Chemical Protection Against Radiation

7/28/2019 Chemical Protection Against Radiation

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Chemical ProtectionAgainst X-Ray, Gamma, and

Neutron Radiation

Petersburg Nuclear Physics Institute

Gatchina, Leningrad District, Russia

Head of Laboratory

S. A. GrachevLeading Research Worker

A. G. Sverdlov

Published by

Armed Forces Radiobiology Research Institute

Bethesda, Maryland, USA

Editor and NIS Initiatives CoordinatorGlen I. Reeves, M.D.

Scientific DirectorE. John Ainsworth, Ph.D.


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Cleared for public release; distribution unlimited.

AFRRI Contract Report 97-1Printed December 1997

Defense Nuclear Agency Contract DNA001-93-C-0155

For information about this publication, write Armed Forces Radiobiology Research

Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA, or telephone

011-301-295-0377, or send electronic mail to [email protected]. Find

more information about AFRRI on the Internets World Wide Web at

http://www.afrri.usuhs.mil.

This and other AFRRI publications are available to qualified users from the Defense Technical

Information Center, Attention: OCP, 8725 John J. Kingman Road, Suite 0944, Fort Belvoir, VA

22060-6218; telephone (703) 767-8274. Others may contact the National Technical Information

Service,5285 PortRoyal Road, Springfield,VA 22161; telephone (703) 487-4650. AFRRI publications

are also available from university libraries and other libraries associated with the U.S. Governments

Depository Library System.


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Preface

One of the major long-term research goals of theArmed Forces Radiobiology Research Institute(AFRRI) has been the study and development ofagents, either singly or in combination, that wouldprotect personnel exposed to either photon or neu-tron radiation, or both. There are several scenarios,besides the obvious one of a nuclear weapon(s)detonation, where military personnel could be sub-jected to a single or mixed radiation field; they in-clude cleanup operations after a reactor accident,such as that at Chernobyl, or a weapons accident orincident.

Criteria fora preventive regimen should include (1)significant dose modification factor (dose reduc-tion factor, or DRF); (2) minimal if any side effectsand no long-term toxicity; (3) oral administration,preferably no more than once daily; and (4) mini-mal reduction in effectiveness when administeredsoon after exposure rather than prior to exposure.

Someof the most effective agents todate have beenaminothiols and their derivatives. Unfortunately,most of these agents have side effects such as nau-sea, vomiting, hypotension, weakness, and fatigu-ability that, while not precluding their use inclinical radiation therapy, have rendered them un-suitable for a military operations scenario. Re-searchers at AFRRI (Weiss et al. 1993; Landauer etal. 1993) demonstrated that administration of caf-feinemitigatedthe neurotoxicitycaused by admini-stration of WR-3689 and WR-2721, though otherauthors have found that caffeine in higherdoses ag-gravated these symptoms.Clearly, theneed fora ra-

dioprotector that is both effective and safe stillexists.

Dr. Joseph F. Weiss visited, on behalf of AFRRI,the authors of the present report in their laboratoryat Gatchina, Russia. He was impressed by the workthey were doing in this field, and how it supple-mented AFRRIs research along different lines to-ward this same goal. Their approach, spelled out in

the section Introduction, will not be repeatedhere.

Briefly, theauthors used a nontoxic thiolcompoundto block the biochemical receptors in cells of thetarget tissues for the side effects while not simulta-neously lowering the DRF. They also tested a newcompound that they synthesized for efficacy andtoxicity protection. These combinations weretested against both neutron and photon irradiationusing a mouse model. The authors recommendedthat these successful preparations be used in a largeanimal (canine) model, and, if successful, be fol-lowed by human toxicity studies. Realizing that theparenteral routes of administration used in theirstudy are unsuitable for a field situation, they alsooutlined steps for development of oral regimens.

While this document does not reflect the opinion ofAFRRI or theDepartment of Defense regarding thesuitability of the described regimens in an opera-

tional situation, it doespresent a thought-provokingstep toward the development of an effective yetnontoxic means of radiation protection and maystimulate further research along these or perhapsslightly different lines.

Grateful acknowledgment is given to the followingscientists at AFRRI whose advice and constructivecriticism were of immense value in the editing ofthis manuscript: Drs. E. John Ainsworth, RameshBhatt, K. Sree Kumar, and Terry Pellmar and Mr.Henry Gerstenberg.Anyerrors inediting, however,are entirely my responsibility.

Glen I. Reeves, M.D.NIS Initiatives CoordinatorArmed Forces Radiobiology Research Institute

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Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Radioprotectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Effect of Unithiol on Toxicity of Radioprotectors . . . . . . . . . . . 7

Effect of Unithiol on the Effectiveness ofChemical Protection in Mice Against X Rays . . . . . . . . . . . . . . 8

Protection Against Neutron Irradiation . . . . . . . . . . . . . . . . . 10

Mechanism of Action of Unithiol. . . . . . . . . . . . . . . . . . . . 11

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Suggestions for Further Research. . . . . . . . . . . . . . . . . . . . . . . 15

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

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Abstract

Experiments in mice showed that intraperitoneal(i.p.) injection of unithiol (sodium salt of 2,3-dimercapto-l-propanesulfonic acid) diminishedtoxicity of several aminothiol radioprotectors, in-creasing the LD50 of cystamine by 40% and amino-ethanisothiuronium bromide hydrobromide (AET)by64%. The optimum ratio for the doses is 0.5 molarequivalent of unithiol per radioprotective thiol. Anew radioprotector (mixed disulfide of cysteamineand unithiolMDCU) has a weak toxicity: the LD50is750 mg/kg i.p.The use ofunithiolmakes itpossibleto increase the dose of the SH-radioprotectors,

enhancing the dose reduction factor (DRF) of cys-tamine and AET by 30% for x-ray irradiation. Asomewhat lessereffect is observed with fission neu-tron irradiation. The DRF of MDCU is equal to 1.6for x-ray irradiation and is 1.1 for neutron irradia-tion. The mechanism of antitoxic action of unithiolcouldnotbe detected inChinese hamster fibroblasts.It may be caused by the competition of unithiol andthe SH-radioprotectors for certain, as yet undeter-mined, biochemical structures in brain neurons. It isalso possible that unithiol may decrease penetrationof SH-radioprotectors into the brain.

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Introduction

Extensive studies of chemical protection againstionizing radiation hazard have led to the develop-ment of efficient radioprotectors that significantlydiminish radiation injury in living organisms. Themost effective radioprotectors developed so far areaminothiols and their derivatives: cysteamine,cystamine, AET (aminoethanisothiuronium bro-mide hydrobromide), WR-638 (cystaphos), andWR-2721 (gammaphos). Some of these com-pounds have been successfully used to preventcomplications of radiation therapy in patients withcancerandareconsidered as a protection against ra-diation hazard in space flights and in accidental ra-diation exposure scenarios as well as in clinical use(Bacq 1964; Mozzhukhin and Rachinskii 1979;Monig 1990; Thompson 1964). Unfortunately, allof the aminothiols have toxic side effects that limittheir use in medical practice. Thus, scientists havelong searched for ways to decrease their toxicity.

