logy 557 (2007) 58–65www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Anticlastogenic activity of morin against whole bodygamma irradiation in Swiss albino mice

Vipan Kumar Parihar a, Koiram Rajanna Prabhakar a, Veeresh Prabhakar Veerapur a,Kavirayani Indira Priyadarsini b, Mazhuvancherry Kesavan Unnikrishnan a,⁎,

Chamallamudi Mallikajuna Rao a

a Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal-576104, Karnataka, Indiab Radiation and Photochemistry Division, Bhabha Atomic Research Center, Mumbai-400 085, India

Received 19 June 2006; received in revised form 19 September 2006; accepted 26 September 2006Available online 19 October 2006

Abstract

Anticlastogenic activity of morin was explored against whole body gamma radiation, at a dose rate of 1.66 Gy/min in Swiss albino micepretreated intraperitoneal or orally. Pretreatment with morin 10, 25, 50, 75, 100, 125, and 150 mg/kg, i.p. delayed and reduced percentagemortality and increased mean survival times in mice irradiated with 10 Gy gamma radiation. Intraperitoneal route was found superior to oral route.An i.p. dose of 100 mg/kg was found to be the most effective dose in preventing radiation-induced weight loss, increasing the mean survival timesand reducing percentage mortality. Morin (100 mg/kg) pretreatment effectively maintained spleen index (spleen weight /body weight×100) andstimulated endogenous spleen colony forming units. Pretreatment with morin (100 mg/kg) significantly reduced dead, inflammatory, and mitoticcells in irradiated mice jejunum along with a significant increase in goblet cells and rapidly multiplying crypt cells. Morin (100 mg/kg) alsomaintained the villus height close to normal, prevented mucosal erosion and basement membrane damage in irradiated jejunum. Nuclearenlargement in epithelial cells of jejunum was lower in morin treated mice compared to radiation control. Morin (100 mg/kg) also significantlyelevated the endogenous antioxidant enzymes viz. glutathione S transferase (GST), superoxide dismutase (SOD) and reduced glutathione (GSH),in normal mice at 2, 4 and 8 h post treatment. Drastic decrease in endogenous enzymes (GSH, GST, catalase and SOD) and total thiols wasobserved in irradiated mice at 2, 4 and 8 h post irradiation, while pretreatment with morin (100 mg/kg) prevented this decrease. Morin (100 mg/kg)also elevated radiation LD50 from 9.2 to 10.1 Gy, indicating a dose modifying factor (DMF) of 1.11.© 2006 Elsevier B.V. All rights reserved.

Keywords: Morin; Radioprotection; Jejunum; Spleen index; Glutathione; GST; SOD catalase; Total thiol; Dose modifying factor

1. Introduction

Clastogen is any agent that causes breaks in chromosomeand/or alters DNA sequences. Clastogens can damage thegenetic material either by direct attack or by formation of freeradicals (Halliwell, 1994). Clastogens can be ionizing radiationsor any chemical (e.g. pesticides) present in our environment.Advances in industry, exposure to electromagnetic radiationsand radioactive materials etc. have drastically increased theexposure to clastogens (De Rosa et al., 2003). It has beenestimated that more than 80% of cancers and genetic abnor-

⁎ Corresponding author. Fax: +91 820 2571998.E-mail address: cheruvaloor@yahoo.com (M.K. Unnikrishnan).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.09.073

malities are due to clastogens (Feigelson et al., 1996). Diabeticmellitus (Type-1), which is increasing alarmingly throughout theworld and especially in India since two decades, has beenattributed to the destruction of beta cells by clastogens (Dennery,2006). Neurodegenerative diseases (Alzheimer's disease, Par-kinson's disease, and Huntington's disease) are stronglycorrelated to the exposure to clastogen (Krantic et al., 2005).Free radicals are endogenous clastogens produced in differentbiochemical processes and are major culprits for chromosomalaberration, lipid peroxidation and finally ageing (Barouki,2006). Nowadays anticlastogenic compounds have been suc-cessfully used in management of different diseases eg. diabeticcomplications, cardiac stress, mutagenesis (Donaldson, 2004),chemoprevention and radioprotection (Sasaki et al., 1990).

