Radioprotection by Tempol Studies on Tissue Antioxidant Levels, Hematopoietic and Gastrointestinal Systems, In Mice Whole Body Exposed to Sub- Lethal Doses of Gamma Radiation

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Radioprotection by tempol: Studies on tissue

antioxidant levels, hematopoietic and

gastrointestinal systems, in mice whole body

exposed to sub- lethal doses of gamma radiation

L. Ramachandran1

and C.K.K. Nair2*

1Amala Cancer Research Centre, Thrissur 680555, Kerala, India2 Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla 689101, Kerala, India

*Corresponding author:Dr. C.K.K. Nair,Dean of research, Pushpagiri Institute of medicalsciences and Research Centre, Thiruvalla 689101,Kerala, India.Fax: +91 469 2731005E-mail:[email protected]

Background: Ionizing radiation induces theproduction of reactive oxygen species (ROS), whichplay an important causative role in cell death. Whole-body exposure of mice to gamma radiation leads to

diminution of tissue antioxidant defense systems;increases the peroxidative damage to membranelipids and damages the haematopoietic and gastroin-testinal systems. Tempol (TPL), a cell membrane-permeable amphilite nitroxide, shown to protectagainst cell injury caused by ROS was studied for itsradioprotective effects. Materials and Methods:Animals were administered with TPL at doses of 100or 200 mg/kg body weight p.o 10 minutes prior tosub- lethal doses (4 or 6 Gy) of whole body gammaradiation exposure. Results: Tempol prevented theradiation induced depletion in RBC and total WBCcounts, glutathione content in blood and bone

marrow cellularity. TPL also protected the tissueantioxidant system and membrane lipids from theradiation-induced damages. An enhanced spleencolony formation and spleen weight recovery werealso observed in radiation exposed mice adminis-tered with TPL. The compound also protected theepithelial cells of the gastrointestinal tract from theradiation-induced structural alterations. Conclusion:These preclinical data indicate that TPL may have itspotential as a radioprotector during radiationexposure scenarios. Iran. J.Radiat.Res.,2012;10(1):1-10

Keywords: Antioxidant defense, radioprotector,hematopoietic system,gastrointestinal mucosa, spleencolony, tempol.

INTRODUCTION

Total-body exposure to ionizing radia-tion in humans and animals can result inmultiple organ dysfunction as a conse-quence of damage to the hematopoietic,

gastrointestinal or cerebrovascular systems,depending on the total dose of radiation

absorbed (1, 2). There remains a need todevelop safe and effective radioprotectorswhich would mitigate the deleteriousconsequences of radiation exposure in theevent of a massive radiological accident, anuclear terrorist attack, or prolonged spacetravel (1-5).

Many natural and synthetic compoundshave also been found to protect biologicalsystems against radiation induced damage (6-8). Cyclic nitroxides are stable free radicalsstabilized by methyl groups at the positionin six membered piperidine ring structures.The methyl groups confer stability to thenitroxide radicals by preventing radical

radical dismutation. 4-Hydroxy-2,2,6,6-tetramethylpiperidine- N-oxyl or tempol(TPL) C9H18NO2, (scheme 1) is a cellmembrane-permeable amphilite nitroxide, aredox cycling agent that can metabolizesuperoxide anion (O2.) and many other ROS(9-12). The action of nitroxides to metabolizereactive oxygen species is ascribed primarilyto cyclic one- or two-electron transfer amongthree oxidation states: the oxammoniumcation, the nitroxide, and the hydroxyl-

amine. Nitroxides undergo a very rapid, one-electron reaction in vivoto the correspond-ing hydroxylamine (13, 14), which has antioxi-dant activity (9, 10, 15, 16). Tempol protectedV79 cells against radiation in a concentra-tion dependent manner (17). Preclinical stud-

