneumonitis and Pulmonary Fibrosis in Non-Small-Cell Lung Cancer: Pulmonary Function, Prediction, and Prevention,” International Journal of Radiation Oncology Biology Physics, Vol. 63, No. 1, 2005, pp. 5-24. doi:10.1016/j.ijrobp.2005.03.047

[3]   C. Tsuji, Shioya, Y. Hirota, N. Fukuyama, D. Kurita, T. Tanigaki, et al., “Increased Production of Nitrotyrosine in Lung Tissue of Rats with Radiation-induced Acute Lung Injury,” American Journal of Physiology Lung Cellular and Molecular Physiology, Vol. 278, No. 4, 2000, pp. 719-725.

[4]   P. G. Tsoutsou and M. I. Koukourakis, “Radiation Pneumonitis and Fibrosis: Mechanisms Underlying its Pathogenesis and Implications for Future Research,” International Journal of Radiation Oncology Biology Physics, Vol. 66, No. 5, 2006, pp. 1281-1293. doi:10.1016/j.ijrobp.2006.08.058

[5]   A. Giaid, S. M. Lehnert, B. Chehayeb, D. Chehayeb, I. Kaplan and G. Shenouda, “Inducible Nitric Oxide Synthase and Nitrotyrosine in Mice with Radiation-Induced Lung Damage,” American Journal of Clinical Oncology, Vol. 26, No. 4, 2003, pp. 67-72. doi:10.1097/01.COC.0000077940.05196.86

[6]   J. A. Royall, N. W. Kooy and J. S. Beckman, “Nitric Oxide-Related Oxidants in Acute Lung Injury,” New Horizons, Vol. 3, No. 1, 1995, pp. 113-122.

[7]   Y. Nozaki, Y. Hasegawa, A. Takeuchi, Z. H. Fan, K. I. Isobe, I. Nakashima, et al., “Nitric Oxide as an Inflammatory Mediator of Radiation Pneumonitis in Rats,” American Journal of Physiology Lung Cellular and Molecular Physiology, Vol. 272, No. 4, 1997, pp. 651-658.

[8]   R. Saini, S. Patel, R. Saluja, A. A Sahasrabuddhe, M. P. Singh, S. Habib, V. K. Bajpai, et al., “Nitric Oxide Synthase Localization in the Rat Neutrophils: Immunocytochemical, Molecular and Biochemical Studies,” Journal of Leukocyte Biology, Vol. 79, No. 3, 2006, pp. 519-528. doi:10.1189/jlb.0605320

[9]   K. J. Park, Y. T. Oh, W. J. Kil, W. Park, S. H. Kang and M. Chun, “Bronchoalveolar Lavage Findings of Radiation Induced Lung Damage in Rats,” Journal of Radiation Research, Vol. 50, No. 3, 2009, pp. 177-182. doi:10.1269/jrr.08089

[10]   U. Costabel and J. Guzman, “Bronchoalveolar Lavage in Intestinal Lung Disease,” Current Opinion in Pulmonary Medicine. Vol. 7, No. 5, 2001, pp. 255-261.

[11]   J. Bousquet, P. Chanez, J. Y. Lacoste, G. Barnéon, N. Ghavanian, I. Enander, et al., “Eosinophilic Inflammation in Asthma,” New England Journal of Medicine, Vol. 323, No. 15, 1990, pp. 1033-1039. doi:10.1056/NEJM199010113231505

[12]   J. Y. Lacoste, J. Bousquet, P. Chanez, T. Van Vyve, J. Simony-Lafontaine, N. Lequeu, et al., “Eosinophilic and Neutrophilic Inflammation in Asthma, Chronic Bronchitis, and Chronic Obstructive Pulmonary Disease,” Journal of Allergy and Clinical Immunology, Vol. 92, No. 4, 1993, pp. 537-548. doi:10.1016/0091-6749(93)90078-T

[13]   D. A. Campbell, L. W. Poulter and R. M. du Bois, “Immunocompetent Cells in Bronchoalveolar Lavage Reflect the Cell Populations Intransbronchial Biopsies in Pulmonary Sarcoidosis,” American Review of Respiratory Disease, Vol. 132, No. 6, 1985, pp. 1300-1306.