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Hence, compounds which antagonize clastogenic effects canplay an important role in clinical practice (Maxwell, 1997).

Exposure to UV rays and radiation in nuclear war, occu-pational exposure and nuclear accidents are the important causesof radiation toxicity. Patients undergoing radiotherapy are themost vulnerable to radiotoxicity due to the nonselective nature ofradiotherapy towards normal and cancerous cells. Though ra-diotherapy prolongs lives of cancer patients and cures a sub-stantial number, many patients develop secondary cancers, someof which are more severe than the primary cancer (Little, 2001).Hence prevention of normal cell damage and improving thequality of life are as important in radiotherapy as killing ma-lignant cells. Radiotherapy requires the co-prescription ofeffective radioprotectors to minimize the undesired effects.Broadly speaking, radioprotectors can protect human beingsfrom radiation toxicity during radiotherapy, space travel, nuclearwar, occupational and accidental radiation exposures.

Radiolysis of water produces a substantial quantity of oxi-dizing species e.g. hydroxyl radical, hydrogen peroxide, mo-lecular oxygen, singlet oxygen, super oxide and peroxinitrite.Central dogma of radiation biology reveals that the radiationtoxicity occurs either by its direct effect on genetic material and/or macromolecules or by formation of free radicals. DNAdouble-strand breaks, denaturation of proteins, and depletion ofantioxidant enzymes are the most prominent features inradiotoxicity. DNA double-strand breaks are considered as themost biologically important radiation-induced damage. Hencethe compound which scavenges free radicals, increasesendogenous antioxidant enzymes and participates in directbiochemical repair can be effective potential radioprotectors.

Flavonoids are ubiquitous and abundant molecules in plantsand are considered important for preventing a wide variety ofdiseases, including allergies, cardiovascular diseases, certainforms of cancer, hepatic diseases, and inflammation (Middletonet al., 2000). Morin [2-(2, 4-dihydroxyphenyl)-3, 5, 7 trihydroxy-4H-1-benzopyran-4-one] a flavonoid constituent of many herbsand fruits, and a light yellowish pigment found in the wood of oldfustic Chlorophora tinctori. Morin is a food preservative (Smithet al., 1992) that inhibits lipid peroxidation in micelles and in ratliver microsomes (Cholbi et al., 1991). Morin is a powerfulchemoprotective, antimutagenic (Kawabata et al., 1999) and anti-inflammatory (Kim et al., 1999; Raso et al., 2001). Morin is apowerful antioxidant and scavenges diphenyl picryl hydrazyl(DPPH) and hydroxyl radicals (Makris and Rossiter, 2002).Morin chelates metal ions (Fe3+/Fe2+, Al, Cd, Zn, Mg, Mn) and isan important analytical tool for the estimation of these metal ions

by HPLC (Schulman, 1985). Morin produces fluorescence withDNA (Song et al., 2002), and alters DNA replication and repair byinteracting with topoisomerase (Boege et al., 1996). Amphipathicnature of morin allows its partition into lipid bilayers and byincreasing its local concentration can prevent oxidative stress inchallenged conditions. Morin can incorporate into vesicle mem-branes and cause stress in the bilayer packing. This can change itsbarrier functions and alter the membrane potential of bilayers(Ollila et al., 2002). Morin inhibits the production of nitric oxide(NO), tumor necrosis factor-alfa (TNF α) and interleukin (IL)-12from lipopolysaccharide (LPS) activated macrophages (Fanget al., 2003). Morin is polyphenolic compound and alsostructurally related to the aglycone residue of orientin and viceninwhich have been established to be potent radioprotectivemolecules (Uma Devi et al., 2000). Natural antioxidants havebeen extensively studied and many compounds have been foundto possess promising radioprotective action. Further, naturalantioxidants show cell-specific activity in radiotherapy, sensitiz-ing cancerous cells while selectively protecting normal cellsfrom radiation (Maxwell, 1997). Therefore, the present study wasdesigned to correlate the antioxidant activity of morin to itsradioprotective ability.

