Evolving Insights and Implications

Evolving

Insights and Implications

Sheldon

Wolff

Department

of

Radiology, University

of

California

San

Francisco,

San

Francisco,

California

The first of the regularly reproducible experiments to show thatvery lowdoses of ionizing radiation, likevery low doses of chemical agents, could induce mechanismswhereby cells become better fitto copewith subsequentexposures tohigh doses werecarried outon the induction of chromosomeaberrations in cultures of human lymphocytes. If cells that had been exposedtoaverylowdose(1 cGy) of Xraysweresubsequently exposedtoarelatively high dose

(1 Gy), approximately halfasmanychromosome breakswere induced. Subsequent experiments showed that this adaptive responseto low doses requiresa certain minimal dose before it becomesactive;occursonly withinarelatively small window of dose; is dose-rate dependent; and depends on thegenetic constitution of the people oranimals exposed, with some being unresponsive. Itwasfurther shown that the response tothe low-dose preexposure was not

instantaneous but tookapproximately4to6 hrtobecomefully active, and could be prevented if during this period protein synthesiswasinhibited, i.e., a necessary protein(enzyme) wasbeing induced. In fact, subsequent experiments with two-dimensional gel electrophoresis showed new proteins in cells irradiated with 1 to2cGy. The adaptation induced by low doses of radiationwas therefore attributedtothe induction ofanovel efficientchromosome break repair mechanism that ifactive atthe time of challenge with high doses would leadto less residual damage. This hypothesiswasstrengthened byaseries ofexperiments in which itwasfound thatinhibitors of

poly(ADP-ribose)polymerase, an enzyme implicated in DNAstrand breakrejoining, couldprevent

theadaptiveresponse.Although the phenomenon is well established in cellularsystems,it is still problematicalastowhetherornotitwillhaveanyutility inestablishingrisks ofionizingradiationto

humans. Newer experiments havenowbeen carriedoutonthe mechanisms underlying the effect andwhetherornotthe effectcanmanifest itselfas adecrease in the number ofinducedcancers and radiation-induced mortality. Experiments with restriction enzymes nowindicate that double-strand breaks inDNAcanbetriggeringeventsinadaptation. Inaddition, preliminary experiments onthe survival ofwhole-body irradiated mice have shown that multipleexposurestolowadapting dosescanhaveprofound effectsonsurvival, and other experiments have shown thatadaptation canaffect the inductionofthymic lymphomainirradiated mice. Itthereforeappearsthatthe initial experiments behind theadaptive responsehave ledtoavigorous worldwide efforttounderstand the basic mechanisms behind it.This effort isstimulated both byadesiretounderstand the basic cell biology behind the response and adesire to see ifindeed this phenomenon affects the estimation of risks of low-level radiation exposure. Environ Health Perspect 106(Suppl 1): 277-283(1998). http.//ehpnetl.niehs.nih.gov/1998/Suppl-1/277-283wolff/abstract.html

Key words: adaptiveresponse,chromosomeaberrations, cytogenetics, adaptation, ionizing radiation, human lymphocytes, restrictionenzymes,repair, radiation risks

Introduction

Because of the uncertainties

surrounding

statistically valid experimental data has been the

shapes

of

dose-response

curves atlow to

extrapolate

the results obtained at

high

levelsof ionizing

radiation,

the usual model dosesto those

expected

in the low-dose for

establishing

risks of radiation atvery region. This

procedure

has been

contro-low doses whereit is not

possible

to obtain versial as to whether the

extrapolation

This paper is basedon apresentationatThe Third BELLEConferenceonToxicologicalDefense Mechanisms and theShapeofDose-ResponseRelationshipsheld 12-14November 1996inResearchTrianglePark,NC. Manuscript receivedatEHP29April 1997; accepted3September1997.

This workwassupportedbycontractDE-AC03-76-SFO1012from theOffice of Health and Environmental Research, U. S. Department ofEnergy.

AddresscorrespondencetoDr.S.Wolff,RadiationEffects ResearchFoundation,5-2HijiyamaPark,

Minami-ku,Hiroshima,732,Japan.Telephone:81082 261 3131.Fax:81 082 2637279.E-mail:[email protected]

Abbreviationused:UNSCEAR,United NationsCommitteeontheEffects of AtomicRadiation.

dose

by

definitionincreases

linearly,

b)

should involve a

threshold,

which could

resultfrom

biological

processes that

modify

the initialresponses or

perhaps

evenreflect thenatureofthe

biological

event

(e.g.,

mul-tistep carcinogenesis),

or

c)

should be

greater than

linear,

because

radiation-induced homeostatic processes could be

inactiveatverylowdoses.