Iran. J. Radiat. Res., 2012; 10(1): 1-10


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ies in guinea pigs revealed that topicalapplication was effective at preventingradiation-induced alopecia (18, 19). A phase Iclinical trial in patients receiving whole-

brain radiotherapy suggested that TPL maybe effective at preventing radiation- inducedalopecia with only mild (grade I and II)toxicity (20). Oral administration of TPL hasbeen shown to prevent the age-dependentrise in blood pressure in the spontaneouslyhypertensive rats (21) and buthionine sulfoxi-mine induced lipid peroxidation, bloodpressure and reduction in cellular levels ofglutathione (22). Tempol also protectedsalivary glands from radiation- induced

damage, but did not protect the tumortissue, suggesting that delivery of the agentprior to irradiation would not alter tumorcontrol (23). Tempol afforded complete protec-tion from the mutagenic effects of hydrogenperoxide and superoxide and was not itselfmutagenic (24). It also provided protectionagainst X-ray- and neocarzinostatin-inducedmutagenicity and double-strand breaks inDNA (25). Studies using comet assayrevealed that TPL and other nitroxides

provided significant protection to trouterythrocytes against oxidative damage (26).In the present study, we explored the

protective effects of TPL against depletion ofantioxidants, and damages to hematopoieticand gastrointestinal systems in mice wholebody exposed to sub-lethal doses of gamma-radiation.

L. Ramachandran and C.K.K. Nair

Agricultural University, Thrissur, Kerala,India. They were kept under standardconditions of temperature and humidity inthe Centres Animal House Facility and

provided with standard mouse chow (SaiDurga Feeds and Foods, Bangalore, India)and water ad libitum. All animal experi-ments in this study were carried out withthe prior approval of the InstitutionalAnimal Ethics Committee, strictly adheringto the guidelines of Committee for thepurpose of Control and Supervision ofExperiments on Animals constituted by theAnimal Welfare Division of Government ofIndia.

Chemicals

Tempol (TPL) C9H18NO2,was purchasedfrom Spectrochem Pvt. Ltd., Mumbai, India.Nitro blue tetrazolium (NBT), reducedglutathione (GSH), 55dithiobis-(2 nitrobenzoic acid) (DTNB), EDTA and riboflavinwere obtained from Sisco Research Labora-tories Ltd., Mumbai, India. TCA (Tri chloroacetic acid) was from Merck Specialties Pvt.Ltd. Mumbai, India. All other chemicals

were of analytical grade procured fromreputed Indian manufacturers.

Exposure to -radiation

Irradiation was carried out using a 60Co-Theratron Phoenix teletherapy unit (Atomicenergy Ltd, Ottawa, Canada) at a dose rateof 1.88 Gy per minute.

Administration of TPL

Solution of TPL was prepared in sterile

distilled water and animals were adminis-tered with TPL by means of oral gavage.

Effect of TPL on -radiation inducedbiochemical and histological alterations invarious tissues of whole body radiation (4Gy) exposed mice

Animals were divided into six groups ofthree animals each, administered with TPLand exposed to whole body gamma radiation(4 Gy) as detailed below.

Group I- 0.2 ml distilled water + Sham

2 Iran. J. Radiat. Res., Vol. 10 No. 1, June 2012

Scheme 1. Chemical structure of tempol.

MATERIALS AND METHODS

Animals

Male Swiss albino mice, 8-10 weeks oldand weighing 22-25 g were obtained fromthe Small Animal Breeding Section, Kerala


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Radioprotection in mice by tempol

irradiation, Group II- TPL, 100 mg/kg.b.wt.+ Sham irradiation, Group III- TPL, 200mg/kg.b.wt. + Sham irradiation, Group IV-0.2 ml distilled water + 4 Gy, Group V- TPL,

100 mg/ kg body weight+ 4 Gy, Group VI-TPL, 200 mg/kg.b.wt. + 4 Gy.

A single dose of TPL (100 mg or 200 mgper kg body weight) was administered toanimals 10 minutes prior to the sub- lethaldose of 4 Gy gamma radiation.

a) Antioxidant status and lipid peroxida-tion:After 24 hours of radiation exposure,the animals were sacrificed by cervicaldislocation and liver, brain and kidney wereexcised. Blood was collected by heart

puncture into heparinised tubes andanalyzed for hemoglobin content byDrabkins method (27) and GSH content (28).Femurs of the animals were dissected outand bone marrow cells were flushed intophosphate buffered saline (pH 7.4) contain-ing 10% fetal bovine serum. The cells werewashed and bone marrow viability wasdetermined by the method of Sredni et al.(29). The results were expressed as number ofbone marrow cells 106/femur. From the