2. Materials and methods

2.1. Animals

Animal care and handling were done according to the guide-lines issued by the World Health Organization, Geneva Swit-zerland and the INSA (Indian National Science Academy, NewDelhi, India). The inbred Swiss albino mice from a stockpurchased from National Institute of Nutrition Hyderabad, Indiawere used for all the experiments. Eight to ten week old Swissalbino mice weighing 26 to 30 g were selected from an inbredcolony maintained under the controlled conditions of temper-ature (23±2 °C), humidity (50±5° %) and light (14 and 10 h oflight and dark, respectively) at Central Animal ResearchFacility MAHE, Manipal-576104, India. The animals wereprovided with sterile food and water ad libitum. Four animalseach were housed in polypropylene cages containing sterilepaddy husk (procured locally) as bedding. The study wasapproved by the Institutional Animal Ethical Committee.

2.2. Irradiation

Gamma radiation (γ-radiation) was delivered from a60Cobalt teletherapy unit (Siemens, Germany) in the Depart-ment of Radiotherapy and Oncology, Shirdi Sai Baba CancerHospital and Research Center, Kasturba Medical College,MAHEManipal. The remote controlled unit has a head made ofa thick absorbent tungsten sheet, which houses a 2000 Ci 60Cosource (half life 5.2 years). Radiation is let verticallydownwards through a collimator directed to a steel table andthe field size can be adjusted from 5×5 cm to 25×25 cm. Thecollimator is fitted with a bulb, the light beam of whichcoincides with the field of irradiation. The timer of the unit waspreset to a calculated time for different doses of exposure. The

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dose rate was calculated every month using the decay tableprovided for 60Co. Source to surface distance (SSD) wasadjusted to get the required dose rate. Whole-body irradiation ofthe adult mice was done by restraining the unanaesthetizedmouse in a well-ventilated perspex box (20 cm×20 cm×4 cm)partitioned into 12 compartments. Each chamber (3 cm×3 cm×10 cm) can accommodate a single mouse. The field size wasmaintained at 25×25 cm and the γ-radiation was delivered at adose rate of 1.6 Gy/min.

2.3. Preparation of the test compound andmode of administration

Morin was procured from Sigma-Aldrich Co, USA, 1%CMC suspension (DDW) was prepared immediately before use,administered either i.p. or orally at a volume 10 ml/kg. Survivalstudy was carried out by using two different routes (i.p. andorally) by administering the graded drug doses (0, 10, 25, 50,75, 100, 125 and 150 mg/kg). Further studies were carriedout only at most effective dose 100 mg/kg i.p. achieved inExperiment-1.

2.4. Experimental design

2.4.1. Experiment 1: survival study (n=10)

Sham control 10 ml/kg 1% CMC, p.o. sham irradiationMorin sham control Morin 150 mg/kg, i.p.; 100 mg/kg p.o.

sham irradiationRadiation control 1% CMC either i.p. or p.o.; 30 min prior to

10 Gy γ irradiationTreated groups (1) Morin 10, 25, 50, 75 and 100, mg/kg; p.o.;

30 min prior to 10 Gy γ-irradiationTreated groups (2) Morin 10, 25, 50, 75 and 100, 125, 150 mg/

kg; i.p; 30 min prior to 10 Gy γ-irradiation.

Animals were monitored daily for a period of 30 days forsymptoms of radiation sickness and mortality (Satyamitra et al.,2001).

2.4.2. Experiment 2: Dose Modifying Factor(DMF)

Radiation control 10 ml/kg 1% CMC i.p.; 30 min prior todifferent dose of radiation 9, 9.5, 10 Gy γ-irradiation

Treated groups Morin 100 mg/kg; i.p.; 30 min prior to differentdose of radiation 9, 9.5, 10 Gy γ-irradiation.

DMF was calculated from the ratio of radiation LD50 in thepresence and absence of morin (Uma Devi et al., 1996).