Although

there

are

underlying

uncertaintiesinallthese

pro-cedures,

the linear

hypothesis

has been the onemost

accepted

by regulatory

agenciesas

being

a

prudent

way toestimateriskand

thuspreventundue harmtothe

public.

As

early

as 1976some

high-dose

experi-mentswith

algae

and desmidsindicated that a

complex

biology

could

possibly modify

a simple linearresponseto ionizingradiation.

Experiments ofthistype,which invoked the inductionof therepairofpotentiallylethal

damage, however,

did notspeaktowhat

might possibly happen

atlow doses where datawere unobtainable and for which the above-mentioneduncertaintiesexisted.

The first of the

regularly

reproducible

experimentstoshow thatverylowdoses of ionizing

radiation,

likeverylow doses of chemical agents,could inducemechanisms

whereby

cellsbecame somewhat

refractory

to theinduction of

damage

by

subsequent

exposuresto

high

doseswerecarriedouton

the induction ofchromosome aberrations

incultures ofhumanlymphocytes.

Asaresult ofthiswork, others carried outsimilarexperiments with other

biologi-cal endpoints and othertypes of

cells,

and theevidencekeptmountingthat thiswasa

newly

discovered phenomenon ofgreat

intrinsic interestandpossibly of

practical

importance in understanding whatmight

actuallyoccur at verylow doses and how this might affect risk estimation. Soon, enough evidenceonthis adaptive response to low doses had been gathered from all

overthe world for the United Nations

Committee on the Effects of Atomic Radiation (UNSCEAR) to point out that therewas no doubt about the phenome-non's occurrence atthe cellularlevel, but that itwasstill problematic ifithad

practi-cal consequences inestimatingdamage to

populations and,inparticular,inestimating

cancerrisks (1).

Early Experiments

Intheinitialexperimentsontheinduction

ofadaptation by low doses of ionizing radiation, humanlymphocytes that had

incorporated tritiated thymidine (2) were

exposedto lowdosesof chronic radiation

as the tritiumdisintegrated, andthe cells

were subsequently exposedtothe relatively highdose of150 cGy ofXrays.

Approx-imatelyhalfas manychromosome

aberra-tions wereinduced asincells that had not

incorporated the radioisotope. This result

with very low doses of radiationwas

remi-niscentof the adaptive response found in

cells treated with low chronic dosesof

alky-latingagents, which made those cells less

sensitivetohigh doses of thesame orother

alkylatingagents (3,4). Subsequent

experi-ments inwhich theamountofradioisotope

wasgreatly reducedsoonshowed that the adapting, orconditioning,doseneednotbe

chronicbecause asingleinstantaneous

dis-integration of incorporated tritium could

bring itabout (5). Further experiments

then showed that preexposureof the

lym-phocytes to X-ray doses as low as 1 cGy

couldcausethecellstoadapt(6).

Inexperimentswith chemical agentsit

was also found that cross-adaptation occurred. That is, exposureof cellstolow dosesof radiomimeticchemicals, alkylating

agents, cross-linkingagents, orionizing

radiations allcouldlead to a decreaseinthe

cell'ssensitivityto thesameagentoranyof

theothers(7).

The adaptation inducedbylowdoses of

radiation was attributed to the inductionof

anovel, efficientchromosome-break repair

mechanism that, if present at the timeof thechallengewithhighdosesofradiation, wouldresult inlessdamage afterthe

expo-sure (2,5).This hypothesis was

strength-enedbyexperiments inwhichit wasfound

that an inhibitor (3-aminobenzamide) of

poly(ADP-ribose) polymerase, anenzyme

implicatedin DNAstrandbreak rejoining,

prevented the adaptiveresponse(5,6).Itdid

this evenwhenit wasadministered afterthe

challenge dose but within thetimebefore inducedchromosome breaksrejoined,which

indicated that thedecreasewascausednot

byachangeinthe initial sensitivityof the cells but

by

apostexposurephenomenon

suchasrepair(5).

It was soonfound thattheresponseto

apreexposure to low doses ofradiation

was not instantaneous but tooksome4 to

6 hrto becomefully active (8), and that

this responsecould be prevented during

thisperiodif protein synthesiswas inhib-ited (9). That is, a necessary protein

(enzyme) was being induced, and

subse-quent experiments with two-dimensional

gel electrophoresis indeed, did show new

proteins incellsirradiatedwith 1 to2cGy

(10) and one-dimensional gel

electrophore-sis indicated thata protein that binds specificallyto radiation-damagedDNAis produced (11).