liver, brain and kidney tissues collected,10% (w/v) homogenates were prepared in icecold phosphate buffered saline (PBS). Thesehomogenates were analyzed for antioxidantstatus. Reduced glutathione (GSH) levelwas measured at 412 nm using DTNB asthe substrate (28). Superoxide dismutaseactivity was determined by the nitro bluetetrazolium (NBT) reduction method ofMcCord and Fridovich (30, 31). Glutathioneperoxidase (GPx) activity was determinedby the method of Hafemann et al., (32) basedon the degradation of H2O2 in the presenceof GSH. The concentrations of malondialde-hyde (MDA) as indices of lipid peroxidationwere assessed according to the method ofBuege and Aust (33). Tissue protein wasestimated according to the method of Lowryet al., (34) using bovine serum albumin asstandard.

b) Histology of intestine: At 72nd hourpost radiation exposure, animals fromdifferent groups were sacrificed. A portion of

the small intestine was removed from eachgroup, washed in PBS, and fixed in 10%formaldehyde solution and embedded inwax. Sections were taken and stained with

hematoxylin- eosin.

Effect of TPL on different blood parame-ters, spleen colony formation and spleenweight recoveryinwhole body radiation (6Gy) exposed mice

Animals were divided into six groupsand administered with TPL, as describedbefore, prior to the sub- lethal dose of 6 Gywhole-body gamma radiation. Blood wascollected from the tail vein of each animal

every third day (till 12th day post radiation),to heparinised tubes and was analyzed forchanges in different peripheral bloodparameters viz. RBC, WBC counts and he-moglobin concentration using Mindray BC-2800 Vet auto hematology analyzer. Theanimals were sacrificed on the 12th day postirradiation by cervical dislocation and thespleen was excised out, weighed and fixed inBouins solution and analyzed for colonyformations (35-37).

Statistical analysis

The results are presented as meanstandard deviation (SD) of the studiedgroups. Statistical analyses of the resultswere performed using ANOVA with Tukey-Kramer multiple comparisons test.

RESULTS

The changes in different antioxidantlevels and extent of lipid peroxidation invarious tissues of mice exposed to wholebody -irradiation are presented in table 1.In liver, GSH levels were decreased from352.20 to 220.45 nano moles/ mg proteinin mice upon exposure to 4 Gy - radiationrespectively. Administration of TPL prior toradiation exposure maintained the GSHlevels to 24.348.55 and 29.360.67 respec-tively in TPL100 and TPL200 administeredgroups. Similar tendency was also observedin other tissues vizbrain and kidney. As can

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be seen in table 1, the activity of both SODand GPx, two of the major enzymes involvedin the antioxidant defense mechanism werealso found to be decreased after irradiationin all the tissues analyzed and the admini-stration of TPL prior to irradiation in allcases prevented the decrease of both SODand GPx levels.

Whole body exposure to -radiationresulted in an increase in the peroxidationof lipids in different tissues. Table 1 depictsthe results on the measurement ofperoxidation of lipids in terms of thiobarbi-turic acid reacting substances monitored asmalondialdehyde (MDA) in the brain, liver

and kidney of mice exposed to whole body 4Gy -radiation. In liver, the extent ofperoxidation of lipids quantified as MDA(nanomoles/ mg protein) were increasedfrom 1.060.062 to 4.762.35 and admini-stration of TPL prior to radiation exposure

showed lower MDA levels, 2.290.03 and1.400.15 respectively in TPL100 andTPL200 administered groups. Similartendency was also observed in other tissuesalso viz brain and kidney where the MDAlevels were found to be decreased signifi-cantly in the TPL administered animals in aconcentration dependent manner.

The protective effect of TPL on thehematopoietic system against deleteriouseffects of ionizing radiation is evident fromthe data on bone marrow cellularity (figure1) and GSH content (figure 2) in blood. Theun-irradiated control animals had 15.00 106 cells/ femur whereas in the irradiated

group this dropped drastically to 6.64 106cells/ femur. The irradiated animals admin-istered with TPL showed 9.14 106 cells/femur and 10.57 106 cells/ femur in 100mg/kg.b.wt. and 200 mg/kg.b.wt. groupsrespectively as compared to the irradiated

Table 1. Changes in antioxidant (GPx, GSH, SOD) and lipid peroxidation levels in 4 Gy whole body irradiated mice (with and withoutoral administration of Tempol (TPL) C9H18NO2, (100 or 200 mg/kg body weight, 10 minutes prior to irradiation) in liver, brain and

kidney homogenates.