Judging by their side effects (nausea, vomiting, as-

thenia, loss of working capacity), the toxic effectsof these radioprotectors are primarilyon the centralnervous system (CNS). Therefore, various drugsand anticonvulsive substances have been tried tomitigate the toxic effects. For example, pentobarbi-tal narcosis reduced hypersalivation, vomiting, andcramps caused by intravenous administration ofcystamineto dogs (Mundyand Heffer1960).Lumi-nal (25 mg/kg), medinal (50100 mg/kg), and lib-rium(30 mg/kg)reduced the death ratefrom80% to2030% in mice that were administered 300 mg/kgof cystamine (Strelnikov et al. 1969). The anticon-vulsive preparation benzonal (50 mg/kg) increased

the LD50 of cystamineby 12%(Zherebchenko et al.1974). In their experiments, Weiss and coauthors(Landauer et al. 1993; Weiss et al. 1993) showedthat caffeine (40 mg/kg) mitigated the behavioraldeficits caused by administration of WR-3689 andWR-2721. Conversely, Strelnikov and associates(1969) found that caffeine (however in a higherdose: 50 mg/kg) as well as other CNS stimulators(phenamine, corasole, strychnine) intensified

cramps, shortenedtheir latentperiod, andincreasedthe lethal effect of cystamine and cystaphos inmice.

However, the toxicity of aminothiol radioprotec-tors may be reduced not only by neurotropic prepa-rations. The toxicity of cystamine is reduced by aconcurrent administration of ACTH, cortisone, orhydrocortisone (Stern et al. 1965; Strelnikov et al.1969). Recently, various metals (zinc, copper) andselenium were found to reduce the toxicity of WR-2721; the effect depended on the interval betweenadministration of themetals and the radioprotectors(Weiss et al. 1987, 1990). It is important that thesemetals and selenium increase the radioprotectiveefficacy of cysteamine, AET, and WR-2721(Brown et al. 1988; Floersheim and Floersheim1968; Weiss et al. 1987, 1990).

One effective way of reducing the toxicity of ami-nothiol radioprotectors could be to use them in

combination. Many authors have found that suchcombinationsas WR-2721 andcystaphos, AETand2-mercaptopropionylglycine, mercaptoethylgua-nidine and cysteamine, cystamine and AET, andcystamine and cystaphos reduce the toxicity andsome side effects caused by these preparationswhen applied separately (Zherebchenko et al.1974;Maisinand Mattelin 1967; Vladimirov et al. 1989).However, it is not advisable to combine prepara-tions, each of which is toxic. The question iswhether similar, if not better, radioprotective ef-fects can be reached using a nontoxic thiol.

The protective properties of the thiol radioprotec-tors areassociated with their effecton thestem cellsof the hematopoietic system and cells of the intesti-nal epithelium. Their toxicity appears to be due totheir effect on cells in another crucial system of thebody, the CNS, and is a result of their interactionwith some biochemical structures in thebrain cells.The functions of these cells may be disturbed bysuch interaction.

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A nonradioprotective and nontoxic thiol with astructure similar to the aminothiol protectors maybecapable ofblocking thebiochemicalstructures inthe nerve cells. This might hinder access to them bythe SH-radioprotectors and thus reduce damage tothe neurons functions, thereby reducing the toxic

action of the radioprotectors. If this should provetrue, the toxicity of aminothiol radioprotectorscould not only be diminished using such nontoxicthiols, but the dose tolerance could be increased,thus increasing the efficacy of the chemical protec-tion against radiation hazard.

One of the most promising candidates for this roleis unithiol, a sodium salt of 2,3-dimercapto-1-propanesulfonic acid. It contains two SH-groups,CH2(SH)CH(SH)CH2SO3Na. It is a nontoxic phar-maceutical preparation that was used in medicalpractice in the former USSR as an antidote against

Lewisiteandotherarsenic compoundsas well as forcases of poisoning by ions of some heavy metals

(Mashkovskii 1967). It was tested as a radioprotec-tor but did not show appreciable radioprotectiveaction.

To determine the potential antitoxic action ofunithiol, we studied its effect on the toxicity of cys-

tamine and AET in mice. We also studied the toxic-ity of a mixed disulfide of cysteamine and unithiol(MDCU), which we synthesized. We further exam-ined thepossibility of increasing theprotective effi-cacy of the aminothiol radioprotectors bycombining them with unithiol and testing the com-binationson mice exposed to irradiationfrom x raysand also from fission neutrons.

The authors greatly appreciate the contributions ofDrs. E.V. Kropachev, N.G. Nikanorova, L.I. Kot-lovanova, I.K. Koroleva,andG.T. Bojko in variousphases of this work. The authors are grateful toDrs.

E.J. Ainsworth, G.I. Reeves, and J.F. Weiss forfruitful discussions and support of this work.

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Contract Report CR97-1


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Materials and Methods

Radioprotectors

In conformity with the contract, two new radiopro-tective combinations were prepared and MDCUwas synthesized.

Cystamine plus unithiol. Preparations of cysta-mine dihydrochloride and unithiol were purchasedfrom commercial suppliers and used without fur-

ther purification. Thepurityof unithiol was not lessthan 98% as shown by SH-group spectrophotomet-ric analysis with Ellman reagent. The purity of cys-tamine detected by SH-group spectrophotometryafter disulfide bond reduction by NaBH

4was 96

3%.

AET plus unithiol. AETwasprepared according toa published procedure (Doherty et al. 1957). Thepurity of AET detected by SH-group spectropho-tometry following the transguanidination reactionwas 96 3%.

MDCU. MDCU was synthesized by the reaction ofcystamine dioxide with unithiol. This synthesis ofCH2(SSCH2CH2NH2)CH(SSCH2CH2NH2)CH2SO3Na HCl 4H2O is described below.

A solution of unithiol (1.75 g, 7.66 mmole) in 2.5ml of 1 mM HCl was added dropwise to a suspen-sion of cystamine dioxide (4 g, 15.5 mmole) in 3 mlof 1 mM HCl. A solution of 2.5 ml of 4 M HCl wasadded to theobtained solution,andthe total volumeof the reaction mixture was subjected to columnchromatography using a 17 x 2 cm column packed

with cationite Amberlite CG-120, 100200 mesh,in H+

form, balanced by a 1 M HCl solution. Thecolumn was washed by a 1 M HCl solution to re-movethe hypotaurine, thenbya 2 M HCl solutionata flow rate equal to 1.3 ml/minute.