[14]   P. J. Haslam, C. W. G. Turton, B. Heard, A. Lukoszek, J. V. Collins, A. J. Salsbury, et al., “Bronchoalveolar Lavage in Pulmonary Fibrosis: Comparison of Cells Obtained with Lung Biopsy and Clinical Features,” Thorax, Vol. 35, No. 1, 1980, pp. 9-18. doi:10.1136/thx.35.1.9

[15]   G. W. Hunninghake, O. Kawanami, V. J. Ferrans, R. C. Jr Young, W. C. Roberts and R. G. Crystal, “Characterization of Inflammatory and Immune Effector Cells in the Lung Parenchyma of Patients with Interstitial Lung Disease,” American Review of Respiratory Disease, Vol. 123, No. 4, 1981, pp. 407-412.

[16]   I. L. Paradis, J. H. Dauber and B. S. Rabin, “Lymphocyte Phenotypes in bronchoalveolar Lavage and Lung Tissue in Sarcoidosis and Idiopathic Pulmonary Fibrosis,” American Review of Respiratory Disease, Vol. 133, No. 5, 1986, pp. 855-860.

[17]   G. Semenzato, M. Chilosi, E. Ossi, L. Trentin, G. Pizzolo, A. Cipriani, et al., “Bronchoalveolar Lavage and Lung histology: Comparative Analysis of Inflammatory and Immunocompetent Cells in Patients with Sarcoidosis and Hypersensitivity Pneumonitis,” American Review of Respiratory Disease, Vol. 132, No. 2, 1985, pp. 400-404.

[18]   M. B. Gotway, “Interstitial lung Diseases: Imaging Evaluation,” Applied radiology, Vol. 29, No. 9, 2000, pp. 31-46. doi:10.1016/S0160-9963(00)80215-0

[19]   M. D. Rossman, J. A. Kern, J. A. Elias, M. R. Cullen, P. E. Epstein and O. P. Preuss, “Proliferative Response of Broncho Alveolar Lymphocytes to Beryllium,” Annals of Internal Medicine, Vol. 108, No. 5, 1988, pp. 687-693. doi:10.7326/0003-4819-108-5-687

[20]   M. Lata, J. Prasad, S. Singh, R. Kumar, L. Singh, P. Chaudhary, et al., “Whole Body Protection against Lethal Ionizing Radiation in Mice by REC-2001: A Semi-Purified Fraction of Podophyllum hexandrum,” Phytomedicine, Vol. 16, No. 1, 2009, pp. 47-55. doi:10.1016/j.phymed.2007.04.010

[21]   M. L. Gupta, V. Gupta, S. K. Shukla, S. Verma, S. Sankhwar, A. Dutta, et al., “Inhibition in Radiation Mediated Cellular Toxicity by Minimizing Free Radical Flux: One of the Possible Mechanisms of Biological Protection against Lethal Ionizing Radiation by a Subfraction of Podophyllum hexandrum,” Cellular and Molecular Biology, Vol. 56, Suppl. OL1341-9, 2010. doi:10.1170/153

[22]   S. Sankhwar, M. L. Gupta, V. Gupta, S. Verma, K. A. Suri, M. Devi, et al., “Podophyllum hexandrum Mediated Survival Protection and Restoration of Other Cellular Injuries in Lethally Irradiated Mice,” Evidence Based Com- plementary and Alternative Medicine, 2011, Article ID: 175140. doi:10.1093/ecam/nep061

[23]   A. Dutta, S. Verma, S. Sankhwar, S.J. Flora and M. L. Gupta, “Bioavailability, Antioxidant and Non Toxic Properties of a Radioprotective Formulation Prepared from Isolated Compounds of Podophyllum hexandrum: A Study in Mouse Model,” Cellular and Molecular Biology, Vol. 58, 2012.

[24]   S. K. Shukla and M. L. Gupta, “Approach Towards Development of a Radioprotector Using Herbal Source Against Lethal Irradiation,” International Research Journal of Plant Science, Vol. 1, No. 1, 2010, pp. 118-125.