(a indicates p


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control group. The radiation exposure alsobrought about drastic drop in blood GSHlevel and administration of TPL helped to

maintain their levels to a considerableextent.A close microscopic examination of the

stained sections of the intestine of radiationexposed animals reveals the alteredstructures of mucosa and sub- mucosalayers. The irradiated mice exhibited thegastrointestinal damage as crypt epithelialcell necrosis, blunting of the villi and dif-fused lymphatic and plasmacellular infiltra-tion. The administration of mice with TPLprior to irradiation protected the intestinalepithelial cells from radiation-inducedstructural alterations as seen in figure 3.

Whole body exposure of mice to gammaradiation resulted in significant depletion ofdifferent hematological parameters.Significant increase in total erythrocyte andleukocyte counts, hemoglobin concentration

were observed in TPL treated radiationexposed animals as compared to controlirradiated animals (figure 4). TPL treated

un-irradiated groups showed normal levelsof all the hematological parameters (datanot shown). Formation of endogenousspleen colonies is an index of hematopoieticstem cell proliferation. TPL administrationsignificantly enhanced the spleen colonyformation in animals exposed to a sub-lethal dose of 6 Gy whole body gammaradiation (table 2) in a concentrationdependent manner. The control irradiatedanimals developed an average of 30.4colonies, whereas TPL treated groupsdeveloped 17 0.9 and 26.5 2.12 coloniesfor TPL100 and TPL200 respectively. Asignificant loss in spleen weight wasobserved in the animals of radiation alonegroup. On the contrary, the spleen weightswere comparatively higher in animals ofTPL treated radiation exposed groups.

Figure 1. Effect of Tempol (TPL) C9H18NO2, administration onbone marrow cellularity in mice exposed to 4 Gy whole-body

gamma radiation. (a indicates p


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Figure 3. Effect of Tempol (TPL) C9H18NO2, ongastrointestinal injury of mice upon 4 Gywhole-body radiation exposure. (A) 0 Gy controlgroup; (B) 4 Gy irradiated group showing alteredstructures of mucosa and sub- mucosa layers.Mice exhibited gastrointestinal damage as cryptepithelial cell necrosis, blunting of the villi anddiffused lymphatic and plasmacellular infiltration;Mucosal structure was preserved in the (C)TPL100+4 Gy and (D) TPL200+4 Gy treatedgroups and pretreatment significantly preventeddecrease in villous number and villous height.Magnification X960 (Objective 40X, Eyepiece 10Xand Camera zoom 2.4X).

Figure 4. Effect ofT e m p o l ( T P L )C9H18NO2, on differenthaematological pa-rameters in mice ex-posed to a sub-lethaldose of 6 Gy gammaradiation.



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DISCUSSION

It is well known that most of the dam-ages induced by ionizing radiation to living

cells are due to the generation of aqueousfree radicals. The bodys innate mechanismhas many enzymes and non- protein com-pounds that protect from the free radicalsand reactive oxygen species produced insidethe body during normal metabolism andalso due to external stimuli. Administrationof mice with TPL prior to radiation exposureeffectively helped to maintain their levelsfrom depletion. The basic effect of radiationon cellular membrane is believed to be the

peroxidation of membrane lipids. Lipidperoxidation can be initiated by radiolyticproducts, including hydroxyl and hydroper-oxyl radicals (38). This highly destructiveprocess results in the formation ofmalondialdehyde (MDA) and alters the totalfunction of the cellular membranes. TPL hasbeen shown to protect lipids (39, 40) and DNA(41-43) from oxidative damages. The presentstudy revealed the efficiency of TPL in in-hibiting the radiation-induced lipid peroxi-dation in different tissues of whole body

irradiated mice.The gastro-intestinal system is one of

the major targets for the somatic injuriesassociated with radiation and chemother-apy. Because of this, radiation-inducedgastrointestinal syndrome (RIGS) is animportant cause of host vulnerabilitywhether in medical therapeutics or innuclear accidents or terrorism. RIGS is duein part to the killing of clonogenic crypt cellswith eventual depopulation of the intestinal

villi(44, 45)

. Crypt epithelial cells proliferaterapidly and are highly sensitive to cytotoxicagents and irradiation. Loss of this regener-ating population of clonogenic cells followingirradiation prevents the normal re- epitheli-alization of the intestinal villi. This impair-ment leads to varying degrees of villousblunting and fusion, with attenuation andhypertrophy of the villous epithelial cells (46).These changes result in the acute RIGSpresenting with malabsorption, electrolyteimbalance, diarrhea, weight loss and death.