Fractionscontaining themixed disulfidewere com-bined and concentrated via rotary evaporation to avolume of 5 ml.Theobtained viscous yellowishso-lution with 2 ml water was transferred to a small

glass and then lyophilized; 2.4 g of a powderedcompound were obtained.

HPLC analysis showed the presence of a singlepeak. Analysis for Cl

-

ions showed that the productobtained by lyophilization represents a mixture ofthemonohydrochlorideanddihydrochloride forms.Aqueous solutions of this preparation were shownto be acidic and therefore have to be neutralized

prior to their administration to animals. Thus, it isadvisable to transform this compound to the mono-hydrochloride. For this purpose, dry resin Amber-lite CG-400, 200400 mesh in OH

-

form was addedto2.2 g of the compoundin20ml ofwater, resultingin a final pH of 6.2. The obtained solution was fil-tered from the resin, the resin was washed by 10 mlof water, and the united filtrate was evaporated to 5ml viarotaryevaporator, then lyophilized;1.8g of apowdered compound were obtained. HPLC analy-sis showed the presence of a single peak.

Disulfide groups in the compound were analyzedby SH-group spectrophotometry after reduction ofthe disulfide bond by NaBH4, as described in Ha-beeb (1973). Analysis showed 102 3% content of-S-S-group against the calculated value for theMDCU molecule.

Calculated values for the elemental analysis ofC7H8N2O3S5 HCl 4 H2O (M.W. 447) were:

C H S Cl.

18.81% 6.09% 35.85% 7.93%

We found: 18.71% 6.15% 35.50% 7.90%

The aqueous solution of the MDCU monohydro-chloride was found to be near neutral; pH for the0.01 M solution was 6.8.

The absorption spectrum of theaqueous solution ofthe mixed disulfide has a maximum absorption of245nm, characteristic of anyorganic disulfide. The

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calculated MDCU extinction coefficient was 850l/mole cmtwo times higher than that of a com-pound with onedisulfidebond (E = 300400 l/molecm). These data show that a molecule of MDCUcontains two disulfide groups.

The doses of the radioprotectors were calculatedfrom their salts.Toxicity was evaluatedby theLD50value, and their protective effect was derived fromthe percentage of the animals that survived 30 daysafter irradiation.

Subjects

The studies were carried out in mice, isolated cul-tured fibroblasts (V79 cells) of Chinese hamsters,and neurons in slices of rat hippocampi.

Mice. Male (CBAC57BLF1) F1 mice age 2.53months and weighing 1822 g were used. The pro-tectors were administered prior to irradiation:intraperitoneally, 15 minutes before irradiation, ororally by buffered feeding needles, 40 minutes be-fore. Mice were housed in cages holding five each.They were carefully observed for signs of fighting.Bedding was wood chips. Illumination was consis-tent with the season. The standard laboratory chowmixture used in theformerUSSR wasused; compo-nents included oats, sunflower seeds, white bread,milk, carrots, green grass, meadow haw, fish oil,fish flower, yeast, and salt.

For the toxicity studies, six mice were used for eachdose level, andeach determination wasrepeated sixtimes. Results were combined and the mean arith-metic value and its error calculated. For the irradi-ated groups, 20 animals were used in each group,and irradiation was repeated three times.

Chinese hamster cells. To study the influence ofcysteamine, unithiol, and their combination, weused an asynchronous culture of Chinese hamsterV79 cells in the exponential phase of growth. Cys-

teamine and unithiol were dissolved in the culturemedium, and the cells were incubated for 20 min-utes at room temperature. Then the cells werewashed, and a fresh medium was added. The settledcellswere incubatedfor67days thereafter, andthenumber of colonies was counted. Survival was de-termined relative to untreated cells. Different con-centrations of cysteamine and unithiol were used.

Rat hippocampal neurons. Hippocampal sliceswere prepared by the standard method (Teyler1980). Two slices from each of 10 rats were used.Cystamine was studied in 10 slices, unithiol in theother 10. Cystamine was not rinsed off the slices.The methods used in our experiments are described

elsewhere (Peimer et al. 1986). Recording of thepopulation spike was conducted from the layer ofpyramidal cells of the CA 1-2 sectorusing thesectors of the hippocampal formation described byLorente de No (1934)by the usual method, usingmetallic or glass microelectrodes with resistance25milliohm. Forstimulationof theSchaffers col-laterals, we used bipolar platinum electrodes withtotal diameterupto 100 ina glass or lac insulation.The electric pulse duration was 100 seconds, withamplitude up to 10V. The results were analyzed bycomputer.

Irradiation

X rays. Irradiation conditions were as follows:RUM-17 x-ray machine, 220 kV, 15 mA, filtration0.5 mm Cu + 1.0 mm Al. The dose rate was 1.8Gy/minute. A VA-J-18 dosimeterwasused. Duringx irradiation the mice were placed in separate cellsof Plexiglas cages. Ten mice were irradiated simul-taneously.

Fission neutrons. Irradiation by fission neutronswas carried out in a vertical biochannel of the Pe-tersburgNuclear Physics InstituteWWR-Mreactor(Sverdlov 1974). The neutron mean energy was0.85 MeV; the contribution of gamma quanta to thetotal dose was 25%, dose rate was 14 cGy/min, andirradiation was circular. Dosimetry was carried outusing tissue equivalent graphite and polyethyleneionization chambers filled with ethylene and CO2.A threshold detector was used to determine the en-ergy spectrum. During neutron irradiation the micewere placed in duralumin (alloy) cages in separatecells. There were 10 mice per cage, and 5 cages intheirradiationchamber.A rotaryplatform wasused

to give uniform exposure. The temperature insidethe chamber was 20 C; ambient air was providedthrough a ventilation system. Dose exposure levelswere determined for each cage. After exposure, themice were returned to the same conditions andcages as before irradiation.

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Results

Effect of Unithiol on Toxicity ofRadioprotectors

The influence of unithiol on the toxicity of cysta-mine and AET was investigated, and the toxicity ofMDCU, in which the radioprotector is chemicallybound to unithiol, was determined. The amount ofunithiol in combination with cystamine and AETwas estimated from the thiol equivalents of thesecompounds. Molar thiol equivalents of cysteamineand AET are equal to their molecular weightssince they contain one free or potential SH-group,whereas those of cystamine and unithiol are equaltohalf of their molecular weights since they containtwo free or potential SH-groups. Thus, every doseofan aminothiol protectorhasa corresponding doseof unithiol. For example, using 0.5 molar equiva-lent of unithiol, 300 mg of AET requires 61 mg ofunithiol, while for 400 mg, 82 mg of unithiol wouldbe used, and so on.