2.4.3. Experiment 3: intestinal toxicity (n=6)Experimental design was according to Experiment No. 1,

except that 15 Gy radiation dose was used in treatment group.Only the most effective dose 100mg/kg i.p. was studied. Animalswere sacrificed by cervical dislocation 72 h post irradiation,jejunum removed; sections of 5 μm prepared and scored forfollowing parameters: number of goblet cells, mitotic cells,inflammatory cells, dead cells, and crypt cells/crypt section. The

status of mucosal erosion, basement membrane and villus height,was also assessed (Samarth et al., 2002).

2.4.4. Experiment 4: hemopoietic system (n=8)Experimental design was according to Experiment No. 1,

except that only the most effective dose 100 mg/kg i.p. was usedfor study, animals were sacrificed by cervical dislocation the11th day post irradiation(11 Gy), spleen removed, spleen indexcalculated by dividing the spleen weight by body weight andmultiplying by a factor of 100. Spleen colonies were countedmanually (Uma Devi and Prasanna, 1995).

2.4.5. Experiment 5: endogenous antioxidant defense (n=6)Experimental design was according to Experiment No. 1.

Only the most effective dose was used in treatment group100 mg/kg. Animals were sacrificed by cervical dislocation at2nd, 4th and 8th h, post irradiation (4.5 Gy) and liver dissectedout after transcardial perfusion with ice-cold saline. The wholeliver was blot dried, weighed and a 10% homogenate wasprepared with ice-cold potassium chloride (150 mM) using ahomogenizer (Yamato LSG LH-21, Japan). The homogenatewas used for the estimation of total protein (Lowry et al., 1951)glutathione (Moran et al., 1979) catalase, (Aebi, 1984), totalthiols (Miao, 1994), glutathione S transferase (Jakoby, 1985),lipid peroxidation (Konings and Driver, 1979). Amount of anti-oxidant enzymes is expressed as Units/mg of protein μMol/mg ofprotein.

2.5. Statistical analysis

Data represent the mean±standard error (S.E.M.) of theindicated number of experiments. Graphs were prepared byMicrocal Origin 6.0 software. Statistical analysis of the data wascarried out by one way ANOVA (Graph PAD Instat Software)followed by Tukey post hoc test. A value of Pb0.05 was con-sidered to be significant.

3. Results

3.1. Survival studies

Oral route showed a maximum percentage survival of 30% atmorin 25 mg/kg compared to radiation control (Zero %). Nomortality was observed in sham control and morin shamirradiation groups. Doses above 25 mg/kg decreased percentagesurvival (Fig. 1A). In radiation control, (vehicle by i.p. route)first mortality was observed on the 8th day and all animals diedwithin 17 days, while no mortality was observed in the shamcontrol and morin sham irradiation control. The maximumsurvival (60%) in i.p. route was observed at morin 100 mg/kgcompared to radiation control (Zero %). Linear dose responseactivity was observed from 10 mg/kg to 100 mg/kg, furtherincreasing the dose above 100 mg/kg, biphasic effect wasobserved (Fig. 1B). Reduction in body weight in irradiatedgroups was observed on day 4th which continued up to day20th. Morin pretreatment kept the body weight close to normalin all treated groups.

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3.2. Dose modifying factor

Exposing animals to 9, 9.5 and 10 Gy γ-radiation caused 30,70 and 100% mortality in mice respectively. Pretreatment withmorin 100 mg/kg i.p. reduced this mortality to 10, 20, and 40%at similar radiation dose. Pretreatment with morin 100 mg/kgelevated radiation LD50 from 9.2 to 10.1 Gy, indicating a dosemodifying factor of 1.11(Fig. 1C).