The late side effects and the sequelae ofsevere acute intestinal radiation injuryinclude varying degrees of intestinalinflammation, mucosal thickening, collagen

deposition, and fibrosis, as well as impair-ment of mucosal and motor functions (47-49).Tempol protected the intestinal epithelialcells from radiation induced structurallesions to a considerable extent.

Peripheral and lymphoid organlymphocytes are among the most radiation-sensitive cells (50, 51). Damage to bonemarrow is known to be the main cause ofdeath in animals following whole body dosesof radiation between about 2 and 10Gy (52).Radiation death in the mid- lethal doserange is due to impairment of bone marrowhematopoietic function such as leukopenia,erythropenia and thrombocytopenia whichwill ultimately lead to whole body infection,hemorrhage and even death (53). Theprotective effect of TPL against radiationinjury to hematopoietic tissues was assessedin terms of bone marrow cellularity, bloodGSH levels, peripheral blood counts,endogenous spleen colony assay and spleenweight. The decrease in hematological

constituents may be attributed to a directdamage by radiation dose and hematopoieticrecovery after whole body irradiation isdependent on the presence of spared hema-topoietic stem and progenitor cells in thebone marrow (54). The administration of TPLprior to the radiation exposure wasassociated with significant protective effectsagainst radiation-induced depletion indifferent hematological parameters. Asignificant increase was found in spleen

weights as well as number of spleen coloniesin the TPL and radiation combined groupsthan the irradiated control group. TPLadministration prior to sub- lethal dose ofradiation, resulted in higher WBC, RBC andbone marrow cell counts in TPL treatedanimals compared to the animals of irradi-ated control group. These findings suggestthat prior administration of TPL resulted inprotection of hematopoietic stem cells at thetime of the radiation exposure, therebyleading to increased recovery of the bone




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marrow and subsequently the peripheralblood counts.

High exposures to radiation may occurdue to accidents or during nuclear war.Radiation also poses a major, un-resolvablerisk for astronauts, especially forlong-duration space flights (55). The mosteffective in vivo radioprotectors studied sofar are effective when administered beforeirradiation, as they must be present in thesystem at the time of irradiation. Hence,they can be used only when the eventualityof the exposure is known and are notsuitable against unplanned exposures, e.g.accidents, spillage, warfare and terrorist

attack. Because conditions of elevatedoxidative stress can exist in cells even afterirradiation, nitroxides and hydroxylaminescan exert protective effects by scavengingsecondarily generated ROS resulting fromradiation-induced damage (56).

Radiotherapy is being frequently usedas part of cancer treatment to achieve tumorcontrol. A major problem associated withcancer radiotherapy is the severe sideeffects resulting from normal tissuedamage. Agents which protect normal tissueagainst radiation damage can increase thepatient tolerance to radiotherapy. Tempolhas been shown to differentially protectbone marrow and not tumor cells. Bioreduc-tion of TPL to its corresponding hydroxyl-amine (which is not a radioprotector)occurred to a greater extent in RIF-1 tumorcells compared to bone marrow (57).

Our present study demonstrates thatTPL has capacity to protect the antioxidant,hematopoietic and gastrointestinal systems

from radiation induced deleterious effectswhen administered prior to radiation expo-sure scenarios. Hence our results coupledwith the available literature on the radio-protective effects of TPL suggest that TPLcan be a potential candidate for clinicalradioprotection.

CONCLUSION

Present study demonstrates that TPLhas capacity to protect the antioxidant,

hematopoietic and gastrointestinal systemsfrom radiation induced deleterious effectswhen administered prior to radiationexposure scenarios.

ACKNOWLEGMENT

The authors express their gratitude to

BRNS, Department of Atomic Energy,

Government of India, Mumbai for the

Research grant to CKKN.