As shown in table 1, the toxicity (LD50) of the radio-protectors in ourexperiments wasclosely related tothat described by others (Mozzhukhin and Rachin-skii 1979; Thompson 1964). At the same time, tox-icity was sharply decreased by simultaneous i.p.administration of an0.5equivalent dose of unithiol.The LD

50ofcystamine roseby40% and that ofAET

by 64%. This effect of unithiol far exceeded the an-titoxic action of other agents (thiol and nonthiol)tested by other authors (Zherebchenko et al. 1974;Jacobus 1959; Kalistratov et al.1972; Pugachevaetal.1972;Suvorov andShashkov1975;Takagyetal.1971).

A decrease in toxicity wasnot observed by simulta-neous oral administration of unithiol andeithercys-tamine or AET (table 2). Perhaps this is related tothe way in which unithiol is absorbed from thestomach. When unithiol is used as a drug, it is givenas pellets instead of a solution (Mashkovskii 1967).However, when oral administration of cystaminewas combined with i.p. administration of unithiol,

then all the mice were able to survive a universallylethal dose of protector; even a 1600 mg/kg (LD100)dose was no longer lethal.

Toxicity of the orallyadministered AETwas mark-edly unaffected by unithiol, regardless of themethod of administration. The reason for this

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Table 1. Toxicity of radioprotectors and its changeunder the influence of unithiol (intraperitonealadministration)

ProtectorDose ofunithiol

LD50,mg/kg

Change(%)

Cystamine 0.5 equivalent0.75 equivalent

392550533

40*36*

AET 0.5 equivalent0.75 equivalent

358588496

64*38*

MDCU 750

*p < 0.05

Table 2. Toxicity of radioprotectors and its changeunder theinfluenceofunithiol(oral administration)

ProtectorDose ofunithiol

LD50,mg/kg

Change(%)

Cystamine 0.5 equivalent0.5 equivalent

(i.p.)

139213961600

< 0.3 (N.S.)All mice

survived*

AET 0.5 equivalent0.5 equivalent

(i.p.)

130013501450

4 (N.S.)11 (N.S.)

MDCU 1900 All micesurvived*

*This doseis normally100%lethal,so further increasingthedose was of no practical importance.


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remains a mystery. However, it should be re-membered that the radioprotective effect of orallyadministered AETis highlyconjectural. It has beenobserved by only a few researchers and as a rule,quite some time (a few hours) after administration.The weak efficacy of oral administration of AET

and i.p. injection of unithiol may be explained bythe different rates of absorption and a discrepancybetween the times of resorptive action of the twopreparations.

remains a mystery. However, it should be re-membered that the radioprotective effect of orallyadministered AETis highlyconjectural. It has beenobserved by only a few researchers and as a rule,quite some time (a few hours) after administration.The weak efficacy of oral administration of AET

and i.p. injection of unithiol may be explained bythe different rates of absorption and a discrepancybetween the times of resorptive action of the twopreparations.

As a whole, our experiments showed a high anti-toxic effectiveness of unithiol with thiol protectorsand demonstrated the most effective relationshipsbetween doses of radioprotective compounds andunithiol. As judged from our data, the best radio-protective combinationusesapproximately0.5 mo-lar equivalent of unithiol. Higher proportions (0.75equivalent and more) were less effective, perhaps

because of theadditional toxic influenceof unithiolitself when administered in such large amounts.

Effect of Unithiol on the Effectiveness ofChemical Protection in Mice Against X Rays

Since combining unithiol with aminothiol radio-protectors reduced their toxicity, it may be possibleto increase the radioprotective effect by increasing

the dose. It is known that the protective effect of thethiol radioprotectors depends on the dose; increas-ing the dose increases the protection (Mozzhukhinand Rachinskii 1979; Thompson 1964). In earlierstudies, unithiol showed no radioprotective effect.It is nontoxic at 168 mg/1000 g, or 1.5 equivalent.Its effect appears to be solely in decreasing the tox-icity of aminothiol radioprotectors.

Efficacy of intraperitoneal administration ofprotectors. Table 3 shows that the radioprotectiveeffect of cystamine and AET given in their usualdoses (150 mg/kg)and administeredi.p. wasclearly

exhibited in our experiments. In both magnitude ofeffect and dependence on dose, our data agree with

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Table 3.Survival percentage (M SD) of x-irradiatedmice protectedby cystamine, AET, their combinationwith unithiol, and MDCU (intraperitoneal administration)

Irradiation dose, Gy

Experimental conditions 6 7 8 9 10 11 12

Control (irradiationwithout protectors) 100 55 3 25 4 0 0 0 0

Irradiation + cystamine

(150 mg/kg) 100 95 5 84 3 85 2 63 3 10 3 4

Protection efficacy* 0 40

59

85

63

10

4

Irradiation + cystamine(300 mg/kg) + unithiol(152 mg/kg) 100 100 100 100 88 5 65 5 55 5

Protection efficacy* 0 45 75 100

88

65

55

Irradiation + AET(150 mg/kg) 100 95 4 70 6 66 5 40 7 20 4 10 6


45

66

40

20

10

Irradiation + AET

(300 mg/kg) + unithiol(64 mg/kg) 100 95 5 100 100 95 5 85 5 55 7


75

100

95

85

55

Irradiation + MDCU(350 mg/kg) 95 5 94 5 85 5 80 3 60 4 40 3 10 4

Protection efficacy* 5 39 60 80 60 40 10

*Difference between survival of irradiated animals unprotected and protected by radioprotectors

Difference significant at the p < 0.05 level


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numerous published data (Bacq 1964; Mozzhukhinand Rachinskii 1979; Thompson 1964).

Because the addition of unithiol reduced the toxic-ity of the radioprotector, doses twice as high asusual were tried. This considerably enhanced theradioprotectiveeffect at every radiationdose given.

The differences in protective action between pro-tectors alone and their combinations with unithiolare best shown at the higher radiation doses. At 11and 12 Gy, the protective effect of cystamine andAET given in their usual doses isverysmall (10 and4.5%, respectively, for cystamine and 20 and 10%

forAET), whereas for thecombination,where dou-bledoses of theprotectors were used, cystaminein-creased survival by 65 and 55%, respectively, andAET by 85 and 55%. MDCU (350 mg/kg) was alsomore effective than cystamine or AET at 150 mg/kg;survival was60and40%higherwith irradiationdoses of 10 and 11 Gy, and 10% higher at 12 Gy.