New Work

Inspiteof all these advances attempts are

stillbeing madeto seeifexplanationsother thaninduced repair can accountforthe

phenomenon, and todefineatthe

molecu-lar level the exact mechanism(s) behind

adaptation. In additionattempts are also being madeto seeiftheadaptiveresponse canaffect the survival of animals after whole-body irradiation and if fewercancers areinducedinpreexposed animals. During thelast severalyears mylaboratoryhas

car-riedout several experiments designedto

obtain further information on various

aspectsoftheadaptiveresponse.

Differential

Display

of

mRNAs

Forexample, furtherattempts have been

madeto find amethod that could leadto

identification ofthe molecular processes

involvedintherepairmanifestedas

adapta-tion.BecauseBoothmanand his colleagues

have foundchangesingene transcription

levelsafter exposure to ionizingradiations,

(12,13), experiments nowhave been

car-riedout todetermineif thelow dosesofX

raysthat result in adaptation can indeed

inducechangesinthe transcription level of

genes involved inregulation ofthe DNA

repairpostulatedto beinvolved in

adapta-tion. In these experiments, human lym-phocytes exposedto an adapting dose of

2cGyofXrays wereanalyzed to seeif

dif-ferentspecies ofmRNAs werepresent in

the irradiatedcells, aswould betrueif gene

activitywere induced. Asuitable set of

primers isused.Oneof theseis anoligo dT

that anchors to the polyA tails of the

mRNAs; the other is one ofagroup of

arbitrarydecamers thatanneals at various

positions relativetotheattachedoligo dT

primer. In this technique, when four

anchored dTprimers areused in

conjunc-tionwithatleast 25 upstreamdecamers, almost allof the expressed mRNAspecies

can be reverse transcribed to cDNA

(14,15). These cDNAs are thenamplified bythepolymerasechainreactionwith

arbi-trarydecamer primers, andtheDNA

frag-ments representing the 3' termini of

mRNAs areseparated bysize on a

denatur-ingpolyacrylamide gel. Any cDNA

frag-ments corresponding to differentially

expressed genes thatproduced mRNA in

irradiated cellswere compared with the

cDNAfragments found in unirradiated

(control) cells. It has been found (Figure 1)

that the irradiated cells havesome mRNAs

not present in control cells. The

cor-responding cDNAs of these represent

appropriate candidate genesthatcould be

involved in the repairmanifestedas

adapta-tion. The irradiated cells also lacksome

mRNAs present inthe control cells. In

addition some cDNAs madefrommRNAs

were found in control cells but not in

adapted cells. These, then, could represent

candidatesuppressor genes that could be

involved inthe regulation of the (DNA)

repair. Theseexperiments areproviding

access tothe DNA involved in theadaptive

responseand indicate that the response

might be attributable eithertothe

induc-tionofnewenzymes or to the loss ofa

repressor intheirradiatedcells.

Induction ofthe

Adaptive

Response

by

DNA

Double-strand

Breaks

All the chemicals and radiations that

induce the adaptive response are not

spe-cific in thatthey produceaspectrum of

lesionsin DNAsuchas basedamage,

sin-gle-strand breaks, and double-strandbreaks

thatin principle couldberesponsible for thephenomenon. Because ionizing

radia-tions areefficient inducersofDNA dou-ble-strandbreaks andtheadaptiveresponse is mediated by poly(ADP-ribose)

poly-merase, anenzymestimulated inresponse

tosuch breaks, it isthought that

double-strandbreaks alone might be the lesions

responsible.Toseeiftheyalonecaninduce adaptation, wecarried outexperiments withrestrictionenzymes,which unlike

ion-izing radiations and radiomimetic

chemi-cals, only induceonespecifictypeoflesion,

DNAdouble-strand breaks, bybindingto

specific DNAsequences,or recognition

sites,andcleavingtheDNA atthesesites.

The experimentswerecarried outwith

humanlymphocytes inwhichrestriction

enzymeswereintroduced into thecellsby

electroporation toproduce different

num-bersofDNAdouble-strand breaks of

vari-oustypes.Cellsintowhichonly thestorage

buffer for theenzymeswaselectroporated servedasthe controls. AluI,whichinduces

alarge number of blunt-endDNA double-strand breaks at the recognition site

AG/CT; DraI,which inducesfewer

dou-ble-strand breaks at therecognition site

TTT/AAA;

and NotI,whichinducesonly

very fewstaggered-end double-strand breaksattherecognition siteGC/GGCGC,

wereused.