REFERENCES

1. Coleman CN, Blakely WF, Fike JR, MacVittie TJ, Metting

NF, Mitchell JB, et al. (2003) Molecular and cellularbiology of moderate-dose (1-10 Gy) radiation and poten-tial mechanisms of radiation protection: report of aworkshop at Bethesda, Maryland, December 17-18,2001, Radiation research, 6: 812-34.

2. Mettler FA Jr and Voelz GL (2002) Major radiation expo-sure--what to expect and how to respond. The New Eng-land journal of medicine, 20: 1554-61.

3. Coleman CN, Stone HB, Moulder JE, Pellmar TC (2004)Medicine. Modulation of radiation injury. Science, 5671:693-4.

4. Moulder JE (2004) Post-irradiation approaches to treat-ment of radiation injuries in the context of radiologicalterrorism and radiation accidents: A review. Int J RadiatBiol, 1: 3-10.

5. Wilson JW, Cucinotta FA, Shinn JL, Simonsen LC, DubeyRR, Jordan WR, et al. (1999) Shielding from solar parti-cle event exposures in deep space. Radiation research,3: 361-82.

6. Nair CKK, Parida DK, Nomura T (2001) Radioprotectorsin radiotherapy.Journal of radiation research, 2137

7. Upadhyay SN, Dwarakanath BS, Ravindranath T (2005)Chemical Radioprotectors. Defence Science Journal, 4:403-25.

8. Weiss JF, Landauer MR (2003) Protection against ioniz-ing radiation by antioxidant nutrients and phytochemi-cals. Toxicology, 1-2: 1-20.

9. Krishna MC, DeGraff W, Hankovszky OH, Sar CP, Kalai T,Jeko J, et al. (1998) Studies of structure-activity rela-

tionship of nitroxide free radicals and their precursorsas modifiers against oxidative damage. Journal of me-dicinal chemistry, 18: 3477-92.

10. Krishna MC, Grahame DA, Samuni A, Mitchell JB, RussoA (1992) Oxoammonium cation intermediate in thenitroxide-catalyzed dismutation of superoxide. Proceed-ings of the National Academy of Sciences of the United

States of America, 12: 5537-41.11. Krishna MC, Russo A, Mitchell JB, Goldstein S, Dafni H,

Samuni A (1996) Do nitroxide antioxidants act as scav-engers of O2-. or as SOD mimics? The Journal of biologi-cal chemistry, 42: 26026-31.

12. Li WG, Zhang XY, Wu YJ, Gao MT, Zheng RL (2006) Therelationship between structure and antioxidative activityof piperidine nitroxides. Journal of Pharmacy and Phar-



9/10

macology, 7: 941-9.13. Okajo A, Matsumoto K, Mitchell JB, Krishna MC, Endo K

(2006) Competition of nitroxyl contrast agents as an invivo tissue redox probe: comparison of pharmacokinet-ics by the bile flow monitoring (BFM) and blood circulat-

ing monitoring (BCM) methods using X-band EPR andsimulation of decay profiles. Magn Reson Med, 2: 422-31.

14. Swartz HM (1990) Principles of the metabolism of ni-troxides and their implications for spin trapping. FreeRadic Res Commun, 3-6: 399-405.

15. Hahn SM, Krishna MC, DeLuca AM, Coffin D, Mitchell JB(2000) Evaluation of the hydroxylamine Tempol-H as anin vivo radioprotector. Free Radic Biol Med, 6: 953-8.

16. Wu YJ, Li WG, Zhang ZM, Tian X (1997) Antioxidativeactivity of 4-oxy- and 4-hydroxy-nitroxides in tissues anderythrocytes from rats, Zhongguo yao li xue bao = Actapharmacologica Sinica, 2: 150-4.

17. Mitchell JB, DeGraff W, Kaufman D, Krishna MC,Samuni A, Finkelstein E, et al. (1991) Inhibition of oxy-gen-dependent radiation-induced damage by the nitrox-ide superoxide dismutase mimic, tempol.Arch BiochemBiophys, 1: 62-70.

18. Cuscela D, Coffin D, Lupton GP, Cook JA, Krishna MC,Bonner RF, et al. (1996) Protection from radiation-induced alopecia with topical application of nitroxides:fractionated studies. The cancer journal from ScientificAmerican, 5: 273-8.