Thedifferences areparticularlystrikingwhen thera-dioprotective effect is expressed as the dose reduc-tion factor (DRF). As shown in table 4, doubling thedose raises the DRF ofcystamine from1.4 to1.8 andof AETfrom 1.3 to 1.7, that is, the DRF of both pro-

tectors is increased by 30%. The DRF of MDCU ishigh, too, reaching1.6. Thepronounced protectiveac-tion of these combinations and MDCU on radiation-induced, acute intestinaldistress must beemphasized,as the problem is very real and difficult to solve.

Efficacy of oral administration of protectors.Analogousresults wereobtainedin theexperimentswith oral administration of the protectors to mice(table 5).

9

Results

Table 4. LD50/30 (cGy, midline dose) of x-irradiatedmice and DRF of radioprotectors (intraperitonealadministration)

Experimental conditions LD50/30 DRF

Control (irradiation

without protectors) 710

Irradiation + cystamine(150 mg/kg) 1025 1.4

Irradiation + cystamine(300 mg/kg) + unithiol(152 mg/kg) 1268 1.8

Irradiation + AET(150 mg/kg) 937 1.3

Irradiation + AET(300 mg/kg) + unithiol(64 mg/kg) 1192 1.7

Irradiation + MDCU(350 mg/kg) 1122 1.6

Table 5. Survival percentage (M SD) of x-irradiated mice using cystamine, AET, their combination withunithiol, and MDCU (oral administration)

Irradiation dose, Gy

Experimental conditions 8 9 10 11 DRF

Control (irradiation without protectors)

Irradiation + cystamine (300 mg/kg)Protection efficacy*

0

50 450

0

20 420

0

00

0

00

01.1

Irradiation + cystamine (1300 mg/kg)+ unithiol (152 mg/kg)

Protection efficacy*

100

100

52 3

52

31 4

31

10 5

10 1.3

Irradiation + AET (300 mg/kg)Protection efficacy*

80 280

40 340

20 320

30 430 1.2

Irradiation + AET (800 mg/kg)+ unithiol (81 mg/kg)

Protection efficacy*

90 2

90

50 3

50

30 2

30

0

0 1.2

Irradiation + MDCU (1600 mg/kg)Protection efficacy*

45 515

50 250

00

00 1.2

*Differencebetweensurvivalof irradiated animalsunprotectedand protected byradioprotectors.All nonzerovaluesare significant atthe p < 0.05 level.

Differences between protective effects at 8 and 9 Gy are not significant.


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Cystamine alone (300 mg/kg) protects from deathonly 50% of mice subjected to8 Gy ofx irradiation,whereas the combination with a fourfold (1300mg/kg) amount of cystamine permitted 100% sur-vival. At 9 Gy, cystamine given in the usual oraldose (300 mg/kg) enhanced survival by 20%, but a

fourfold dose raised it to 52%. Cystamine at thishigher dose affords protection to 31% of mice evenat10Gy. Toa lesserdegree, unithiolexerts someef-fect on the protection afforded by AET.

MDCU hasa protective effect with oral administra-tion as well as i.p., whereas cysteamine itself isknown tobeineffective whengiven thisway.Asforcystamine, adding unithiol makes it possible to in-crease thedose of cystaminesufficientlytoenhancechemical protection withoral administration. How-ever,unithiol aloneappeared tobe ineffective inourprevious experiments when given orally and must

be administered intraperitoneally when oral dosesof cystamine or AET are used. Perhaps this is be-cause the free SH-groups of unithiol decrease theabsorption of theradioprotectorfromthestomach.

Looking at this part of the study as a whole, it canbe said with confidence that chemical protectionagainst ionizing radiation can be substantially

enhanced by taking advantage of the antitoxic ac-tion of unithiol to increase the dose of the thiol pro-tectors. This possibility is particularly important insituations with high radiation levels where protec-tors in their usual doses are ineffective.

Protection Against Neutron Irradiation

The chemical protection of an organism againstneutron exposure is especially difficult in viewof the high damage potential of these particles(Sigdestadt et al. 1976; Sverdlov et al. 1969; Sverd-lov 1974). The opportunity to enhance such protec-tion in some way or other is all-important. Thus, amajor objective of our study was to evaluate antira-diation activity of a combination of the thiol radio-protectors with unithiol in animals subjected toneutron irradiation.This is especially important be-

cause chemical protection against neutrons, as arule, requires high doses of SH-radioprotectors(Sverdlov 1974).

The effect of the radioprotectors under study andtheir combinations with unithiol in mice irradiatedby fission neutrons is shown in table 6. Cystamineand AET given in their usual doses increased the

10


Table 6. Survival percentage (M SD) of fission-neutron irradiated mice using radioprotectors alone andcombined with unithiol

Total irradiation dose, cGy

Experimental conditions 200 250 300 350 400

Control (irradiationwithout protectors) 94 3 87 2 31 2 0 0

Irradiation + cystamine(150 mg/kg) 100 87 2 60 2* 0 0

Irradiation + cystamine(300 mg/kg) + unithiol(152 mg/kg) 100 94 2* 71 2* 31 2* 0

Irradiation + AET(150 mg/kg) 100 100* 75 2* 27 3* 12 2*

Irradiation + AET(300 mg/kg) + unithiol(64 mg/kg) 100 100* 87 2* 74 3* 19 3*

Irradiation + MDCU(350 mg/kg) 100 100* 60 4* 21 2 0

*Difference from control significant at p < 0.05


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survival rate from a lethal dose of 300 cGy ofgamma-neutron radiation1 (225 cGy from neu-trons)cystamine by about 30% and AET by40%, which corresponds to our previous data(Sverdlov et al. 1969; Sverdlov 1974). The radio-protective effect of cystamine disappeared and that

of AET was decreased to 30% as the radiation dosewas increased to 350 cGy (260 cGy of neutrons).On further increasing the gamma-neutron dose to400 cGy, the effect of AET was decreased to 12%.The addition of unithiol to cystamine and AET,which makes it possible to increase theradioprotec-tor dose, measurably increased the efficacy ofchemical protection: the combination of unithiolwith cystamine defended mice not only at 300 cGybut at 350 cGy as well, when cystamine by itself isineffective.

The combination of AET with unithiol was even

more effective. It protected 75% of irradiated miceat 350 cGy in contrast to 27% for AET alone, andeven at the supralethal dose of 400 cGy, it enabledsurvival of nearly 20% of animals.

Therole ofunithiol isclearly demonstratedbycom-paring the DRF of the protectors taken in their usualdoses and in the enhanced doses made possible bythe use of unithiol (table 7). The DRFs of thecombinations were higher than the DRFs of theradioprotectors alone, taken in their usual dose. Thecombination of cystamine with unithiol increasedthe DRF by nearly 10%, and the combination ofAET with unithiol raised it by 20%.