All three ofthe restriction enzymes

induced adaptation in thatthey reduced

Environmental HealthPerspectives * Vol 106, Supplement * February 1998 278

T12MA

2 3 4 5 6 7

c 2r c 2r c 2r c 2r c 2r c 2r

0

-

Gain

Table 1.Adaptiveresponseinducedby5U ofA/ula

(nativeand heatinactivatedb).

Expected Deletions/ deletionsc/ Treatment 100cells 100 cells

SB 2

-SB+150cGyX rays 69

Alu 35

-Alu + 150cGyX rays 60 102 A/u (heatinactivated) 12

-A/u (heat inactivated) 59 79

+150cGyX rays

SB,storagebuffer.&RecognitionsiteAG/CT. b100 cells/point.cSum of deletions seen in slides of cells

exposedto 150cGy after electroporation in presence ofSB,plusthose seen in slides electroporated with A/u alone. Because both ofthesenumbersinclude the control (SB) level ofdeletions, one control level is subtracted.

12 13 14 15 16

c 2r c 2r c 2r c 2r c 2r

Figure1.Polyacrylamide gelofcDNAsobtainedbythe differentialdisplayof mRNAtechnique.1

oligodTprimerthat boundtothepolydT tails of the mRNA.Columns 1to16wereobtained with1E traryupstream decamerprimers.Abbreviations:c, controlcells;2r, irradiated cells.

the number of chromosome breaks

producedbyasubsequentexposure to 150

cGy ofXrays. When the cells were

pre-treated with Alu I, the yield of chromatid

aberrations found after 150 cGyofXrays

was60%, whereas the sumof the

aberra-tions inducedbyAluIalone and 150cGy

ofXraysalonewas 102%

(Table

1).

Inthese experiments the controls

always

consisted of cells

electroporated

with the

restriction enzymes' storage buffers,which

contain bovine serumalbumir

to introduce a more specific

which heat-inactivated AluI

ducedinto the cells showed th

treated enzyme, too, led to

(Table 1). That the heat-i

enzymewas inactive in cleavin

confirmed by assaying for

enzymeactivity by

incubating

the plasmidpHAZE withact

inactivatedAluI in vitroandr

DNAfragments on an agarosegel. DNA

digestedwith activeAluI showed multiple

bands, whereas DNAexposed tothe

heat-inactivatedenzymedidnot, i.e., was uncut (Figure 2).

That the heat-inactivated enzyme,

which induced adaptation, might have renaturedinsidethe cellandthus regained its abilityto cleave DNAwasshown in

experiments on theinductionof mutations

intheshuttle vector pHAZE,which can

replicatein both mammalianand bacterial

cells. Itwasexposed to heat-inactivated

enzymewhile it wasmaintained as an

epi-*- Loss someinhuman lymphoblastoidcells (Raji)

N1 Loss cells. Theplasmid recovered fromthehost

containedenzyme-induced mutations

selected in Escherichia coli (Table 2).

Therefore, heat-inactivatedAluI, which had

noenzymatic activity in vitro(Figure 2),did

inducemutationsinpHAZEafterits

intro-ductioninto cellsby electroporation. The

experiments indicate thatonce inside the

cell, thedenaturedenzyme can renature

and become abletocutDNA.Theyfurther

indicate the importance ofusingan

unre-[f2MA

was

the latednonenzymatic protein such as bovine 6different arbi- .

serum

albumin

as a proper control to

ascertain whether theeffects observed

after exposureto a restriction enzyme can

n.Attempts be attributed to anonspecific effect of

control in proteins ingeneral.

was intro- Similarexperiments with DraI, which

atthe heat- induces far fewerDNAstrandbreaks and

adaptation thus fewer chromosome aberrations than

nactivated AluI, reduced by approximately45% the

g DNAwas number of chromatid breaks subsequently

restriction induced byXrays (Table 3), indicating

DNAfrom once again that blunt-end double-strand

ive orheat- breaks themselves are lesions capable of

*esolvingthe inducingtheadaptiveresponse.As

before,

l l

c c 2r

8 9 10 11

Table 3. Adaptive response induced by 10 U of Dra la (native and heat-inactivatedb).

Expected

Deletions/ deletions/

Treatment 100 cells 100 cells

None 3

-SB 2

-SB+150cGy X rays 54

-Dra 8

-Dra +150cGyXrays 41 60 Dra (heat inactivated) 4

-Dra (heat inactivated) 37 56 +150 cGy X rays

aRestrictionsiteM/AAA.bl00 cells/point.

Table 4. Frequency of chromatid and isochromatid breaks in humanlymphocytes after electroporationa with Not 1.

Chromatid and isochromatid Treatment breaks/200 cells

Control 2 SB 17 Notl, U 200 1 100 9 25 11 12.5 8

'Electroporationwascarriedout at48 hr of culture (i.e., after stimulation with phytohemagglutinin[PHA]).