19. Goffman T, Cuscela D, Glass J, Hahn S, Krishna CM,Lupton G, et al. (1992) Topical application of nitroxideprotects radiation-induced alopecia in guinea pigs, Int JRadiat Oncol Biol Phys, 4:803-6.

20. Metz JM, Smith D, Mick R, Lustig R, Mitchell J, Chera-kuri M, et al. (2004) A Phase I Study of Topical Tempol

for the Prevention of Alopecia Induced by Whole BrainRadiotherapy. Clinical Cancer Research, 19: 6411-7.21. Nabha L, Garbern JC, Buller CL, Charpie JR (2005) Vas-

cular oxidative stress precedes high blood pressure inspontaneously hypertensive rats. Clinical and Experi-mental Hypertension, 1: 71-82.

22. Banday AA, Fazili FR, Lokhandwala MF (2007) Oxidativestress causes renal dopamine D1 receptor dysfunctionand hypertension via mechanisms that involve nuclearfactor-B and protein kinase C.Journal of the AmericanSociety of Nephrology, 5: 1446-57.

23. Cotrim AP, Hyodo F, Matsumoto K, Sowers AL, Cook JA,Baum BJ, et al. (2007) Differential radiation protectionof salivary glands versus tumor by Tempol with accom-panying tissue assessment of Tempol by magnetic reso-

nance imaging. Clin Cancer Res, 16: 4928-33.24. Degraff WG, Krishna MC, Russo A, Mitchell JB (1992)

Antimutagenicity of a low molecular weight superoxidedismutase mimic against oxidative mutagens. Environ-mental and Molecular Mutagenesis, 1: 21-6.

25. DeGraff WG, Krishna MC, Kaufman D, Mitchell JB(1992) Nitroxide-mediated protection against X-ray-andneocarzinostatin-induced DNA damage. Free RadicalBiology and Medicine, 5: 479-87.

26. Villarini M, Moretti M, Damiani E, Greci L, Santroni AM,Fedeli D, et al. (1998) Detection of DNA damage instressed trout nucleated erythrocytes using the cometassay: protection by nitroxide radicals, Free Radic BiolMed, 7-8: 1310-5.

27. Drabkin DL, Austin JM (1932) Spectrophotometric stud-ies; spectrophotometric constants for common hemo-globin derivatives in human, dog and rabbit blood.J BiolChem, 719-33.

28. Moron MS, Depierre JW, Mannervik B (1979) Levels of

glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica etbiophysica acta, 1: 67-78.

29. Sredni B, Albeck M, Kazimirsky G, Shalit F (1992) Theimmunomodulator AS101 administered orally as a che-moprotective and radioprotective agent. InternationalJournal of Immunopharmacology, 4: 613-9.

30. McCord JM, Fridovich I (1969) Superoxide dismutase.An enzymic function for erythrocuprein (hemocuprein).The Journal of biological chemistry, 22: 6049-55.

31. McCord JM and Fridovich I (1969) The utility of super-oxide dismutase in studying free radical reactions. I.Radicals generated by the interaction of sulfite, dimethylsulfoxide, and oxygen. The Journal of biological chemis-try, 22: 6056-63.

32. Hafeman DG, Sunde RA, Hoekstra WG (1974) Effect ofdietary selenium on erythrocyte and liver glutathioneperoxidase in the rat. The Journal of nutrition, 5: 580-7.

33. Buege JA and Aust SD (1978) Microsomal lipid peroxi-dation, Methods in Enzymology, 302-10.

34. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951)Protein measurement with the Folin phenol reagent.The Journal of biological chemistry, 1: 265-75.

35. Ramachandran L, Krishnan CV, Nair CKK (2010) Radio-protection by -lipoic acid palladium complex formula-tion (POLY-MVA) in mice. Cancer Biotherapy and Radio-pharmaceuticals, 4: 395- 9.

36. Till JE and Culloch EAM (1961) A direct measurementof the radiation sensitivity of normal mouse bone mar-

row cells. Radiation research, 213-22.37. Till JE, Culloch EAM (1963) Early repair processes in

bone marrow cells irradiated and proliferating in-vivo.Radiat Res, 96-105.