Theseprotectorsandtheircombinationswith unithiolare less effective for neutron irradiation than radia-tion with lowlinear-energy transfer (LET).This as-pect of chemical protection against the hazards ofneutron radiationis well known(Bacq 1964; Mozz-hukhinandRachinskii 1979; Sigdestadt et al.1976;Sverdlov et al. 1969). However, it should be pointedout that the essential protective effect against neu-tron irradiation is accomplished by increasing theradioprotector dose and by reducing its toxicity by

using unithiol. This protection is higher than thatafforded by other combinations, for example, com-binations of sulfur-containing protectors with eachother or with 5-methoxytryptamine (5-MOT)(Bogatyrev et al. 1983).

The combination of an enhanced dose of AET withunithiol in our experiments provided protectionagainst neutron radiation with a DRF of 1.4, that is,the same as AET or cystamine in their usual doseagainst much less damaging low LET radiation.Thus, this combination increases the efficacy ofchemical protection not only against x and gammarays,butalsoagainst fission neutrons.Animals irra-diated by neutrons withstand the combination andMDCU as well as x-irradiated animals.

Mechanism of Action of Unithiol

Our next step was to try to determine the mecha-nism by which unithiol diminishes the toxic actionof the aminothiol radioprotectors cystamine andAET. To analyze the phenomenon, we needed toconsider more than one circumstance. First, it wasnecessarytoprovide fixed ratiosof unithiol with theaminothiolsandthen to increase theunithioldoseas

the radioprotector dose was increased. This wouldshow that the described effect of unithiol was notdue to the effect of this dithiol on any regulatorsystems but to its interaction at this concentrationwith the radioprotectors at the cellular level. In

11

Results

Table 7. LD50/30 (cGy, midline dose) for mice irra-diatedby neutrons andgamma rays (approximately75%of dose duetoneutrons)and DRFforradiopro-tectors and their combinations with unithiol

Experimental conditions LD50/30 DRF*

Control (irradiation withoutprotectors) 275

Irradiation + cystamine(150 mg/kg) 312 1.1

Irradiation + cystamine(300 mg/kg) + unithiol(152 mg/kg) 330 1.2

Irradiation + AET (150 mg/kg) 325 1.2

Irradiation + AET (300 mg/kg)+ unithiol (64 mg/kg) 375 1.4

Irradiation + MDCU (350 mg/kg) 312 1.1

*Differences significant from control at p < 0.05

1Technical note: It is impossible, usinga reactor, togive a pure neutron dose. Some gammaandx rays arealwaysmixed in. Hence thecompound word, gamma-neutron.


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connection with this, we studied the influence ofunithiol on the toxic effects of cystamine and cys-teamine in vitro with V79 cells of Chinese hamsterin culture and with slices of rat hippocampal neu-rons.1 Theresultsof theexperiments with cysteamineon the V79 cells are summarized in table 8.2

For the first time, in these experiments (as opposedto the experiments in animals), unithiol by itselfdemonstrateda toxic effect, although it was presentin fairly large concentrations (25 mM and more).The reason for this is a valid avenue for special in-vestigation. However, one fact stands out: in spiteof its toxicity, unithiol did not strengthen the dam-aging action of cysteamine on the cells in any of theexperimental variations.

Preliminary experiments on the hippocampal slicesshowed that, with the addition of cystamine in con-

centrations near 10M into theincubation medium,the population spike resulting from stimulation ofthe Schaffer s collaterals increased in amplitude to100150%.3 This reaction came immediately afteraddition of the radioprotector and lasted about 20minutes. The reaction of the hippocampal neuronslices to unithiol, added to the incubation mediumin the same concentration, was the same. Thus, wefound some variation in the function of the nervecells under the effect of cystamine and unithioltaken in concentrations approaching those in an or-ganism at a protective dose of radioprotectors andanantitoxicdoseofunithiol (taking into account theblood-brain barrier).

Unfortunately, these experiments do not fully ex-plain the influence of the cystamine and unithiol

combination on neurons. However, they appearto be interesting in themselves because they tes-tify to the influence of the radioprotector and uni-thiol on nerve cells and exhibit one type of suchinfluences.

12


Table 8. Survival percentage of V79 cells afteradding cysteamine, unithiol, or their combination

Concentration SurvivalPreparation (mM) M SD

Cysteamine 0 100

50 74 7100 28 12150 16 6

Unithiol 15 10025 66 750 35 875 28 6

Cysteamine 100+ 38 11

Unithiol 50


Unithiol 15


Unithiol 25


Unithiol 75

Note: Differences between the effects of cysteamine alone anda combination of cysteamine and unithiol are not statisticallysignificant at the p = 0.05 level.

1Cystamine only wasusedin animalsandin hippocampalslices.Ascystamine hasno protective effect in vitro, weusedcysteamine inour in vitro experiments.We alsoused cysteaminewhen wereferred to a mixed disulfidebetween cysteamine andunithiol. In thiscase, cysteamine is not a separate compound but part of the molecule MDCU which consists of two parts: cysteamine and unithiolbound by a chemical bond.

2These experiments were carried out by L.I. Kotlovanova.

3These experiments were carried out by G.T. Bozhko.


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Discussion

Our study showed that the toxic action of the ami-nothiol protectors inmice canbedecreasedbycom-bining it with unithiol. This effect is significantenough to be of practical interest. The LD50 of cys-tamineandAETwhen administered i.p. increase by40and64%, respectively, which shows anantitoxicaction much stronger than that of other agents(Zherebchenko et al. 1974; Jacobus 1959; Kalis-tratov et al. 1972; Pugacheva et al. 1972; SuvorovandShashkov1975; Sverdlovet al.1969; Takagyetal. 1971). Unithiol diminishes the toxicity of cysta-mine with either i.p. or oral administration of thera-dioprotector. When AETis administeredorally, theeffectofunithiol is modest,which ispossiblyattrib-utable to AET absorption from the digestive tractand unithiol absorption from the abdominal cavity.It may be that the two compounds are not suppliedto nervous tissue concurrently and, consequently,do not work together.

The antitoxic action of unithiol was only observed

with i.p. injection; oral administration had no suchimpact. This point requires special study.However,it is necessary to stress that the radioprotective ac-tion of such thiols as cysteamine and AET with oraladministration is either not detected or weaklyexpressed. Among the aminothiol radioprotectors,cystamine, which hasno free SH-groups, is theonlyone that is efficient not only with i.p. injection butwith oral administration as well. It is felt that thepresence of free thiol groups in the unithiol mole-cule (as in the cysteamine molecule) creates thecondition that degrades its effectiveness at oral ad-ministration. The use of special medicinal form

(tablets) for oral administration of unithiol may in-directly support this fact (Mashkovskii 1967).