All cultures were harvested at 72 hr. Table 5.Adaptation induced by 12.5 U of Not la(nativeand heatinactivated).

Expected

Treatment Deletions, no deletions, no

Experiment 1: 100 cells scored/point

None 3

SB 4

SB+150cGy X rays 66

Not (heat inactivated) 2

Not (heat inactivated) + 150 cGy X rays 39 66

Experiment2:200cellsscored/point

None 2

SB+150cGyXrays 79

Not1 8

-Not1 +150cGy X rays 51 85

aRecognition

siteGC/GGCCGC. Figure 2.Cleavageof DNA by native but not

heat-inactivatedA/u I. Lane 1, uncut pHAZE; lane 2, A/u I, lane 3, heat-inactivatedA/u I, lane 4, 1-kb ladder. Table 2.Frequency of lac-Zmutantsinduced in intra-cellularpHAZE by previously heat-inactivated A/u l.a Treatment Mutationfrequency,

xlO-4

SB A/u1,200U

Heat-inactivated A/uI,500 U

aRecognitionsiteAG/CT.

2.74

5.89

7.28

thepreviously heat-inactivated enzyme

couldcausethe effect.

Various concentrations of the restriction

enzymeNotI (12.5 to200 U/0.8ml)were

introducedinto the cellsby electroporation

48 hrafter stimulationwith

phytohemag-glutinin, and thecells werescored 6hrlater

to see ifchromatidaberrations could be

induced. This restriction enzyme, which

cuts DNAinfrequently, did notinducea

significant number of chromosome

aber-rations even atthehighest dose (Table 4).

It did, however, induce the adaptive

response incells subsequently exposed to

150cGy(Table 5) evenatthe lowest

con-centration, whichreduced the number of

X-ray-induced aberrations by 40%.

The experiments show that DNA double-strand breaks with either bluntor

staggered ends can be the lesions that

induce the adaptiveresponsewhereby cells

become lesssusceptibletothe inductionof

cytogeneticdamage byexposuretohigher

doses ofradiation. Itcannotyetbe ruled

out, however, if thestaggered ends are

processedinto blunt ends by intracellular

processes. The experiments furthershow

that the responsecan beinducedbyvery

low levels ofbreakage, i.e.,levels thatare

notreflected in anobservable increase in

chromosome aberrations.

Independence of

the

Adaptve

Responseon

Induc

C cle

Delays that Allow

MoreTime

for Repair

End points other than chromatid breaks

have been usedinexperimentsonthe

adap-tive response. Forinstance, experimentson

cellular survivalresponseshave beencarried

out (12,16). Ithas beenproposed that the

adapting dose, even though verylow,

inducesaneffectoncellcycling,perhaps by

signal transductionmechanisms, and that

this is reflected inchangesinsensitivityto

the killingeffects of radiation. For such

mechanisms to accountfor the adaptive response that causesa reduction in the number of chromatid aberrations observed

at metaphase only6 hr after achallenge

dose of 150cGy, however, a2-cGy

adapt-ing dose would havetoinducea G2 (not

G,)

delaythatpurportedlywould allow

more timeforrepairto occurbefore the

cells challengedwith 150 cGy reached

metaphasewheretheycould be scored.

Thishypothesiswastested inexperiments

inwhich humanlymphocytesfrom amale (XYchromosome constitution) and a

female (XX chromosome constitution) weremixed in various combinations after

being exposed toeither no radiation, an

adapting dose,achallenge dose, orbothan

adaptingand achallengedose. The

mix-turesweremade at the time of the

chal-lenge dose andthe cellswerecocultured for

6 hruntilfixation, atwhich time the

pro-portions of cells at metaphase from the

male and the female could be observed

cytologically. Thelymphocytes from both

sexes showedtypical adaptive responses

whenpreexposedto2cGyof Xraysbefore

being challengedwith150 cGy (Table 6).

Regardless of the treatment, the

percentageof cocultivated male andfemale

Environmental HealthPerspectives *Vol 106, Supplement1 * February 1998

1 2 3 4

im .:.,.

cells reaching metaphase stayed the same

(Table 7).That is, when 200 consecutive

metaphaseswereobserved6 hraftermixing, 55 untreated malecells and 145 untreated

female cellswerefound. When bothgroups of cells received 150 cGy ofXrays before mixing, 48male cellsand 152 female cells appearedatmetaphase,which is not signifi-cantlydifferent from the 55 and 145 cells

seenwithnoradiation exposure. Exposing cells to anadapting dosebefore the 150

cGychallenge didnot inanycombination affect the speedatwhich the cellsappeared

at metaphase, so thatthe proportion of male cells and female cells didnotchange.