3.3. Histopathological study

Irradiated mice jejunum showed a decrease in crypt and gobletcells and an increase in mitotic, dead and inflammatory cells/crypt. Reduction in villus height and mucosal erosion was also

Fig. 2. Effect of pretreatment morin 100 mg/kg on mouse jejunum, exposed to15 Gy whole body γ-irradiation. (A) Section showing jejunum of shamirradiated mouse with normal villi and absence of inflammatory and dead cells.(B) Section showing jejunum of radiation control mouse with reduced andirregular villi and presence of inflammatory and dead cells. (C) Section showingjejunum, mouse pretreated with morin 100 mg/kg i.p. with improved villi heightand fewer inflammatory and dead cells.

Fig. 1. (A) Survival studies of different doses of morin administered p.o. 30 minprior to 10 Gy whole body γ-irradiation in Swiss albino mice (n=10).(B) Survival studies of different doses of morin administered i.p., 30 min prior to10 Gy whole body γ-irradiation in Swiss albino mice. (C) Reduction inpercentage mortality by morin 100 mg/kg i.p. in mice, exposed to different dosesof radiation.

significantly higher in irradiated mice (Fig. 2A and B). Pre-treatment with morin 100 mg/kg kept the villus height close tonormal with a significant reduction in dead, mitotic, and inflam-matory cells/crypt in irradiated group. Significant increase inrapidly multiplying crypt and goblet cells was observed in morintreated mice. Extent of nuclear enlargement in epithelial cells wasfound to be lower in morin treated mice (Fig. 2C). No anatomicalchanges were observed in sham control (picture not presented).Overall improvement is evident from the gross anatomy of thesections.

3.4. Hemopoietic study

Significant reduction (Pb0.05) in the spleen index (0.35)and CFU (2.7) was observed in radiation control. Pretreatmentwith morin (100 mg/kg) maintained the spleen index close to

Table 1Effect of MH on spleen index, spleen weight and Colony Forming Units (CFUs)in mice expose to 11 Gy γ-radiation

Parameters Shamcontrol

MH Shamcontrol

Radiationcontrol

MH 100 mg/kg+radiation

Spleen index 0.56±0.08 0.59±0.07 0.35±0.056a 0.61±0.025b

Spleen weight(mg)

134±3.7 132±2.4 106±2.9a 124±3.9b

CFUs 0.19±0.015 0.21±0.019 2.70±0.026a 7.87±0.095b

aPb0.05 compared to sham control.bPb0.05 compared to radiation control.

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normal value (0.61) in irradiated group. Morin was also foundeffective in initiating CFU formation, with a score of 7.87. Nosignificant change in spleen index, spleen weight and CFU wasobserved in morin sham control (Table 1).

3.5. Endogenous antioxidant enzymes

Significant (Pb0.05) reduction in antioxidant enzymes(GSH, GST, catalase and SOD) and elevation in malondialde-hyde (MDA) were observed in radiation control at 2, 4 and 8 hpost irradiation (Figs. 3 and 4). Morin significantly (Pb0.05)increased GSH, GST and SOD concentrations at 2, 4 and 8 hpost sham irradiation while no significant change was observedin the levels of catalase and total thiols. Pretreatment with morin100 mg/kg effectively maintained GSH and GST levels at 2, 4and 8 h post irradiation and significantly (Pb0.05) reduced the

Fig. 3. Effect of pretreatment morin 100 mg/kg on liver endogenous anti-oxidantenzymes estimated at different post irradiation hours. (A) GSH, (B) GST, (1 Unitof GST=amount of enzyme that catalyses formation of 1 μmol GSH-CDNBconjugate per minute). (A) MDA. ⁎Pb0.05 compared to sham control.⁎⁎Pb0.05 compared to radiation control.

Fig. 4. Effect of pretreatment morin 100 mg/kg on liver endogenous anti-oxidantenzymes estimated at different post irradiation hours (A) Catalase, (1 Unit=amountof enzyme required to decompose 1 m mole H2O2 per minute). (B) SOD, (C) Totalthiols. ⁎Pb0.05 compared to shamcontrol ⁎⁎Pb0.05 compared to radiation control.

elevation in malondialdehyde formation (Fig. 3). Morin pre-treatment significantly (Pb0.05), prevented radiation-induceddecrease in SOD, catalase and total thiol levels at all monitoredhours post irradiation (Fig. 4).