A new preparation with radioprotective proper-tiesmixed disulfide of cysteamine with unithiolin which aminothiol and dithiol are chemicallyboundwas studied in our experiments. Reductionof the aminothiol toxicity was detected in this case,too. There is reason to believe that the disulfide

linkage may be broken in an organism and thatunithiol may diminish the toxic effect of the re-leased radioprotector. It is worth noting from apractical standpoint that the preparation has littletoxicity with oral administration. It does notrequirei.p. injectionof unithiol to reducetheradioprotectortoxicity, which is necessary when cystamine is ad-ministered orally.

We also found that in order to achieve the antitoxicaction of unithiol vis-a-vis the aminothiol radiopro-tectors, a specificquantitativeratiobetween thetwosubstances must be maintained. The optimum ratiofor doses is 0.5 molar equivalent of unithiol per ra-dioprotective thiol. These data are liable to be ofinterest in theelaboration of the corresponding pre-scription. They are equally important for under-standing the mechanism of the unithiols antitoxicaction as regards the thiol radioprotectors.

As may be seen from our experiments, unithiols

ability to enhance tolerance to aminothiol permitslarger doses and thus a better radioprotective effectof the SH-compounds. The combination withunithiol under x-ray irradiation increased the DRFof cystamine and AET by 30% with i.p. administra-tion and by 18% with oral cystamine administra-tion. The DRF of MDCU was 1.6 at i.p. injection.Thus, the use of unithiol substantially enhances theeffectiveness of protection provided by the ami-nothiol radioprotectors. The survival rate is in-creased not only in animals irradiated with aminimum universally lethal dose but also in ani-mals subjected to supralethal doses.

A similar effect was found with fission-neutron ac-tion. The DRF of cystamine and AET when com-bined with unithiol increased in comparison withthe DRF of each individual radioprotector: the cys-tamine DRF increased by 10% and the AET DRFby 16%. Along with that, the efficiency of AET-based protection in mice was higher than the effec-tiveness observed by us previously in experiments

13


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using the aminothiol radioprotectors and theircross-combination or combination with indolylal-kylamine (Bogatyrev et al. 1983). Considering thatmodification of a neutron injury is a difficultproblem to resolve, the enhanced effectiveness ofchemical protection against fission neutron hazard

using unithiol is worthy of notice. However, the ef-fect of unithiol in irradiation of animals by neu-trons is of a lesser degree than its protectionagainstlow LET radiation.

Consequently, unithiol extended the radioprophy-lactic capabilities of traditional aminothiol radio-protectors used in conditions of both low and highLET radiation, although toa lesser degree in the lat-ter case. This effect ofunithiol does not appear tobespecific to mice. We have detected the effect inother biological species, for example, in rats (Gra-chev et al. 1994), that indicates the feasibility of

studying it in animals more closely related to hu-man beings, such as dogs and monkeys, and, in thefuture, in humans.

The study of the antitoxic mechanism of unithiolvis-a-vis aminothiol protectors is a complex issuethat may beno less complicated than the problem ofchemical protection itself. The results of our studyof unithiols influence on toxicity of radioprotec-tors of other types(5-methoxytryptamine (indolylal-kylamine) and di-ammonium salt amidothiphos-phoric acid), carried out in parallel, may be usefulforanalyzing theproblem, as we found that unithiolhas no effect on their toxicity. These data show thatthe antitoxic action of unithiol is unlikely to be as-sociated with a nonspecific increase of an organ-isms resistance to chemical agents. We are appar-ently dealing here with a specific interaction ofthiol-containing compounds, SH-radioprotectors,and unithiol, at the cellular level.

The results obtained in this study on the correlationbetween unithiols antitoxic effect and its optimumratio to the SH-containing radioprotectors providemore evidence for this assumption.Such factors are

characteristic of a cell concentration relationship

rather than an organisms reactions, which are typi-cally threshold reactions. Although our attempt todetect the antitoxic effect of unithiol in Chinesehamster V79 cells was indeed unsuccessful, thisdoes notdisprove ourassumption.It is believed thatunithiol only weakens those toxic effects of the

thiol radioprotector that arerelated tocell excitationand has no connection with the structures responsi-ble for cell growth.

This assumption is supported by the fact that thetoxic effects of theSH-radioprotectorson an organ-ism reveal themselves as a disturbance in the func-tion of the nervous system rather than by damage tocertain cells, tissues, or organs or by dysfunction ofspecificorgans. It shouldbe added that inourearlierexperiments on the same Chinese hamster V79 fi-broblasts, although using another technique (culti-vation of cells on glass), the antitoxic action of

unithiol in relation to cystamine was observed. Un-der the radioprotectors effect (10-2 mg/ml), thecells changed their shape, became rounded, andslipped off theglass. Theaddition ofunithiol (1:2 inrelation to cystamine) prevented this effect (Gra-chev et al. 1994). It is therefore worthwhile to con-tinue studying isolated cells in a culture.

However, the results of our tentative experimentson rat hippocampal slices are of a greater interest inrelation to the problem of unithiols mechanism ofaction. The stimulating effect of cystamine as wellas of unithiol in inducing activity in these neuronswas found in these experiments. Further neuro-physiological and neurochemical studies are re-quired to identify the processes underlying the anti-toxic effect of unithiol. In addition, we should notforget that these results were obtained in in vitroex-periments. Another way of influencing toxicity ofthe aminothiol radioprotectors is possible in vivo bylimiting their penetration to the brain through theblood-brain barrier. In general, the information onmechanisms of unithiols antitoxicaction presentedin this report should be considered as tentative andfacilitating development of approaches to this

complex issue.

14



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Suggestions for Further Research

Study the effect of unithiol alone and in combi-nation with cystamine or AET or MDCU on be-havioral toxicity.

Study unithiols antitoxic effect with respect toSH-radioprotectors in dogs andhumans. If suchstudies have a positive outcome, wider use ofaminothiol radioprotective compounds will be

possible in medical practice and other fields.

Study the possibilities of reducing the thiol ra-dioprotectors toxic action using unithiol ad-ministered not only i.p. but also orally (the useof tablets, capsules, and so on). Establish an op-timum timing of unithiolandcystamine admini-stration per os.

Study the mechanism of unithiol antitoxic ac-tion in vitro (neurophysiological and neuro-chemical studies of neurons) and in vivo (studythe influence of unithiol on aminothiol protec-tors penetration to the brain and certain neu-ronal structures using radionuclides).

Study the possibility of using unithiol ana-

logues, other thiol compounds (including otherdithiols), and similar compounds as antitoxicagents.

Conduct systematic studies of the new radio-protector MDCU: its absorption, distribution,elimination, and efficacy in protecting againstirradiation in other biological species.