These experiments indicate that

adapt-ation is notcausedbyachangein therate

of cell progression to mitosisafter a chal-lenge dose, and areafurther indication that cell stage sensitivity isnot afactorin

theadaptive response observedasdecreased cytogeneticdamage.

Worldwide Experments

with Other

End Points

Stimulatedby the

early

work, other groups

soonfound that thisadaptation

phenomenon

Table 6.Adaptive response inhumanlymphocytes

from a male and a female.

Deletions/cells, Deletions/cells,

Treatment male female

Control(unirradiated) 2/100 1/100

2cGy 3/100 5/100

150cGy 39/100 42/100

2cGy+150cGy 27/100 27/100

Theadaptingdose(2cGy)wasadministeredat24 hrof culture(i.e.,after stimulation withPHA),thechallenge

dose(150cGy)at48 hr.Cellswerefixedat54hr.

Table7.Lack of effect ofadaptingdoseof2cGyof X raysoncellprogression of cellschallengedwith 150

cGyof X rays.

X-raydose,cGy Cellsatmetaphase

Male Female Male Female

0 0 55 145 2 0 58 142 2 2 53 147 2 2 54 146 150 150 48 152 2+150 150 52 148 150 2+150 54 146 2+150 2+150 53 147

Theadapting dose(2cGy)wasadministeredat24 hrof culture(i.e.,after stimulation withPHA),thechallenge

dose(150cGy)at48 hr.Equalvolumes of each culture thenweremixed and the cells cocultured for another6 hr.Colcemid (2x10-7M)waspresentfor thelast2 hr.

Two hundredmetaphase cellswerescoredto deter-minewhethertheycamefrom the male(XY)orfrom

thefemale(XX).

was not restricted to the induction of chromatid deletions. For instance, experi-ments with the HPRTlocus in human lymphocytes showed that exposure to triti-ated thymidine (17) or 1 cGyofXrays (18) couldmarkedly decreasethe number of mutations induced by subsequent high doses of radiation. The response tolow X-ray dosesvirtuallyeliminated the effects of thechallenge dose.

Becauseradiation-induced mutations can beeithersmall point mutations or larger chromosomaleventssuch as deletions (which predominate), experiments were now carried

out todeterminethe natureof the mutagenic

eventssubject to adaptation.Inthe normal humanlymphoblastoid cell line AHH-1, Rigaud et al. (19) examined 94 7-ray-induced mutants by Southern blot analysis after digestion of the DNA by the restriction enzymesPstI or Eco RI.They founda4-fold reductioninthe number of mutants induced by4 Gyincellspretreated with 2cGyof

7-rays

(Table 8). Molecular analysis of the natureof the mutations showed that 78% of the mutants induced by4 Gy alonehad detectable changes inthe gene, i.e., had deleted bands or newbands after elec-trophoresis. Incellspreexposed to2 cGy of

7-rays,

however, the proportion of mutants induced by4 Gy that werecharacterized by loss or rearrangement of the gene was reduced to42% (Table 9). As expected, the proportion of mutants that had unchanged restriction fragment patterns increased Table 8.Adaptiveresponseof human lymphoblastoid

cells(AHH-1) inducedbya preexposureto2cGy of y-rays:HPRT mutations.

Mutantfrequency Treatment Observed,x106 Induced, x106

Control 11.5±2.7

0.02Gy 8.9±2.8

-4.0Gy 70.7±21.4 61.8

0.02Gy+4.0Gy 24.4±8.6 15.5

Exponentially growingcellswereexposedtoy-rays

from137Cs.The interval between the lowadapting

dose and thehighchallengedosewas6 hr.Data from

Rigaudetal.(19).

Table9. DistributionoftypesofHPRT mutantsaftery-ra

accordingly. Thus, when the cellswere

preexposed to a low adapting dose, a

changein the molecular spectrumof radia-tion-induced mutationswas found. The preferential decrease in those premutational lesions that led to deletions is consistent with the interpretation of the early work that verylow doses of radiation induce a

chromosome

break/repair

mechanism. Consistent with thisis the workcarried outby Zhou and colleagues (20) on the induction of HPRTmutations as well as the repairof DNA double-strand breaks in

DNA in amousemammary carcinoma cell

line (SR-1).When cells were irradiated with

0.01 Gyof

7-rays

and thenchallenged with

3 Gy18or24 hrlater,approximately halfas

many mutationswereinduced as when cells

were irradiatedwith only 3 Gy of

7-rays.