38. Konings AW, Oosterloo SK (1980) Radiation effects onmembranes. II. A comparison of the effects of X irradia-tion and ozone exposure with respect to the relation ofantioxidant concentration and the capacity for lipid per-oxidation. Radiation research, 2: 200-7.

39. Samuni AM, Barenholz Y (1997) Stable nitroxide radi-cals protect lipid acyl chains from radiation damage.Free Radic Biol Med, 7: 1165-74.

40. Samuni AM, Barenholz Y, Crommelin DJ, Zuidam NJ(1997) Gamma-irradiation damage to liposomes differ-ing in composition and their protection by nitroxides.Free Radic Biol Med, 7: 972-9.

41. Damiani E, Greci L, Parsons R, Knowland J (1999) Ni-troxide radicals protect DNA from damage when illumi-nated in vitro in the presence of dibenzoylmethane anda common sunscreen ingredient Free Radical Biologyand Medicine, 7-8: 809-16.

42. Damiani E, Kalinska B, Canapa A, Canestrari S,Wozniak M, Olmo E, et al. (2000) The effects of nitrox-ide radicals on oxidative DNA damage. Free RadicalBiology and Medicine, 8: 1257-65.

43. Samuni A, Godinger D, Aronovitch J, Russo A, MitchellJB (1991) Nitroxides block DNA scission and protectcells from oxidative damage. Biochemistry, 2: 555-61.

44. Marshman E, Booth C, Potten CS (2002) The intestinal




10/10


epithelial stem cell. Bioessays, 1: 91-8.45. Potten CS (1998) Stem cells in gastrointestinal epithe-

lium: numbers, characteristics and death. Philosophicaltransactions of the Royal Society of London, 1370: 821-30.

46. Potten CS, Merritt A, Hickman J, Hall P, Faranda A(1994) Characterization of radiation-induced apoptosisin the small intestine and its biological implications. Int JRadiat Biol, 1: 71-8.

47. Coia LR, Myerson RJ, Tepper JE (1995) Late effects ofradiation therapy on the gastrointestinal tract. Int J Ra-diat Oncol Biol Phys, 5: 1213-36.

48. Hauer-Jensen M (1990) Late radiation injury of thesmall intestine. Clinical, pathophysiologic and radiobi-ologic aspects. A review. Acta oncologica (Stockholm,Sweden), 4: 401-15.

49. Zimmerer T, Bocker U, Wenz F, Singer MV (2008) Medi-cal prevention and treatment of acute and chronic radia-tion induced enteritis--is there any proven therapy? ashort review. Zeitschrift fur Gastroenterologie, 5: 441-8.

50. Dainiak N (1997) Practical and theoretical issues in1993 concerning radiation effects on the growth of nor-mal and neoplastic hematopoietic cells. Stem cells

(Dayton, Ohio), 75-85.51. Iyoda T, Nagata K, Akashi M, Kobayashi Y (2005) Neu-

trophils accelerate macrophage-mediated digestion ofapoptotic cells in vivo as well as in-vitro. J Immunol, 6:3475-83.

52. Coggle JE. Biological effects of radiation. Taylor andFrancis, London 1983:p. 90.

53. Floersheim GL, Chiodetti N, Bieri A (1988 ) Differentialradioprotection of bone marrow and tumor cells by zincaspartate. British Journal of Radiology, 501- 8.

54. Herodin F and Drouet M (2005) Cytokine-based treat-ment of accidentally irradiated victims and new ap-proaches. Experimental hematology, 10: 1071-80.

55. Maurya DK, Devasagayam TPA, Nair CKK (2006) Somenovel approaches for radioprotection and the beneficialeffect of natural products.

56. Soule BP, Hyodo F, Matsumoto K, Simone NL, Cook JA,Krishna MC, et al. (2007) The chemistry and biology ofnitroxide compounds. Free Radic Biol Med,

11: 1632-50.57. Hahn SM, Sullivan FJ, DeLuca AM, Krishna CM, WerstoN, Venzon D, et al. (1997) Evaluation of tempol radioprotec-tion in a murine tumor model, Free Radic Biol Med,7:1211-6.


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Radioprotection by Tempol Studies on Tissue Antioxidant Levels, Hematopoietic and Gastrointestinal Systems, In Mice Whole Body Exposed to Sub- Lethal Doses of Gamma Radiation