4. Discussion

Radiation causes tissue injury, both in tumors and in normaltissues, by induction of apoptosis or clonogenic cell death trig-gered by free radical-mediated DNA damage (Karran, 2000).Radiotoxicity is strongly influenced by a sequence of overlappingevents that include activation of the coagulation system, inflam-mation, epithelial regeneration, collagen deposition and remodel-ing (Fajardo, 1989). This complex process is orchestrated by a

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large number of interacting molecular signals, includingcytokines, chemokines, and growth factors (Denham and Hauer,2002).

Enterotoxicity is strongly influenced by exocrine secretions ofthe pancreas particularly trypsin, a physiological agonist ofproteinase activated receptor (Morgenstern and Hiatt, 1967).Proteinase activated receptor regulates many pathophysiologicalprocesses relevant to intestinal radiation injury, motility, prolif-eration of epithelial cells, and inflammation (Vergnolle et al.,1999). Conversely, PAR knockout mice exhibit a markedlydelayed inflammatory response after surgical trauma (Lindner etal., 2000). Decreased crypt cells, and villus height, and increasedinflammatory, dead, and mitotic cells have been observed inradiotoxicity. Pretreatment with morin 100 mg/kg significantlyreduces mucosal erosion, inflammatory, mitotic, and dead cellswhile maintaining villus height. Nuclear enlargement in epithelialcells was also lower in morin treated mice. Morin is a trypsinantagonist (Maliar et al., 2004) that may inhibit proteinaseactivated receptor activation, which partly explains its radiopro-tective activity. Morin blocks TNF α and interleukins in manypathological conditions that contribute to radiotoxicity. Endog-enous infections and bacteremia are major causes of mortality, inpost irradiation period and antibiotics are effectively used asadjuncts in radiotherapy (Rosoff, 1963). Morin is also anantibacterial that may also contribute to radioprotection (Arimaand Danno, 2002). All these observations collectively contributeto the protective effect of morin against radiation toxicity.

Sublethal radiation doses cause hemopoietic failure and lead toanimal death in 12–15 days. Morin pretreatment with 100 mg/kgkept the spleen index close to normal and initiated endogenousspleen colony forming units (CFU) that indicates protection of thehemopoietic system. Further, it has been seen that TNF α andinterleukins delay the hemopoietic recovery as they are powerfulproinflammatory agents. As already discussed, morin inhibits theproduction of TNF α, interleukins, and this may be partly re-sponsible for its hemopoietic protection.

Radiotoxicity and repair mechanisms depend on the status ofendogenous antioxidant enzymes on exposure time and in postirradiation period (Sun et al., 1998). Decreased GSH, GST,catalase and SOD have been promptly observed in radiotoxicity.GSH performsmultifunctional activities to attenuate radiotoxicityby scavenging of free radicals, maintaining thiol-disulphidebalance, synthesis of DNA precursors, synthesis of porphyrin,and maintaining the cellular ATP levels (Wang and Ballatoria,1998). Rate limiting enzymes in GSH synthesis are highly radio-sensitive and are considered responsible for decreased level ofGSH. Compounds that increase the endogenous level of GSH areeffective radioprotectors. Pretreatment with morin at 100 mg/kgsignificantly increases GSH levels at all themonitored hours post-irradiation, which may be one of the possible mechanisms forradioprotective activity of morin. Pretreatment with morin100 mg/kg significantly increases GST, the phase II detoxifyingenzyme that promotes combination of GSH with electrophilicmoieties, thus rendering them inactive. Increased GST level mayprovide further protection. Total thiols are important biologicalmarkers and a source of –SH-groups in enzyme formation andDNA synthesis. Radiation toxicity denatures proteins and causes

conformational changes in structure which renders them inactive.Pretreatment with morin 100 mg/kg significantly increases thetotal thiols in irradiated mice, and maintains the desired amountof SH groups in biological systems to carry out the normalphysiological process.