15


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16


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INHALATION TOXICOLOGY RESEARCH INSTITUTEATTN: LIBRARY

INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCESATTN: DIRECTOR

INSTITUTE OF RADIOBIOLOGY, ARMED FORCESMEDICAL ACADEMY

ATTN: DIRECTOR

OAK RIDGE ASSOCIATED UNIVERSITIESATTN: MEDICAL LIBRARY

PETERSBURG NUCLEAR PHYSICS INSTITUTEATTN: HEAD, LABORATORY OF ORGANIC

SYNTHESIS

RESEARCH CENTER OF SPACECRAFT RADIATION SAFETYATTN: DIRECTOR

RUTGERS UNIVERSITYATTN: LIBRARY OF SCIENCE AND MEDICINE

UNIVERSITY OF CALIFORNIAATTN: DIRECTOR, INSTITUTE OF TOXICOLOGY &

ENVIRONMENTAL HEALTHATTN: LIBRARY,LAWRENCE BERKELEYLABORATORY

UNIVERSITY OF CINCINNATIATTN: UNIVERSITY HOSPITAL, RADIOISOTOPE

LABORATORY

XAVIER UNIVERSITY OF LOUISIANAATTN: COLLEGE OF PHARMACY


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REPORT DOCUMENTATION PAGE F o r m A p p r o v e d

O M B N o . 0 7 0 4 - 0 1 8 8

P u b l i c r e p o r t i n g b u r d e n f o r t h i s c o l l e c t i o n o f i n f o r m a t i o n i s e s t i m a t e d t o a v e r a g e 1 h o u r p e r r e s p o n s e , i n c l u d i n g t h e t i m e f o r r e v i e w i n g i n s t r u c t i o n s , s e a r c h i n g e x i s t i n g d a t a s o u r c e s g a t h e r i n g a n d

m a i n t a i n i n g t h e d a t a n e e d e d , a n d c o m p l e t i n g a n d r e v i e w i n g t h e c o l l e c t i o n o f i n f o r m a t i o n . S e n d c o m m e n t s r e g a r d i n g t h i s b u r d e n e s t i m a t e o r a n y o t h e r a s p e c t o f t h i s c o l l e c t i o n o f i n f o r m a t i o n ,

i n c l u d i n g s u g g e s t i o n s f o r r e d u c i n g t h e b u r d e n , t o W a s h i n g t o n H e a d q u a r t e r s S e r v i c e s . D i r e c t o r a t e f o r I n f o r m a t i o n O p e r a t i o n s a n d R e p o r t s , 1 2 1 5 J e f f e r s o n D a v i s H i g h w a y , S u i t e 1 2 0 4 , A r l i n g t o n , V A

2 2 2 0 2 - 4 3 0 2 , a n d t o t h e O f f i c e o f M a n a g e m e n t a n d B u d g e t , P a p e r w o r k R e d u c t i o n P r o j e c t ( 0 7 0 4 - 0 1 8 8 ) , W a s h i n g t o n , D C 2 0 5 0 3 .

1 . A G E N C Y U S E O N L Y ( L e a v e

b l a n k )

2 . R E P O R T D A T E

December 1997

3 . R E P O R T T Y P E A N D D A T E S C O V E R E D

4 . T I T L E A N D S U B T I T L E

Chemical Protection Against X-Ray, Gamma, and Neutron Radiation

5 . F U N D I N G N U M B E R S

6 . A U T H O R ( S )

S. A. Grachev and A. G. Sverdlov

7 . P E R F O R M I N G O R G A N I Z A T I O N N A M E ( S ) A N D A D D R E S S ( E S )

Armed Forces Radiobiology Research Institute

8901 Wisconsin Avenue

Bethesda, MD 20889-5603

8 . P E R F O R M I N G O R G A N I Z A T I O N

R E P O R T N U M B E R

CR97-1

9 . S P O N S O R I N G / M O N I T O R I N G A G E N C Y N A M E ( S ) A N D A D D R E S S ( E S ) 1 0 . S P O N S O R I N G / M O N I T O R I N G

A G E N C Y R E P O R T N U M B E R

1 1 . S U P P L E M E N T A R Y N O T E S

1 2 a . D I S T R I B U T I O N / A V A I L A B I L I T Y S T A T E M E N T

Approved for public release; distribution unlimited.

1 2 b . D I S T R I B U T I O N C O D E

1 3 . A B S T R A C T ( M a x i m u m 2 0 0 w o r d s )

Experiments in mice showed that intraperitoneal (i.p.) injection of unithiol (sodium salt of2,3-dimercapto-l-propanesulfonic acid) diminished toxicity of several aminothiol radioprotectors, increasing theLD

#

of cystamine by 40% and aminoethanisothiuronium bromide hydrobromide (AET) by 64%. The optimum ratiofor the doses is 0.5 molar equivalent of unithiol per radioprotective thiol. A new radioprotector (mixed disulfide ofcysteamine and unithiolMDCU) has a weak toxicity: the LD

#

is 750 mg/kg i.p. The use of unithiol makes itpossible to increase the dose of the SH-radioprotectors, enhancing the dose reduction factor (DRF) of cystamineand AET by 30% for x-ray irradiation. A somewhat lesser effect is observed with fission neutron irradiation. The DRFof MDCU is equal to 1.6 for x-ray irradiation and is 1.1 for neutron irradiation. The mechanism of antitoxic action ofunithiol could not be detected in Chinese hamster fibroblasts. It may be caused by the competition of unithiol and the

SH-radioprotectors for certain, as yet undetermined, biochemical structures in brain neurons. It is also possible thatunithiol may decrease penetration of SH-radioprotectors into the brain.

1 4 . S U B J E C T T E R M S 1 5 . N U M B E R O F P A G E S

321 6 . P R I C E C O D E

1 7 . S E C U R I T Y C L A S S I F I C A T I O N

O F R E P O R T

UNCLASSIFIED


O F T H I S P A G E

UNCLASSIFIED


O F A B S T R A C T

UNCLASSIFIED

2 0 . L I M I T A T I O N O F

A B S T R A C T

ULN S N 7 5 4 0 - 0 1 - 2 8 0 - 5 5 0 0 S t a n d a r d F o r m 2 9 8 ( R e v . 2 - 8 9 )

P r e s c r i b e d B y A N S I S t a 2 3 9 - 1 8


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C L A S S I F I E D B Y :

D E C L A S S I F Y O N :

S E C U R I T Y C L A S S I F I C A T I O N O F T H I S P A G E


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This publication is a product of the AFRRI Information Services Division.

Editor: JaneMyers, JanusAssociates. Desktoppublisher:CarolynWooden.

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Chemical Protection Against Radiation