[25]   A. Kawana, S. Shioya, H. Katoh, C. Tsuji, M. Tsuda and Y. Ohta, “Expression of Intercellular Adhesion Molecule-1 and Lymphocyte Function-Associated Antigen-1 on Alveolar Macrophages in the Acute Stage of Radiation-Induced Lung Injury in Rats,” Radiation Research, Vol. 147, No. 4, 1997, pp. 431-436. doi:10.2307/3579499

[26]   S. Shioya, C. Tsuji, D. Kurita, H. Katoh, M. Tsuda, M. Haida, et al., “Early Damage to Lung Tissue after Irradiation Detected by the Magnetic Resonance T2 Relaxation Time,” Radiation Research, Vol. 148, No. 4, 1997, pp. 359-364. doi:10.2307/3579521

[27]   Q. Zhang, Y. Kusaka, Q. Zhang, L. He, Z. Zhang and K. Sato, “Dynamic Changes of Constituents in Bronchoalveolar Lavage Fluid in Experimental Silicotic Rats,” In- dustrial Health, Vol. 34, No. 4, 1996, pp. 379-388. doi:10.2486/indhealth.34.379

[28]   M. Natiello, G. Kelley, J. Lamca, D. Zelmanovic, R. W. Chapman and J. E. Phillips, “Manual and Automated Leukocyte Differentiation in Bronchoalveolar Lavage Fluids from Rodent Models of Pulmonary Inflammation,” Comparative Clinical Pathology, Vol. 18, No. 2, 2009, pp. 101-111. doi:10.1007/s00580-008-0772-9

[29]   A.B. Thompson, H. Teschler, Y. M. Wang, N. Konietzko and U. Costabel, “Preparation of Bronchoalveolar Lavage Fluid with Microscope Slide Smears”. European Respiratory Journal, Vol. 9, No. 3, 1996, pp. 603-608. doi:10.1183/09031936.96.09030603

[30]   P. Sharma, S. A. V. Raghavan, R. Saini, and M. Dikshit, “Functional Role of Ascorbic Acid in the Regulation of Free Radical Generation and Phagocytosis by Polymorphonuclear Leukocytes: A No-Mediated Effect,” Journal of Leukocyte Biology, Vol. 75, No. 6, 2004, pp. 1070-1078. doi:10.1189/jlb.0903415

[31]   B. Vojtesek, J. Bartek, C.A. Midgley and D. P. Lane, “An Immunochemical Analysis of the Human Nuclear Phosphoprotein P53. New Monoclonal Antibodies and Epitope Mapping Using Recombinant P53,” Journal of Immunological Methods, Vol. 151, No. 1-2, 1992, pp. 237-244. doi:10.1016/0022-1759(92)90122-A

[32]   P. Seth, R. Kumari, M. Dikshit and R. C. Srimal, “Modulation of Rat Peripheral Polymorphonuclear Leukocyte Response by Nitric Oxide and Arginine,” Blood, Vol. 84, No. 8, 1994, pp. 2741-2748.

[33]   M. Bradford, “A Rapid And Sensitive Method for the Quantitation of Microgram Quatities of Protein Utilizing the Principle of Protein-Dye Binding,” Analytical Biochemistry, Vol. 72, No. 1-2, 1976, pp. 248-254. doi:10.1016/0003-2697(76)90527-3

[34]   Y. Yoshida, E. Furuya and K. Tagawa, “A Direct Colorimetric Method for the Determination of Phospholipids with Dithiocyanatoiron Reagent,” Journal of Biochemistry, Vol. 88, No. 2, 1980, pp. 463-468.

[35]   J. A. Buege and S. D Aust, “Microsomal Lipid Peroxidation,” Methods Enzymology, Vol. 52, 1978, pp. 302-310. doi:10.1016/S0076-6879(78)52032-6

[36]   E. Beutler, “Reduced Glutathione-GSH, U: Beutler E. (Ur.) Red Cell Metabolism: A Manual of Biochemical Methods,” Grane and Straton, New York, 1975.

[37]   I. Carlberg and B. Mannervik, “Glutathione Reductase,” Methods Enzymology, Vol. 113, 1985, pp. 484-490. doi:10.1016/S0076-6879(85)13062-4

[38]   A. K. Sinha, “Colorimetric Assay of Catalase,” Analytical Biochemistry, Vol. 47, No. 2, 1972, pp. 389-394.