Furthermore, the rate of repair ofDNA

double-strand breaks-the lesions responsi-ble for chromosomal breaks (21-23) increasedincells thathad been preexposed.

The data on induced mutations illustrate two otherknown aspects of

adap-tation. That is, after the adapting dose it

takes time for theinduction to occur and

onceinduceditdisappears with time. The

SR-1 data showthat in this system the effect takes more than 6 hr to become operable; it thendisappears if 48 hrelapse between thetwodoses, i.e., theeffect lasts between 30 and 41 hr. As noted earlier,

previous experiments on the induction of chromatid deletions in human

lympho-cytes showed thatin those cells it took 4 to 6 hr foradaptation to become fully operable (8) and the effectwas foundto

lastfor three cellcycles,

about

40 hr.

Inother mutational studies, Fritz-Niggli and Schaeppi-Buechi (24) showed that

dominant lethalmutationsinduced in vivo

in the classicalgenetic organism Drosophila

melanogasterwere alsosubject tothe

adap-tiveresponse.Repair-proficient (yw) aswell

as repair-deficient (mei 41, mus 302)

strainsshowed theeffect.That is, notonly

doesadaptation occur in vivoand ingerm cells, butasearlier noted itis unrelated to

3ys.

Mutants,no

Rearranged:

partial deletion Total

Treatment Analyzed Unchanged Deletions ±newband changed

Control 24 17(71%) 1(4%) 6(25%) 7(29%)

0.02Gy 17 10(59%) 2(12%) 5(29%) 7(41%) 4.0Gy 27 6(22%) 7(26%) 14(52%) 21(78%)

0.02Gy+4.0Gy 26 15(58%) 6(23%) 5(19%) 11(42%)

thewell-known standard postreplication

andexcision repairmechanisms.

Otherend points that are the result of

chromosomal damage have also been

observed. Ikushimafoundareduction in

the number ofmicronucleiinducedinV79

Chinese hamstercells byradiation ifthe challengedoseof1 cGy was preceded by

an exposure toeither tritiated thymidine

(25) or 5 cGyof7-rays (26).

Azzam

et al.

(27) alsofound this same end point to be

reducked

in anormalhumanfibroblast line

(AG1522) exposedto chronic radiation

(4.25 Gy at 0.003 Gy/min) beforebeing

challenged immediately with 4.25 Gy

given inless than2min.

As might beexpected from the results obtained in Drosophila, adaptation does

not appear toberestrictedtocultured cells;

italso canoccurwhen animals are

irradi-ated in vivo.Thus, CaiandLiu (28)first

showed that adult male Kunming mice

preirradiated with 1 cGy of X rays had

fewer chromatid deletions induced in their

somaticcells (bonemarrow)and germcells

(spermatocytes) byachallenge dose of75

cGy given 2.5 to 3hr later. When female

C57B16 mice were used, adapting doses as

low as 2 cGy were effective, although in

this experiment no germcellswerescored.

Experimentswithyetanotherstrain of

mice(white SHK) byGazievand

co-work-ers (29)showed that chronicirradiation,as

used inAzzam etal.'s experiments with

human fibroblasts (27), could also be effectiveinwholeanimals; i.e.,the number

ofmicronucleiinduced inthe bone

mar-rowby 1 Gyof

y-rays

was reduced when

the mice werechronically irradiatedwith

dosesrangingfrom12to500cGy.

Ingeneral, however, notallindividuals

or strains of animalsareequallyresponsive

to adapting doses. Inhumans, individual variabilitywasfound by Sankaranarayanan

et al. (30), who observed the adaptive

response in 8 of9 subjects tested, and by

Bosi and Olivieri (31), who observed the

response in 14 of 18 subjects tested.

Similarly, strain differences have been foundinexperimentswithmice.Wojciket

al. (32) observed adecrease inthe

aberra-tions found inlymphocytes ofonly

one-thirdtoone-half of theC57B16 micethat had beenpreexposed in vitro to 10 cGyof 7-rays at 32hr of culture andwerethen challenged with 1.5 Gyat48hr. Noother combinations of dosesor times weretested. When, however, they carriedoutsimilar

experimentswithvariousprotocols in the

Heiligenberger inbredstrain ofmice, no

sucheffectwas found. Whentheseresults

arecompared with those of Cai and Liu

(28), Gaziev et al. (29), and Zhou et al.