Superoxide radical is radiolytic product of water and initiatesthe cascading chain reaction by generating peroxynitrate andhydroxyl radical. SOD dismutizes superoxide into hydrogenperoxide and minimizes the radiotoxicity. Morin pretreatmentsignificantly elevates the SOD levels in post irradiation, whichindicates potential radioprotective activity. Hydrogen peroxide isalso formed in radiolysis of water, which is easily accessible toDNA and lipids. Presence of trace amounts of metal ions in DNAand lipids enhanced this toxicity. Elevating the levels of catalase isan ideal strategy to minimize radiation toxicity. Morin pretreat-ment significantly increases endogenous catalase, an enzymewhich converts hydrogen peroxide into water and prevents theformation of hydroxyl radicals, which may be one of the con-tributing factors for its radioprotective activity. Malondialdehydehas been extensively observed in radiotoxicity and is an importantmarker to assess radiotoxicity. Morin (100 mg/kg) pretreatmentsignificantly reduces malondialdehyde formation at every moni-tored hour post irradiation, which shows its radioprotectiveactivity. Similar results have been reported by Prasad et al. (2005)where sesamol reduced radiation toxicity in cultured humanlymphocytes by elevating endogenous antioxidant enzymes, pre-venting DNA damage and reducing malondialdehyde formation.

Topoisomerases are among the enzymes responsible forthe DNA damage and micronuclei formation upon radiationexposure. Cells lacking topoisomerases are radioresistant andtopoisomerase inhibitors are effective radioprotectors (Borehamet al., 1990). The active metabolite of amifostine viz, WR-1065,inhibits normal phosphorylation of topoisomerases, renderingthem catalytically inactive. This property renders the moleculeanticlastogenic (Murley et al., 1997). Topoisomerase-inactiva-tion delays cell cycle, providing more time for DNA repair(Murley et al., 1997) which protects cell against the ioni-zation radiation. Morin forms a ternary complex with topoisom-erase and renders it inactive and in this respect, it might actlike WR 1065. In DNA replication, during cleavage, morininhibits the DNA re-ligation step. This could also be one ofthe possible mechanisms of its radioprotective effect (Boegeet al., 1996).

Damaged DNA in radiotoxicity triggers biological signalswhich activate checkpoints and repair systems that delay cellcycle progression by allowing more time for repair (Zhou andElledge, 2002). Compounds like NSAIDS and prostaglandinsanalogs, which delay cell cycle progression or delay the G1/Sphase, are effective radioprotectors (Lee and Stupans, 2002).Morin delays the cell cycle progression in G1/S phase (Brownet al., 2003) thereby increasing the repair time for damagedDNA. Morin also prevents Fenton induced calf thymusDNA damage by scavenging hydroxyl radicals (Makris andRossiter, 2002). Morin reduces DNA damage, which in turncould prevent deleterious signaling by damaged DNA leadingto apoptosis. 5-Aminosalicylic acid, a powerful antioxidant,prevents in vivo and in vitro DNA damage and has been shown

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to be effective as a radioprotector (Sudheer et al., 2003). It hasalso been observed that compounds which prevent DNA damageare promising radioprotectors, further supporting the radiopro-tective potential of morin.

Ionizing radiation activates NF-kB, MAP kinase cascadeand Jun N-terminal kinase (Chen et al., 1996). These factorssubstantially contribute in radiotoxicity. Morin inactivates cellcycle kinase, and also has pleiotropic effects on kinase signallingpathways, including inhibition of protein kinase B, Jun N-terminal kinase and p38 kinase (Brown et al., 2003). Morincould have produced some protective effect by minimizing all ofthe above, though this requires to be experimentally confirmed.

Efficacy of any radioprotector is usually expressed in termsof DMF, which indicates extent of the radioprotective activity ofcompound. Morin (100 mg/kg) elevated radiation LD50 from9.2 to 10.1 Gy, indicating DMF of 1.11. DMF of 1.11 clearlydemonstrates the radioprotective ability of morin. Thus freeradical scavenging, antioxidant properties and protection to thehemopoietic system are to be likely mechanisms for the radio-protective activity of morin.

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