[39]   Y. Z. Fang, S. Yang and G. Wu, “Free Radicals, Anti- oxidants, and Nutrition,” Nutrition Journal, Vol. 18, No. 10, 2002, pp. 872-879. doi:10.1016/S0899-9007(02)00916-4

[40]   M. Kirsch and H. de Groot, “Formation of Peroxynitrite from Reaction of Nitroxyl Anion with Molecular Oxygen,” The Journal of Biological Chemistry, Vol. 277, No. 16, 2002, pp. 13379-13388. doi:10.1074/jbc.M108079200

[41]   S. J. Hosseinimehr, “Trends in the Development of Ra- dioprotective Agents,” Drug Discovery Today, Vol. 12, No. 19-20, 2007, pp. 794-805. doi:10.1016/j.drudis.2007.07.017

[42]   S. Verma, M. L. Gupta, A. Dutta, S. Sankhwar, S. K. Shukla and S. J. Flora, “Modulation of Ionizing Radiation Induced Oxidative Imbalance by Semi-Fractionated Extract of Piper Betle: An in Vitro and in Vivo Assessment,” Oxidative Medicine and Cellular Longevity, Vol. 3, No. 1, 2010, pp. 44-52. doi:10.4161/oxim.3.1.10349

[43]   D. P. Uma Devi, A. Ganasoundari, B. S. Rao and K. K. Srinivasan, “In Vivo Radioprotection by Ocimum Flavonoids: Survival of Mice,” Radiation Research, Vol. 151, No. 1, 1999, pp. 74-78. doi:10.2307/3579750

[44]   M. L. Gupta, S. Tyagi, S. J. Flora, P. K. Agrawala, P. Choudhary, S. C. Puri, et al., “Protective Efficacy of Semi Purified Fraction of High Altitude Podophyllum hexandrum Rhizomes in Lethally Irradiated Swiss Albino Mice,” Cellular and Molecular Biology, Vol. 53, No. 5, 2007, pp. 29-41.

[45]   H. Y. Reynolds, “Bronchoalveolar Lavage,” American Review of Respiratory Disease, Vol. 135, No. 1, 1987, pp. 250-263.

[46]   R. K. Sagar, R. Chawla, R. Arora, S. Singh, B. Krishna, R. K. Sharma, et al., “Protection of Hematopoietic System by Podophyllum hexandrum against Gamma Radiation-Induced Damage,” Planta Medica, Vol. 72, No. 2, 2006, pp. 114-120. doi:10.1055/s-2005-873148

[47]   Y. Erbil, C. Dibekoglu, U. Turkoglu, E. Ademoglu, E. Berber, A. Kizir, et al., “Nitric Oxide and Radiation Enteritis,” European Journal of Surgery, Vol. 164, No. 11 1998, pp. 863-868. doi:10.1080/110241598750005291

[48]   E. Giannopoulou, P. Katsoris, D. Kardamakis and E. Papadimitriou, “Amifostine Inhibits Angiogenesis in Vivo,” Journal of Pharmacology and Experimental Therapy, Vol. 304, No. 2, 2003, pp. 729-737. doi:10.1124/jpet.102.042838

[49]   P. V. Limaye, N. Raghuram and S. Sivakami, “Oxidative Stress and Gene Expression of Antioxidant Enzymes in the Renal Cortex of Streptozotocin Induced Diabetic Rats,” Molecular Cell Biochemistry, Vol. 243, No. 1-2, 2003, pp. 147-152.

[50]   I. Zelen, P. Djurdjevic, S. Popovic, M. Stojanovic, V. Jakovljevic, S. Radivojevic, et al., “Antioxidant Enzymes Activities and Plasma Levels of Oxidative Stress Markers in B-Chronic Lymphocytic Leukemia Patients,” Journal of Balkan Union of Oncology, Vol. 15, No. 2, 2010, pp. 330-336.

[51]   C. H. Foyer, H. Lopez-Delgado, J. F. Dat and I. M. Scott, “Hydrogen Peroxide- and Glutathione-Associated Mechanisms of Acclamatory Stress Tolerance and Signalling,” Physiologia Plantarum, Vol. 100, No. 2, 1997, pp. 241- 254. doi:10.1111/j.1399-3054.1997.tb04780.x