(20), it appearslikely that the genetic

con-stitution ofthe mouse strains can be a

determiningfactorintheir responsiveness. The apparent genetic variability that

determines whetheror not a person or a

strain ofanimal will react to alow dose of

radiation is consistent with the hypothesis

thatgenetically competent cells have a

dam-age-inducible repairmechanism that can

affecthow thecellresponds to a subsequent

insult. Experiments have shown thatin

human lymphocytes (10), V79 cells (26),

and human Ul-Mel cells (12), new

pro-teins areinduced afterexposure to low

dosesof radiation, presumably byactivation

ofgenes.

The mechanisms proposed for the

adaptiveresponsehave also been invoked

toexplainthe finestructure nownoted in

dose-effectcurves forsurvival of irradiated

mammalian cells in culture (33). When

carefulplating techniques are used to

define the low-doseregionof the survival

curves, thecells exhibitextremesensitivity

to low doses that is not predicted by

extrapolatingthe response from higher doses backward. As the radiation doses

increasebeyond about 0.3 Gy,

radioresis-tanceappears; this becomes maximal at

dosesofabout 1 Gy. It isthoughtthat it is

this induced radioresistance that

character-izestheusual ordinary cell survivalcurves.

Experimental treatments that inhibit the

adaptiveresponse also inhibit the

induc-tion of this radioresistance, strengthening the relationtoadaptation.

Cytogeneticists longhave known that

chromosome aberrations constitute anend

pointthat could be relatedtothe induction

ofcancer, althoughtherewas norationale

to connectthemcausally. Thediscovery of

oncogenesandthe exquisite control

mecha-nismsbywhich translocationsofoncogenes

topromoter regionsonotherchromosomes

could lead toactivation, as in thecase of Burkitt'slymphoma, orby which deletions

ofasuppressorlocuscouldalsoleadto

acti-vation, asin the case ofretinoblastoma,

gave adirect mechanism throughwhich

chromosomal orcytogenetic damagecould

lead to cancer. Therefore, itisnot

unrea-sonable to think thatanadaptiveresponse

that reducescytogeneticdamage mightalso

reducecancer. Because of thelonglatent

period forcancerinduction,however,ithas

notyetbeen determined whether

adapta-tion does, indeed, lowercancer rates in

humanpopulations preexposed to low

doses,andastheUNSCEARreportnoted,

it is notclear whether or not the adaptive

responsewill have any utility for the

devel-opment ofradioprotectionguidelines.As a

result a worldwideeffort isbeing carried

out to see if low-dose preexposures can

modify the amounts ofcancerinduced in

irradiated animals or even increase the

survival ofthoselethallyirradiated.

Prelim-inaryexperiments nowindicate thatboth

possibilities mightoccur.

In India, Bhattarcharjee (34) found

that when hepreirradiatedSwiss mice for

5 dayswith7-raysattherateof1 cGy/day, thymic lymphomawas induced in 16%

(8/50) oftheanimals. Ahigh 2-Gydose

inducedlymphomasin46%(23/50)ofthe

mice,whereas ifthe animalswere

preirradi-atedbefore exposure to the 2-Gydose, only 16%ofthemdevelopedthe cancers;

i.e.,thepreirradiationseemed to cancelthe

induction ofthymic lymphoma bythe

high dose(Table 10).

In Japan, Yonezawa and co-workers

(35) havecarriedoutexperimentsinwhich

theylethally irradiated21-ICRmice with

8GyofXrays.About30%survived30days

afterthe irradiation. When the animals werepreirradiated with 5 cGyofXrays, the survival rate increased to about 70%.

Conclusion

Much remains to be learned about the

adaptiveresponsewherebyexposuretovery

low dosesofradiation resultsinless

dam-agebeing inducedby subsequentexposures

tohigh radiationdoses. Uncertaintiesstill

existabout many aspectsofadaptation and

its underlyingmechanisms. What is

cer-tain, however,is that thephenomenon is

real,andthatavigorousworldwide effortis nowunderway to understand the basic

mechanismsinvolved. Thiseffortis

stimu-lated both by a desireto understand the

basic cell biology behind theadaptive

responseand adesireto seeif, indeed, this

phenomenonaffects theestimationofthe

risksoflow-levelradiation exposure.

Table 10.Adaptive response of60Co y-ray-induced

thymiclymphomain mice.

Micewiththymic

Dose Mice,no lymphoma,no

0 50 0

5xlcGy 50 8(16%)

2Gy 50 23(46%)

(5x1

cGy)

+2Gy 50 8(16%)

Data fromBhattacharjee(34). Eight-toten-week old Swiss miceweresacrificed 240to260daysafter y-ray exposure: 1cGy/dayfor 5days,2Gy24 hrafterlast adapting dose.

EnvironmentalHealthPerspectives *Vol 106,Supplement 1 * February1998 282

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